A SYSTEM FOR THE CAPTURE OF A BIOLOGICAL OR CHEMICAL ENTITY I

- 1 -

Technical Field

The present invention relates to complexes of silica-containing materials and peptide linkers for the capture of biological and chemical entities. In particular, the present invention relates to the use of complexes of silica-containing materials and peptide linkers comprising capture moieties for the capture of biological and chemical entities.

Background

Various systems are available for the capture of biological and chemical entities

j

from a sample. However, such systems typically require the production of reagents specific

i

for particular entities such as bacteria, proteins or organic compounds.

Silica has been used widely as an inert and stable support for enzyme immobilization owing to its high surface area and controllable pore diameter which can be tailored to the dimension of a specific enzyme. Zeolites are inorganic materials containing silica in a highly ordered structure and can be synthesized as nano- and microcrystals. They offer desirable characteristics as support materials for the immobilization of proteins such as high mechanical and chemical resistance coupled with a high surface area. Several proteins have been shown to adsorb to zeolites and retain their biological activity. However, absorbing proteins to mesoporous silicates typically results in decreased biological activity of the protein.

The present invention is predicated on the inventor's finding^ that a complex of a silica-containing material and a linker peptide in combination with a Rapture moiety, can be used to capture biological and chemical entities.

Summary of the Invention

In a first aspect there is provided a method for capture of a biological or chemical entity comprising:

(a) complexing a silica-containing material with a peptide linker optionally comprising a capture moiety, wherein said peptide linker is capable of interacting, directly or indirectly, with the entity thereby forming a complex of the silica-containing material and the peptide linker; and

(b) contacting said complex with a sample comprising the entity under conditions permitting interaction of the peptide linker with the entity. I

In a second aspect there is provided a method for capture of a biological or chemical entity comprising: contacting a complex of a silica-containing material and a peptide linker optionally comprising a capture moiety, wherein said peptide linker is capable of interacting, directly or indirectly, with the entity under conditions permitting interaction of the peptide linker with the entity. The entity may be present in a fluid, such as a liquid or a gas.

In a third aspect there is provided a method for reducing oad of a biological or chemical entity in a sample comprising: contacting a complex of a silica-containing material and a peptide linker optionally comprising a capture moiety, wherein said peptide linker is capable of interacting, directly or indirectly, with the entity under conditions permitting interaction of the peptide linker with the entity.

The sample may be a fluid, suspension or emulsion. The fluid may be a gas or an aqueous solution such as water or waste water. The suspension may be a cell culture.

The sample may be an enzyme-catalysed chemical reaction. The sample may be a biological sample selected from the group comprising: blood, plasma, urine, cerebrospinal fluid, sweat, tears, saliva, faeces, exudate, excretions and secretions.

The complex of the silica-containing material and the peptide linker may be present in a liquid or disposed on a solid support. The solid support may comprise a substrate selected from the group comprising: a particle, a bead, a powder, a fibre, a coating and a surface of a vessel or chamber. The solid support may be a substantially planar substrate

i

such as a strip or dipstick.

In a fourth aspect there is provided a complex comprising a biological or chemical entity, a peptide linker and a silica-containing compound.

The peptide linker may comprise the sequence (VKTQATSkEEPPRLPSKHRPG) _n where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The peptide linker may further comprise a fragment of the sequence VKTQATSREEPPRLPS HRPG. For Example, the peptide linker may comprise the sequence (VKTQATSREEPPRLP|3KHRPG) ₃ VKTQATS or (VKTQATSREEPPRLPS HRPG) V TQATS.

The capture moiety may be a protein. The protein may be selected from the group comprising: an antibody or fragment thereof, an antibody-binding protein, an enzyme, an aptamer and any combination or complexes thereof. The antibody-binding protein may be selected from the group comprising: protein A, protein A G, proteim G, protein L and any combination thereof. The capture moiety may be a complex of proteinA or protein G with an antibody or antigen-binding fragment thereof.

3623084-1 The silica-containing material may comprise at least about 1% Si0 ₂ , at least about 5% Si0 ₂ , at least about 10% Si0 ₂ , at least about 15% Si0 _2> at least about 20% Si0 ₂ , at least about 25% Si0 ₂ , at least about 30% Si0 ₂ , at least about 35% Si0 ₂ , at least about 40% Si0 ₂ , at least about 45% Si0 ₂ , at least about 50% Si0 ₂ , at least abdut 55% Si0 ₂ , at least about 60% Si0 ₂ , at least about 65% Si0 ₂ , at least about 70% Si02, at least about 75% Si0 ₂ , at least about 80% Si0 ₂ , at least about 85% Si0 ₂ , at least abc t 90% Si0 ₂ , at least about 95% Si0 ₂ , or at least about 99% Si0 ₂ .

The silica-containing material may comprise a zeolite. The zeolite may be selected from for example: amicite, analcime, barrerite, bellbergite, bikitaite,'boggsite, brewsterite, chabazite, clinoptilolite, cowlesite, dachiardite, edingtonite, epistilbite, erionite, faujasite, ferrierite, garronite, gismondine, gmelinite, gobbinsite, gonnajdite, goosecreekite, harmotome, herschelite, heulandite, laumontite, levyne, maricopaite, mazzite, merlinoite, mesolite, montesommaite, mordenite, natrolite, offretite, paranatrolite, paulingite, pentasil, perlialite, phillipsite, pollucite, scolecite, sodium dachiardite, stellerite, stilbite, tetranatrolite, thomsonite, tschernichite, wairakite, wellsite, illhendersonite and yugawaralite. The silica-containing material may be magnetic.

The entity may be released from the silica-containing material. For example, the entity may be released from the capture moiety or the entity, the peptide linker and the capture moiety may be released from the silica-containing material.

The entity may be released by a buffer having a pH between 3.0 and 10.0. The buffer may be a solution comprising at least one agent selected from the group comprising: betaine, imidazole, MgCl ₂ , NaCl, ammonium sulphate, cetyl trimethylammonium bromide (CTAB), polyethyleneimine (PEI), KC1, guanidine hydrochlbride (CN ₃ H ₅ *HC1), ammonium thiocyanate (NH ₄ SCN), sodium deoxycolate, I, L-histidine monohydrochloride and L-arginine monohydrochloride. The concentration of the agent may be at least about 250mM, at least about 500mM, at least about 750mM or at least about 1M, at least about 1.5M, at least about 2M, at least about 2.5M or at least about 3M. In a particular embodiment the agent may be NaCl. ^!

The entity may be a biological entity. For example the biological entity may be selected from the group comprising: a lipid, a protein, a peptide, a polypeptide, a metabolite, an organelle, a pathogenic or non-pathogenic organism, an archaeon, a virus, a parasite, an alga, a fungus, a cell, a nucleic acid, a toxin, a contaminant or any combination thereof. The peptide may be, for example, a phosphopeptide or a glycopeptide.

3623084-1 The pathogenic organism may be selected from a bacterium, a virus, a fungus or an apicomplexan. The bacterium may be selected from the group of genera comprising: Actinobacillus, Actinomyces, Bacillus, Campylobacter, Clostridium, Enterococcus, Esherichia, Fusobacterium, Haemophilus, Legionella, Listeria Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Salmonella, Shigella, Staphylococcus and Streptococcus.

The entity may be a chemical entity. For example the chemical entity may be selected from the group comprising: a radionuclide, a drug, a toxin, a petrochemical, an organic compound, an inorganic compound, a heavy metal and an industrial pollutant a metal ion, mono-saccharide, an oligo-saccharide, a poly-saccharide, a mineral, a catalyst, a volatile compounds, stereoisomers, an amino acid, a salt, a carcinogen, a teratogen, an allergen, and an explosive.

In a fifth aspect there is provided an assembly for the capture of a biological or chemical entity comprising;

(a) at least one chamber having at least two apertures defining a flow path therethrough; and

(b) a complex of a silica-containing material and a peptide linker optionally comprising a capture moiety, wherein said peptide linker is capable of interacting, directly or indirectly, with the entity; and

wherein the complex is disposed within the at least one chamber and in the flow path.

In a sixth aspect there is provided a kit for the capture of biological or chemical entity, comprising a silica containing material with a peptide linker capable of interacting, directly or indirectly, with the entity. The kit may further comprise a buffer having a pH between 3.0 and 10.0. The buffer may be a solution comprising at least one agent selected from the group comprising: betaine, imidazole, MgCl ₂ , NaCl, ammonium sulphate, cetyl trimethylammonium bromide (CTAB), polyethyleneimine (1}EI), KC1, guanidine hydrochloride (CNaHs'HCl), ammonium thiocyanate (NH ₄ SCN), sodium deoxycolate, KI, L-histidine monohydrochloride and L-arginine monohydrochloride. The concentration of the agent may be at least about 250mM, at least about 500mM, at least about 750mM or at least about 1M, at least about 1.5M, at least about 2M, at least about 2.5M or at least about 3M. In a particular embodiment the agent may be NaCl. ,

In a seventh aspect there is provided an expression vector comprising a nucleotide sequence encoding a peptide linker and a capture moiety.

3623084-1 Brief Description of the Drawings

Particular embodiments of the present invention will now be described by way of example only, with reference to the accompanying drawings wherein:

Fig. 1 illustrates the binding affinity of purified LPG to Castle Mountain and Zeolite Australia natural clinoptilolite (5 mg each). Binding was performed as described in the General Methods. Unbound and wash fractions (3 xlOO μΐ) were not loaded onto the SDS-PAGE gels. S, starting LPG (80 μg); U, unbound LPG fraction; B, zeolite-bound LPG fraction; LPG, Linker-Protein G.

, Fig. 2 illustrates the binding affinity of purified LPG to Commercial synthetic zeolites (5 mg each). Binding was performed as described in the General Methods. Unbound and wash fractions (3 lOO μΐ) were not loaded onto the SDS-PAGE gels. S, starting LPG (30 μg); U, unbound LPG fraction; B, zeolite-bound LPG fraction; LPG, Linker-Protein G.

Fig. 3 illustrates the binding affinity of purified LPG to commercial silica materials (5 mg each). Binding was performed as described in the General Methods. Unbound and wash fractions (3 xlOO μΐ) were not loaded onto the SDS-PAGE gels. S, starting LPG (30 μg); U, unbound LPG fraction; B, silica-bound LPG fraction;LPG, Linker-Protein G.

Fig. 4 illustrates the results of a truncated linker-sequence LPG binding assay. Purified truncated LPG (Linker-ProteinG) derivatives were incubated with 5 mg of zeolite (A) or silica (B) as described in the General Methods. Unbound and bound fractions were resolved by SDS-PAGE and visualised by staining with Coomassife brilliant blue. PG\ truncated Protein G without linker sequence (Sigma- Aldrich P468j ); IxLPG, truncated LPG carrying a single linker sequence; 2xLPG, truncated LPG carrying two linker sequence repeats; 3xLPG, truncated LPG carrying three linker sequence repeats; 4xLPG, LPG carrying four linker sequence repeats (LPG). S, starting protein (30 μg); U, unbound protein fraction; B, bound protein fraction. Wash (3 xlOO μΐ) fractions were not loaded onto the gels.

Fig. 5 illustrates binding of IgG to zeolite-bound LPG. (A) Rabbit anti-mouse IgG (20 μg) was incubated with zeolite (5 mg) according to the standard binding assay. Unbound, wash and bound fractions were resolved by SDS-PAGE and visualised by staining with Coomassie brilliant blue. (B) Two zeolite samples (5 mg each) each were incubated with LPG (30 μg). One tube was treated as described in, the standard binding assay and the second tube according to the procedure in the binding of IgG to zeolite- 3623084-1 bound LPG section. Unbound and bound fractions were resolved^ by SDS-PAGE and visualised by staining with Coomassie brilliant blue. Wash (3 x 100 μΐ) fractions were not loaded onto the gel B. IgG, rabbit anti-mouse IgG conjugated with FITC; S, starting protein; U, unbound protein fraction; W, wash protein fraction; B, bound protein fraction; LPG, Linker-Protein G; HC, IgG heavy chain; LC, IgG light chain, j

Fig. 6 illustrates the LPG-IgG mediated binding of Cryptosporidium oocysts to zeolite. (A) Fluorescence micrographs of oocysts after binding assay with CRY 104 IgG but without LPG. (B) Fluorescence micrographs of oocysts after binding assay with CRY 104 IgG and LPG. (C) Confocal microscope close up imagej of A. (C) Confocal microscope close up image of B. Left panel shows CRY104-FITC IgG fluorescence; middle panel shows corresponding DIC image and right panel shows the FITC fluorescence image overlayed on the DIC image. LPG, Linker-Protein G.

Fig. 7 illustrates the elution of zeolite-bound LPG and IgG by Class 1 buffers. The LPG and IgG were bound to the zeolite as described in the General Methods. Unbound and wash fractions (3 xlOO μΐ) were not loaded onto the SDS-PAGE gels. S, starting LPG and IgG (20 μg each); El, first elution fraction; E2, second elution fraction; B, zeolite- bound fraction. LPG, Linker-Protein G; HC, IgG heavy chain; LC, IgG light chain.

Fig. 8 illustrates the elution of zeolite-bound LPG and IgG by Class 2 buffers. The LPG and IgG were bound to the zeolite as described in the General Methods. Unbound and wash fractions (3 xlOO μΐ) were not loaded onto the SDS-PAGE gels. S, starting LPG and IgG (20 ^g each); El, first elution fraction; E2, second elutiorji fraction; B, zeolite- bound fraction;LPG, Linker-Protein G; HC, IgG heavy chain; LC, IgG light chain.

Fig. 9 illustrates the elution of zeolite-bound LPG and IgG by Class 2 buffers at different LPG: IgG molar ratios. The LPG and IgG were bound to the zeolite at different molar ratios as described in the General Methods. Unbound and wjash fractions (3 xlOO μΐ) were not loaded onto the SDS-PAGE gels. El, first elution fraction; E2, second elution fraction; B, zeolite-bound fractionjLPG, Linker-Protein G; HC, IgG heavy chain; LC, IgG light chain.

Fig. 10 illustrates the elution of zeolite-bound LPG and IgG by Class 3 buffers. The LPG and IgG were bound to the zeolite as described in the General Methods. Unbound and wash fractions (3 xlOO μΐ) were not loaded onto the SDSrPAGE gels. S, starting LPG and IgG (20 μg each); El, first elution fraction; E2, second elution fraction; B, zeolite-bound fraction. LPG, Linker-Protein G; HC, IgG heavy ^chain; LC, IgG light chain.

3623084-1 Fig. 11 illustrates the elution of zeolite-bound LPG and IgG by Class 3 buffers at different pHs. The LPG and IgG were bound to the zeolite as described in the General Methods. Unbound and wash fractions (3 xlOO μΐ) were not loaded onto the SDS-PAGE gels. S, starting LPG and IgG (20 μg each); El, first elution fraction; E2, second elution fraction; B, zeolite-bound fraction. LPG, Linker-Protein G; HC, IgG heavy chain; LC, IgG light chain.

Fig. 12 is a series of confocal microscope images of the binding of QDot605 to zeolite particles at several stages of the LPG-CRY104 IgG-Cryptosporidium binding process. (A) Zeolite without QDot605. (B) Zeolite after incubation with QDot605. (C) Zeolite-bound LPG after incubation with QDot605. (D) Zeolitq-bound "LPG-CRY104 IgG-Cryptosporidium" complex after incubation with QDot605. Left panel shows QDot605 fluorescence; middle panel shows the corresponding DIC image and right panel shows the QDot605 fluorescence image overlayed on the DIC image.

Fig. 13 is a series of confocal microscope images of the binding of QDot605 to zeolite particles after arginine Class 3 elution buffer action on zeolite-bound "LPG- CRY104 IgG-Cryptosporidium" complex. Left panel shows the QDot605 fluorescence; middle panel shows the corresponding DIC image and right panel shows the QDot605 fluorescence image overlayed on the DIC image.

Fig. 14 illustrates the universal capture platform and multiplex capturing ability. In step 2, the zeolite solid matrix is loaded with L-ABP (Linker-Antibody Binding Protein, e.g., protein G, protein A, protein A/G, protein L) to form a zeolite-ABP complex. Since the ABP display general affinity to different types of antibodies (β , IgG, IgM, IgD, IgA), this step represents a universal capture platform. The capture specificity for the target organisms is mediated by the antibodies selected and can also comprise a mix of antibodies targeting different organisms. In step 3, an example of a multiplex capture platform is displayed in which the use of antibodies with different specificities mediates the capture of different target organisms.

Fig. 15 illustrates the results of a micro BCA protein assay of LPG and CRY 104 loaded onto zeolite columns. (A) Starting and unbound LPG fractions loaded. (B) Starting and unbound CRY 104 IgG fractions loaded. Grey bars represent starting LPG or CRY 104, black bars represent unbound LPG or CRY 104.

Fig. 16 illustrates a single pre-filtration and capture columns/cartridges for use in the capture of single target organisms.

■j

3623084-1 i Fig. 17 illustrates a single pre-filtration and capture columns/cartridges for use in the capture of two or more target organisms.

Fig. 18 illustrates a pre-filtration and modular capture columns/cartridges for use in the capture of two or more target organisms. The captured organisms (target 1 and 2) are eluted a single elution step. j

Fig. 19 illustrates a pre-filtration and modular capture columns/cartridges for use in the capture of two or more target organisms. The captured organisms (target 1 and 2) are eluted separately from each individual capture column.

Fig. 20 illustrates modular capture column system for use ijn the methods of the invention. Three different perspectives are shown

Fig. 21 illustrates the further processing of a sample after zeolite column capture. The captured organism(s) is eluted from the column directly onto a membrane. Upon fixation, a one- two- or three-color fluorescence in situ-hybridization (FISH) analysis can be performed.

Fig. 22 illustrates the application concept of linker-peptide technology for general gas-phase catalysis reaction using a zeolite and/or silica solid matrix.

Fig. 23 illustrates the application concept of linker-peptide tejchnology for specific removal of odorous volatile compounds in gas-phase catalysis reaction using a zeolite and/or silica solid.

Fig. 24 illustrates truncated derivatives of LPG carrying the synthetic (GGGGS)n sequence. !

Fig. 25 illustrates the results of partially purified Link2X-(GGGGS)4-PG derivative binding assay. The Link2X-(GGGGS) ₄ -PG derivative was incubated with 5 mg of zeolite as described in the General Methods section. Unbound and bound fractions were resolved by SDS-PAGE and visualised by staining with Coomassie brilliant blue.

Fig. 26 illustrates the results of purified LinklX-(GGGGS)i ₂ -PG derivative binding assay. The LinklX-(GGGGS)i ₂ -PG derivative was incubated with 5 mg of zeolite as described in the General Methods section. Unbound and bound fractions were resolved by SDS-PAGE and visualised by staining with Coomassie brilliant blue. '

Fig. 27 illustrates a prototype column.

Fig. 28 illustrates a double column capture system using the prototype column shown in Fig. 27.

Fig. 29 illustrates a triple column capture system using the prototype column shown in Fig. 27.

3623084-1 Fig. 30 illustrates the results of premixed LPG and CRY 104 binding zeolite CBV100. Fractions were resolved by SDS-PAGE and visualised by staining with Coomassie brilliant blue. HC; CRY014 heavy chain, LC; CRYO 14 light chain, LPG; Linker-Protein G.

Fig. 31 illustrates the incorporation of the protease recognition sequence between the Linker and the MekB sequence of Linker-MekB to create the Linker-rTEV-MekB fusion recombinant protein.

Fig. 32 illustrates the results of Linker-rTEV-MekB binding assay. Unbound, wash, elution and bound fractions were resolved by SDS-PAGE and visualised by staining with Coomassie brilliant blue. * PreScission Protease, ** MekB (after protease treatment).

Definitions

In the context of the present specification, the terms "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

In the context of the present specification, the term "comprising" means including principally but not necessarily solely. Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.

In the context of this specification, the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

As used herein, the term "between" when used herein in reference to a range of numerical values encompasses the numerical values at each endpoint of the range. For example, a pH of between 3.0 and 10.0 is inclusive of the values 3.0 and 10.0.

As used herein, the term "substantially" means "approximately" and may be applied to modify any representation (quantitative or otherwise) that could permissibly vary without resulting in a change in the basic function to which it is related.

As used herein the term "silica" is used to encompass any substance or compound comprising Si and/or Si ions. Therefore, the term silica is to be construed broadly to include for example, silicates and Si0 ₂ .

The term "sequence identity" or "percentage of sequence identity" may be determined by comparing two optimally aligned sequences or subsequences over a comparison window or span, wherein the portion of the polynucleotide sequence in the comparison window may optionally comprise additions or deletions (i.e., gaps) as 3623084-1 compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino ¹ acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Aspj Glu, Asn, Gin, Cys and Met) or the identical nucleic acid base (e.g., A, T, C, G) in the, nucleic acid sequence encoding the amino acid sequence in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

As used herein an "aptamer" comprises a nucleic acid or peptide sequence that has the ability to selectively or specifically bind one or more ligands. Aptamers can bind nucleic acid, proteins, prions, small organic compounds, or entire organisms. Preferred aptamers herein are peptide sequences.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as, an acknowledgement or admission or any form of suggestion that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

For the purposes of description all documents referred to herein are incorporated by reference in their entirety unless otherwise stated.

Detailed Description

It is to be understood at the outset, that the figures and examples provided herein are to exemplify and not to limit the invention and its various embodiments.

Methods and assemblies are provided for the capture of a biological or chemical entity. The methods generally comprise the use of a complex of a peptide linker and a silica-containing material wherein the peptide linker is capable of interacting, directly or indirectly, with the biological or chemical entity. The assemblies generally comprise a chamber containing a complex of a silica-containing material and a peptide linker capable of interacting, directly or indirectly with the biological or chemical entity. Typically the peptide linker comprises a capture moiety which may be selective or specific for the biological or chemical entity to be captured.

3623084-1 i

Peptide Linker

The repeating sequence (VKTQATSREEPPRLPSKHRPG, SEQ ID NO: 1) was fused to the lamB gene of Escherichia coli that encodes the maltose outer membrane porin protein. Thus, the fused polypetide was displayed on the surface of Escherichia coli allowing the bacterium to bind to zeolites. It has also been demonstrated that a fusion protein of alkaline phosphatase and the repeating peptide sequence binds to the zeolite and the alkaline phosphatase is enzymatically active (Nygaard et al 2002, Adv. Mater. 14: 1853-1856).

It will be apparent to the skilled person that as exemplified herein a peptide linker according to the present invention is able to form a complex with a silica-containing material.

Typically the peptide linker comprises SEQ ID NO:l (VKTQATSREEPPRLPSKHRPG). The peptide linker may comprise more than one occurrence of this sequence, for example (VKTQATSREEPPRLPSKHRPG)n where n can be any number. Typically n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15.

In some embodiments the linker may comprise or further comprise a fragment of SEQ ID NO:l . The term "fragment" as it relates to SEQ ID NO:l | refers to an amino acid sequence that comprises a subset of the amino acid sequence of SEQ ID NO: 1. A fragment of SEQ ID NO:l can be a peptide in which amino acid residues are deleted as compared to SEQ ID NO :1 itself, but where the remaining amino acid sequence is typically identical to the corresponding positions in SEQ ID NO:l . Such deletions can occur at the amino- terminus or carboxy-terminus of SEQ ID NO:l or alternatively at both termini. Fragments are typically at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In one embodiment the peptide linker may comprise the sequence (VKTQATSREEPPRLPSKHRPG) ₄ VKTQATS (SEQ ID NO: 2).

The peptide linker may comprise a derivative of SEQ ID NO:l . The term "derivative" as it relates to SEQ ID NO:l, refers to a sequence comprising at least one instance of SEQ ID NO:l, for example (VKTQATSREEPPRLPSKHRPG),, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or a fragment thereof halving one or more amino acid substitutions, deletions and/or additions wherein the derivative will display at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about

99% similarity or identity to SEQ ID NO: 1, or (VKTQATSREEPPRLPSKHRPG) _n where 3623084-1 n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The term "derivative" as it relates to the peptide linker also refers to SEQ ID NO:l or a fragment thereof having one or more amino acid residues chemically modified, e.g., by alkylation, acylation esterification or amidation and/or having one or more amino acid residues biologically modified, e.g. by lipidoylation such as myristoylation, glycation, glycosylation, phosphorylation,! acetylation, acylation, methylation, hydroxylation, biotinylation or ubiquitinylation.

The person skilled in the art will understand that in embodiments where the peptide linker comprises more than one repeat of a sequence, for example SEQ ID NO: 1, one or more of the repeats may be a derivative or fragment of that sequence

In exemplary embodiments the linker may further comprise the sequence (GGGGS)n, where n is, for example, between 1 and 20, between 1 and 15 or between 4 and 12. In particular embodiments n is 4, 8 or 12.

In one embodiment the peptide linker may further comprise an enzyme cleavage sequence. Suitable enzyme cleavage sequences will be known to those skilled in the art and include but are not limited to the recognition sequences for trypsin, chymotrypisin, thrombin, Factor Xa, and tobacco etch virus (TEV) protease. The enzyme cleavage site may be located between, for example (VKTQATSREEPPRLPSKjHRPG)n and a capture moiety.

In some embodiments a peptide linker may be attached to another molecule by any means known in the art. For example the peptide linker may be covalently attached to another molecule or may be expressed as a fusion protein with another molecule.

Capture moiety

In some embodiments, the peptide linker may comprise a capture moiety. The skilled person will understand that the term "capture moiety" refers to any moiety that binds selectively or specifically to a biological or chemical entity. The capture moiety may selectively or specifically bind to one or more biological and/or chemical entities of interest.

Capture moieties are typically proteins, for example immunoglobulins, antibody- binding proteins, antibodies, antigen binding fragments of antibodies, enzymes, enzyme complexes, regulatory proteins, DNA-binding proteins, RNA-binding proteins, receptors, hormones, glycoproteins, lipoproteins or any combinations or complexes thereof. In one embodiment the capture moiety is a complex of an antibody-binding protein, for example, protein G and an antibody.

3623084-1 In one embodiment the capture moiety is a complex of any number of proteins. For example 2, 3, 4, 5 or more proteins may form a capture moiety. For example the capture moiety may be an antibody-binding protein and an antibody, or for example, protein G and an antibody. ¹

Where the capture moiety is a protein, the protein may be expressed as a fusion protein with a peptide comprising SEQ ID NO: 1 or a fragment or derivative thereof. For example, the peptide may be fused to either or both of the amino-terminus or the carboxy- i

terminus of a capture moiety. In some embodiments the peptide may be expressed within a protein sequence, for example in a solvent-accessible loop region. The peptide also may be used to join capture moieties derived from two chains of a miilti-chain protein or two separate capture moieties with different specificities, affinities or selectivities.

In some embodiments the capture moiety may be a non-polypeptide moiety such as a toxin, drug, radioisotope, metabolite, contaminant, enzyme substrate, organic compound or inorganic compound, antibody-binding protein. The antibody-binding protein may be protein A, protein G, protein A G and or protein L.

In some embodiments, the peptide linker is fused to protein G expressed from the . wild-type sequence provided in SEQ ID NO: 3. The linker-protein G protein expressed from the codon-optimised linker-protein G nucleic acid sequence (OPT_LPG (SEQ ID NO. 4)) is referred to herein as Linker-Protein G (LPG). LPG comprises the sequence (VKTQATSREEPPRLPS HRPG) ₄ VKTQATS sequence fused to a truncated recombinant Protein G from of Streptococcus strain G148 reported by Goward et al, Biochem. J. 267:171-177, 1990.

In some embodiments the capture moiety comprises a protein complex. For example, the protein complex may be an antibody and an antibody-ibinding protein, such as protein A, or protein G, protein A G or protein L.

The capture moiety may be covalently or non-covalently coupled to a peptide comprising SEQ ID NO: 1 or a fragment or derivative thereof. For example, the capture moiety may be covalently bound to the peptide linker by way of a crosslinking reagent, for example formaldehyde, imidoester crosslinkers such as dimethyl suberimidate, N- Hydroxysuccinimide-ester crosslinkers such as BS3(Bissulfosucciriimidyl suberate). Other crosslinking reagents include, for example carbodiimide crosslinkers such as EDC (1- ethyl-3-(3-dimethylaminopropyl) carbodiimide)).

3623084-1 Entity

The methods and assemblies disclosed and exemplified herein are suitable for the capture of any entity, for example a biological entity or a chemical entity. The entity may be in a fluid state, such as a gaseous or liquid state or may be in a solid state.

Biological entity

It will be apparent to a person skilled in the art that the methods and assemblies disclosed and exemplified herein are suitable for the capture of any biological entity.

For example, the biological entity may be an organism such as a parasite, a virus, a bacterium, a yeast, a fungus, an archeon or an alga.

In another embodiment the biological entity may be a eukaryotic cell, a bacterial (prokaryotic) cell or an archaeon or fragments thereof. Examples of eukaryotic cells include mammalian cells, amphibian cells, reptilian cells, insect cells, avian cells, plant cells and fungal cells. Examples of prokaryotic cells include bacteria such as pathogenic and non-pathogenic bacteria.

In other embodiments the biological entity may be an organelle for example mitochondria, nuclei, chloroplasts, endoplasmic reticulum, golgi, acrosomes, autophagosomes, centrioles, cilia, glycosomes, glyoxysomes, lysosomes, melanosomes, myofibrils, thylakoid membranes, vacuoles and vesicles or any component or fragment thereof. The biological entity may be a macromolecular structure such as flagella, cytoskeleton, extracellular matrix or ribosomes.

In a further embodiment the biological entity may be ia protein such as a glycoprotein, phosphoprotein, lipoprotein, enzyme, cell surface protein, receptor, hormone or toxin. In another embodiment, the biological entity may be a peptide, polypeptide or fragment thereof, a nucleic acid such as RNA or DNA, a lipid or a carbohydrate.

Chemical entity ,

It will be apparent to a person skilled in the art that the methods and assemblies disclosed and exemplified herein are suitable for the capture of chemical entities. It will be apparent to a person skilled in the art that any chemical entity may be detected.

For example, the chemical entity may be an organic compound or an inorganic compound. The entity may be a radionuclide or any radioactive compound, a drug, a toxin, a petrochemical, an organic compound, an inorganic compound, a heavy metal and an industrial or environmental pollutant, a metal ion, a monosaccharide, an oligo-saccharide, 3623084-1 a poly-saccharide, a mineral, a catalyst, a volatile compound, an ajromatic compound, a stereoisomer, an amino acid, a salt, a carcinogen, a teratogen, an allergen, and an explosive.

In some embodiments it is envisaged that the present invention may be utilised to, for example, capture traces of illicit drugs, or metabolites thereof in waste water or sewerage.

It is envisaged that the present invention may be utilised to capture and thus detect trace explosives, capture or catalyse the destruction of chemicals (for example, those contributing to odours or poisoning), capture of reaction by-prjoducts or inhibitors, filtration of blood, plasma or urine to capture contaminants for purification or further analysis, capture and thus remove dyes or other colouring agents from liquids, selective concentration of active components from mixtures (e.g. Taxol from a Pacific Yew crude bark extract), screening blood or urine for indicators of minimal residual disease, capture and removal of circulating cancer cells, virus or bacteria associated with disease or infection states (for example such an application may be used in conjunction with a dialysis or heart/lung machine). Other applications of the invention may include use within a toothpaste formulation to deliver active components, incorporation within a wound dressing to deliver active components or to capture contaminants (microbial or chemical), colorometric detection of a toxin, a microorganism, gas or other chemical or biological entity using modified fibres, papers or surfaces. Further, the invention may find application in the functionalisation of surfaces used in semi-conductor industries for example in the development of biocomputing applications. The invention may also find application in biosensors, surgical applications relating to the repair of or delivery of active/remedial agents to, for example, cartilage, bone, connective tissue, hair and nails, functionalisation of silicon based medical implants to allow targeted post surgical delivery of drugs. Still further, the invention may find application in the manufacture of sealants, adhesives or lubricants that may be functionally improved by the inclusion of a complex of a peptide linker and a silica-containing material.

i

Silica-Containing Material

As exemplified herein a peptide linker of the present invention can form a complex with silica-containing materials. As used herein the term "silica-containing material" refers to any substance or surface comprising silica. It is envisaged that the peptide linkers of the present invention will form a complex with any silica-containing materials for example, 3623084-1 sand, quartz, diatoms, silica, colloidal silica, silica gel, aerogel, tridymite, cristobalite or zeolite. Other examples of silica-containing materials include paints, papers, emulsions, plastics and laminates. In particular, the peptide linkers of the present invention form a complex with silicas, glasses and zeolites. The silica-containing material may be synthetically produced, may be naturally occurring or derived from a natural source containing the material.

i ^'

In some embodiments the binding of a peptide linker may be at least partially dependent on the nature of the silica-containing material. For example, peptide linkers of the present invention may for example complex more efficiently with materials containing greater than 70% Si0 ₂ than materials containing less than 70% Si0 _2, however a strong binding may not be essential for all anticipated applications. j

The silica-containing material may comprise at least about 1% Si0 ₂ , at least about 5% Si0 ₂ , at least about 10% Si0 ₂ , at least about 15% Si0 ₂ , at least about 20% at least about 25% Si0 ₂ , at least about 30% Si0 ₂ , at least about 35% Si0 ₂ , at least about 40% Si0 ₂ , at least about 45% Si0 ₂ , at least about 50% Si0 ₂ , at least about 55% Si0 ₂ , at least about 60% Si0 ₂ , at least about 65% Si0 ₂ , at least about 70% Si0 ₂ , at least about 75% Si0 ₂ , at least about 80% Si0 ₂ , at least about 85% Si0 ₂ , at least about 90% Si0 ₂ , at least about 95% Si0 ₂ , or at least about 99% Si0 ₂ .

Silicas

In some aspects the silica-containing material may be silica As used herein the term "silica" refers to any substance or surface consisting essentially of silicon dioxide (Si0 ₂ ). The person skilled in the art will understand that any silica will be suitable for use in the present invention.

For example, silicas typically comprise Si0 ₂ in a crystal lattice. The lattice may be orthorhombic, tetragonal, monoclinic, cubic or hexagonal.

The silica may be in a particulate form of any particle size. For example the silica particles may be microparticles or nanoparticles. ^!

Silica nanoparticles may have a mean diameter of about lnm to about 10 nm, about lOnm to about 20 nm, about 20 nm to about 50 nm, or about 50nm to about 100 nm, about lOOnm to about 150 nm, about 150nm to about 200 nm, about 200nm to about 250 nm, about 250nm to about 300 nm, about 300nm to about 350 nm, about 350nm to about 400 nm, about 400nm to about 450 nm, about 450nm to about 500 nm, about 500nm to about 550 nm, about 550nm to about 600 nm, about 600nm to about 650 nm, about 650nm to 3623084-1 i about 700 nm, about 700nm to about 750 nm, about 750nm to about 800 nm, about 800nm to about 850 nm, about 850nm to about 900 nm, about 900nm to about 950 nm, about 950 nm to about 1000 nm.

Silica microparticles may have a mean diameter of about Ιμηι.ίο about 10 μιη, about ΙΟμηι to about 20 μπι, about 20μm to about 50 μπι, or about 50μιίι to about 100 μm, about ΙΟΟμπι to about 150 μπι, about 150 m to about 200 μιη, about 200μm to about 250 μπι, about 250μm to about 300 μπα, about 300μιη to about 350 μιη, about 350μπι to about 400 μπι, about 400 m to about 450 μιη, about 450μm to about 500 μηι, about 500μm to about 550 μιη, about 550μπι to about 600 μm, about 600μηι to about 650 μιη, about 50μm to about 700 μπι, about 700 m to about 750 μπι, about 750μm to about 800 μιη, about 800μπι to about 850 μm, about 850μιη to about 900 μπι, about 900μm to about 950 μηι, about 950μιη to about 1000 μηι.

The silica may be granular. Silica granules typically have ^ mean diameter of about 1000 μπι or greater. One example of granular silica is silica gel. Silica granules typically have a mean diameter of at least about 1 mm, or at least about 1.5 mm, or at least about 2 mm, or at least about 2.5 mm, or at least about 3.0 mm, or at least about 3.5 mm, or at least about 4 mm, or at least about 4.5 mm, or at least about 5 mm, or t least about 5.5 mm, or at least about 6 mm, or at least about 6.5 mm, or at least about 7 mm, or at least about 7.5 mm, or at least about 8 mm, or at least about 8.5 mm, or at least about 9 mm, or at least about 10 mm.

The silica may comprise silicon dioxide (Si0 ₂ ) at about 75% to about 80% (w/w), or about 85% to about 90% (w/w), or about 90% to about 95% (w/w), or about 95% to about 97%, or about 97% (w/w) or about 98% (w/w) or about 99% (w/w).

The silica may be fumed silica, amorphous silica or mesoporous silica. The silica may be commercially available. Examples of commercially available silicas include silica gel, Silica LC60A, Silica Gel60, Silica Gel646, Silica Rhodoline, Silica Tixosil 38, Silica Tixosil 38A, Silica Tixosil 68, Silica Perkosil SM 660, Silica Perkosil KS 300-PD, Silica Elfadent SM514 and Silica Durafill 200.

Glasses

As exemplified herein the silica containing material may be a glass. As used herein the term "glass" refers to a non-crystalline material containing silicon dioxide (Si0 ₂ ) which is solid at room temperature and pressure. The skilled person will understand that any glass may be useful in the present invention.

3623084-1 Typically the glass comprises silicon dioxide (Si0 ₂ ) at about 15% to about 25% (w/w), or about 25% to about 35% (w/w), or about 35% to about 45% (w/w), or about 45% to about 55% (w/w), or about 55% to about 65 (w/w), or about 65% to about 75% (w/w), or about 75% to about 85% (w/w), or about 85% to about 95% (w/w).

The glass may be in the form of a powder, nanoparticles, microbeads or beads. Examples of glass useful in the present invention are flint glass, borosilicate glass, soda- lime glass, lead-glass and alumino-silicate glass.

Zeolites \

Zeolites are a large group of natural and synthetic microporous hydrated aluminosilicates comprising varying proportions of Silicon (Si), typically as silicon dioxide (Si0 ₂ ) and Aluminium (Al), typically aluminium oxide (Al ₂ 03)in a crystalline matrix. The nature of zeolites can be modified by varying the Si/Al ratio or by introducing different metals into the crystalline framework and then by changing the Si/metal ratio. Accordingly, as zeolites may contain metals, some zeolites are magnetic. The acidity of zeolites can be modified by exchanging extra-framework metal cations with H*. Zeolites are known to be stable both in wet and dry conditions and well-tolerated by biological entities such as microorganisms.

Zeolites differ in the arrangement of the 3-dimensonal framework and their Si0 ₂ /Al203 ratios. Dye-exclusion assays of proteins binding to zeolite have previously indicated that the affinity of the linker-peptide zeolite is a consequeiice of the polypeptide being able to recognise and distinguish subtle differences in the spatial orientation of the crystal surface of the zeolite. On that basis it would be expected that the peptide linker would have a very narrow and selective affinity to particular zeolites (Nygaard et al 2002, Adv. Mater. 14:1853-1856). However, in contrast, and as exemplified herein the peptide linker's affinity for a silica-containing compound is related to the Si0 ₂ component (content and/or arrangement) rather than to spatial orientation of the crystal surface of a zeolite.

Accordingly, in a particular embodiments the silica-containing material is a zeolite comprising at least about 15% Si0 _2> at least about 20% Si0 ₂ at leas about 25% Si0 _2, at least about 30% Si0 ₂ , at least about 35% Si0 ₂₎ at least about 40% Si0 _2, at least about 45% Si0 ₂ , at least about 50% Si0 _2j at least about 55% Si0 ₂ , at least about 60% Si0 _2, at least about 65% Si0 _2> at least about 70% Si0 _2> at least about75% Si0 ₂ , at least about 80% Si0 ₂ , at least about 85% Si0 ₂ , at least about 90% Si0 ₂ , at least about 95% $i0 ₂ , or at least about 99% Si0 ₂ . The silica-containing material may comprise a zeolite.

3623084-1 Examples of zeolites include amicite, analcime, barrerite, bellbergite, bikitaite, boggsite, brewsterite, chabazite, clinoptilolite, cowlesite, dachiardite, edingtonite, epistilbite, erionite, faujasite, ferrierite, garronite, gismondine, gmelinite, gobbinsite, gonnardite, goosecreekite, harmotome, herschelite, heulandite, laumontite, levyne, maricopaite, mazzite, merlinoite, mesolite, montesommaite, mordenite, natrolite, offretite, paranatrolite, paulingite, pentasil, perlialite, phillipsite, pollucite, scolecite, sodium dachiardite, stellerite, stilbite, tetranatrolite, thomsonite, tschernichite, wairakite, wellsite, willhendersonite and yugawaralite. In some embodiments the zeolite may be magnetic. Complexes of silica-containing material and peptide linker.

As exemplified herein a peptide linker of the invention iomplexes with a silica- containing material. For example, a peptide ¹ linker comprising (VKTQATSREEPPRLPSKHRPG) ₄ V TQATS (SEQ ID NO: 2) fused with Protein G such as the LPG protein provided herein (see e.g., Example 5) complexes with silica-containing materials such as synthetic and natural zeolite and multiple types of silica.

In some embodiments an antibody*binding protein (e.g., Protein G, Protein A, Protein A/G, Protein L, etc) attached to a peptide linker (e.g. (VKTQATSREEPPRLPSKHRPG) ₄ VKTQATS) (e.g., LPG) can be used as a universal capture platform for capture of pathogens and other organisms of interest. Figure 14 illustrates ^' one example of the universal capture platform strategy. For simplicity, zeolite is shown as the substrate. However, silica and other silica-containing materials as described above may be used. !

In one embodiment a universal capture platform is provided wherein a complex of a silica-containing material and a peptide linker are formed by contacting a silica- containing material (e.g. zeolite) and a peptide linker, for example a fusion protein of (VKTQATSREEPPRLPS HRPG) ₄ VKTQATS and a capture moiety such as an antibody binding protein e.g. Protein G. In some embodiments a further capture moiety may further comprise at least a further protein for example a pathogen (organism)-specific antibody in complex with theantibody-binding protein. The complex of the silica-containing material, peptide linker comprising a capture moiety (the antibody binding protein and antibody) represents the capture of the entity (pathogen in this example).

In other embodiments it is envisaged that the complexes of the present invention may be used to perform an in situ sandwich ELISA diagnostic. For example, after binding

3623084-1 i . of a biological or chemical entity to the complex, the complex may be contacted with a reagent that selectively or specifically binds the entity. This reagent may be for example a secondary antibody conjugated to a fluorescent dye (e.g FITC, ALEXA, TEXAS, or the like) wherein the secondary antibody (e.g., Anti-mouse IgG F(ab') ₂ fragment Alexa 488 conjugate) interacts with the entity.

With reference to Figure 14, it is envisaged that the universal capture platform will function in a multiplex format. That is, peptide linkers specific or selective for a plurality of biological entities may be used in parallel or in series to capture a plurality of biological entities. For example, in Figure 14 the plurality of complexes for capture of a plurality of biological entities are represented by the zeolite-antibody binding protein (ABP) complexes. Selection of a plurality of different entity-selective dr entity-specific capture moieties (for example, pathogen or organism-specific antibodies) allows for a multiplex capture system for example, as illustrated in step 3 of Figure 14 _| which shows that each zeolite particle is complexed with a single peptide linker and capture moiety. However, it is envisaged that a single zeolite particle may be complexed with a plurality of linker peptides, for example the plurality of peptide linkers may each have a different capture moiety. ^!

Methods using complexes

The person skilled in the art will appreciate that a complex of a silica-containing material with a peptide linker, which may further comprise a capture moiety, will be useful in any method or process where it is desirable to capture a biological or chemical entity, such as a protein or organic compound, respectively.

In one aspect, complexes are envisaged to be useful for purification and/or recovery of a biological or chemical entity, such as a protein of a microorganism or organic compound. For example, a complex of a silica-containing material and a peptide linker comprising a capture moiety specific or selective for a protein, or number of proteins may be used to purify or recover the protein(s) from a sample. The complex is contacted with a sample containing the protein(s) of interest, for example either by passing the sample over the complex, or by adding the complex to the sample under ¹ conditions that allow interaction of the protein(s) of interest with the complex. In Some embodiments the complex may comprise a silica-containing material and a peptide, linker comprising for example an antibody-binding protein such as protein G, protein A, protein A/G or protein L capture moiety for use in the purification of antibodies from a sample.

3623084-1 The captured entity or entities may be released from the complex by elution, for example using an elution buffer described below. The released entity is thereby available for re-use.

In another aspect, a complex of a silica-containing material and a peptide linker may be used to reduce the rate of a reaction mediated by a biological or chemical entity or may be used to stop a reaction mediated by a biological or chemical entity, such as an enzyme or a catalyst.

The reaction rate may be reduced or the reaction stopped by contacting the entity with a complex of a silica-containing material and a peptide linker capable of interaction with the entity under conditions that allow the binding of the entity to the peptide linker, for example, either by passing a sample in which the reaction is occurring over the complex, which may be present in a column or assembly, or by adding the complex to the sample under conditions which allow binding of the entity of interest to the peptide linker thereby separating the entity from the environment in which it is active. In embodiments where the complex is added to the sample, the complex (with bound entity) may be removed from the reaction mixture by any means known in tihe art for example by filtration, centrifugation or magnetic separation.

The skilled person will understand that any reaction mediated by a biological or chemical entity can be stopped or its reaction rate reduced using a complex of the invention. Examples of such reactions include, but are not limited to those reactions catalysed by amylases, arabinoxylanases, amyloglucosidases, beta-glucanases, cellulases, glucoamylases, glucanases, lactases, lyases, mannanases, pectinases, polysaccharide lyases, pullulanases, xanthanases, xylanases and other glycoside hydrolases. Further examples of such reactions include, but are not limited to those reactions catalysed by acetolactate decarboxylases (ALDC), proteases, restriction enzymes, DNA ligases, DNA

I

polymerases, trypsin, rennin, lipases, papain, glucose isomerases, ligninases, catalases, glycosyl transferases and carbohydrate esterases.

In another aspect, complexes of the present invention are envisaged to be useful for removing a biological or chemical entity ^' , such as a protein or microorganism from a sample. For example, a complex of a silica-containing material may be complexed with a peptide linker specific or selective for a microorganism, or a number of microorganisms, for example, by way of a capture moiety or moieties. The complex typically is contacted

Ί

with a sample containing the microorganism, for example either by passing the sample over the complex or by adding the complex to the sample under conditions that allow 3623084-1 interaction of the microorganism of interest. The microorganism may be recovered from the complex by elution, for example using an elution buffer described below.

In one embodiment the biological entity may be a lipid, protein, peptide, polypeptide, a metabolite, an organelle, a bacterium, an archaeon, a virus, a parasite, an alga, a fungus, a cell, a nucleic acid, a toxin, a contaminant or any combination thereof. The biological entity may be a pathogenic or non-pathogenic organism such as a bacterium, virus, fungus, parasite, or an apicomplexan.

Examples of parasitic microorganisms include microorganisms of the genera Cryptosporidium, Cyclospora, Entamoeba, Giardia, Microsporidium, Toxoplasma, and Trichinella.

Examples of pathogenic bacteria include bacteria from the genera Actinobacillus, Actinomyces, Bacillus, Borrelia, Bordatella, Brucella, Burkholderia, Campylobacter, Chlamydia, Clostridium, Coxiella, Enterococcus, Esherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Legionella, LeptospiraListeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio and Yersinia.

Examples of non-pathogenic organisms include those used to express biological products such as proteins and antibiotics, those used for brewing, those used for biofuel production and those used for the production of chemicals.

Examples of apicomplexans include organisms of the genera Babesia, Cyclospora, Eimeria, Isospora, Cryptosporidium, Plasmodium and Toxoplasma.

Examples of fungi include those of the genera: Aspergillus, Candida, Cryptococcus, Histoplasma, Pneumocystis and Stachybotrys.

Examples of viruses include those from the families Adenoviridae (e.g. Adenovirus); Picomaroviridae (e.g. Cocksackievirus, Hepatitus A virus, Poliovirus and Rhinovirus), Hepnadaviridae ( e.g. Hepatitis B virus), Flaviviradae (e.g. Hepatitis C virus, dengue virus, yellow fever virus and West Nile virus), Retroviridae (e.g. HIV), Orthomixoviridae (e.g. measles, mumps and parainfluenza viruses), Papillomaviridae (e.g. Human papilloma virus), Rhabdoviridae (e.g. Rabies virus), Togaviridae (e.g. Rubella virus), Parvovitridae (e.g. Human bocavirus and Parvovirus B19) and Reoviridae (e.g. Rotavirus).

In some embodiments the sample may be fluid, suspension, emulsion or a gas. For example the fluid may be an aqueous solution such as water or waste water. The suspension may be a cell culture for example used in production of therapeutics expressed 3623084-1 from eukaryotic or prokaryotic cells e.g. antibiotics and antibodies. The sample may be an enzyme-catalysed chemical reaction. ¹

Assemblies

Biological or chemical entities may be captured according to the methods of the invention using an assembly typically comprising a chamber containing a complex of a silica-containing material and a peptide linker.

In one aspect an assembly for the capture of a biological or chemical entity comprises a chamber having at least two apertures defining a flow path through the chamber. A complex of a silica-containing material and a peptide linker capable of interacting directly or indirectly with a biological or chemical entity is disposed within the chamber and in the flow path such that when a fluid contacting the biological or chemical entity is passed through the chamber, the biological or chemical entity contacts the peptide

i

linker.

The chamber may further comprise a connecting means to allow the chamber to be connected to at least a further chamber. For example, in one embodiment a plurality of chambers may be present in series. In other embodiments a plurality of chambers may be connected with a single chamber. In some embodiments, it is envisaged that each chamber may contain a mixture of silica-containing materials, each with the capability to capture a different biological or chemical entity. Alternatively, a single silica-containing material, may be complexed, for example, covalently linked to a plurality of peptide linkers of the present invention wherein each of the plurality of peptide linkers has the capability to capture a different biological or chemical entity.

A person skilled in the art will appreciate that the connection between assemblies can be formed by any known means for example by friction fitting pf surfaces, by screw- threaded portions, by snap-fitting or quick-release fasteners. A plurality of assemblies may be formed with at least one frangible zone between at least two assemblies to facilitate separation of the at least two assemblies.

An exemplary embodiment of an assembly of the present invention is illustrated in Figure 20. In this embodiment, the assembly comprises a first and a second column. The first column comprises a threaded portion at one end, the opposing end comprises a portion adapted to form a friction fit with the second column. The second column comprises a threaded portion at each end. Typically the columns comprise a wider bore at one end to enable a minimally restricted flow of fluids through the column.

3623084-1 Each threaded portion is capable of engaging an end cap. In some embodiments the end cap may engage with the column by way of a luer lock fitting or by a friction fit. In some embodiments, end cap is adapted to contact media contained within the column for example a frit or a silica-containing material.

The columns illustrated in Figure 20 are designed to be linkable with each column being connectable to another. It is thus envisaged that such assemblies may comprise any number of first columns fitted together in series. The series may terminate with a second column. Alternatively the assembly may comprise a second column with one or more end caps.

Methods using assemblies

The methods of the present invention may be performed using at least one of the assemblies as described above.

For example, with reference to Figure 16, a single entity capture system is provided. This system utilises a pre-filtration column or cartridge (PC) containing a suitable filtration material, for example, a solid matrix (e.g., zeolite and/or silica) to remove insoluble and interfering particles present in the sample. In some embodiments pre-filtration increases the efficiency of capture of a biological or chemical entity. It is envisaged that any suitable prefilter material known in the art may be used, for example, mesh, fibres siich as glass fibres or paper. In this example, the PC is connected directly to an assembly of the present invention represented as a capture column/cartridge (CC) which typically contains a complex of a solid matrix of a silica-containing material (e.g., zeolite and/or silica) complexed to a peptide linker of the present invention, for example the Linker-ABP (antibody-binding protein) and an antibody specific or selective for a biological entity of interest. For example the antibody may be specific for a parasite, or pathogen.

A sample containing the biological or chemical entity of interest, for example a sample of water containing a pathogen or an organic compound, may be passed through the system either by gravity flow or under pressure whereby on contact with the peptide linker (in this example the peptide linker comprises the sequence (VKTQATSREEPPRLPSKHRPG) VKTQATS) fused to an antibody-binding protein) the entity (the pathogen in this case) interacts either directly or indirectly with the peptide linker and is captured as the peptide linker is complexed to the silica-containing material. Upon capture, the PC is removed from the CC. The CC may then be discarded or the 3623084-1 pathogen eluted from the CC such that the CC can be reused. By elution of the pathogen it can thus be purified or recovered from the sample. In some embodiments the pathogen may subsequently be identified by any means known in the art, for example fluorescence antibody staining, fluorescence in situ hybridisation (FISH), 16S R A gene sequencing or any method known in the art. ι

With reference to Figure 17, a double entity capture system using a single capture assembly is provided. The pre-filtration column/cartridge (PC) a solid matrix (e:g., zeolite and/or silica) to remove insoluble and interfering particles present in the sample. However, it is envisaged that any suitable prefilter material known in the art may be used. An assembly of the present invention is represented in Figure 17 as a capture column/cartridge (CC) and typically contains a complex of a solid matrix (e.g., Zeolite and/or silica) and peptide linkers of the present invention. For example the peptide linkers may be a Linker- ABP (ABP is an antibody-binding protein)and an antibody specific or selective for a biological entity of interest (target 1 in Figure 17) and another peptide linker may be a Linker-ABP and antibody specific or selective for a second biological entity of interest (target 2 in Figure 17). For example the antibody may be specific for a parasite and a pathogen, or two or more different pathogens.

A sample containing targets 1 and 2 (or more) may be passed through the system either by gravity flow or under pressure whereby on contact witjh the linkers the targets bind to the linkers and are captured. Upon the capture of the targets, the PC module is removed and they are eluted from the CC. By elution of the targets they can thus be purified or recovered from the sample. In some embodiments, the entities subsequently may be identified by any means known in the art.

With reference to Figures 18 and 19, a double entity captuite system using a double capture module is provided. The pre-filtration column/cartridge (PC) contains a solid matrix to remove insoluble and interfering particles as above. Assemblies of the present invention are represented as capture column/cartridges (CC1 and CC2) CC1 in this example contains a complex of a solid matrix of a silica-containing material (e.g., zeolite and/or silica) and a peptide linker of the present invention for example a Linker-ABP and a single target-specific antibody (target 1). CC2 in this example contains a complex of a solid matrix of a silica-containing material (e.g., zeolite and/or silica) and a peptide linker of the present invention for example a Linker-ABP and a single-|target specific antibody (target 2). It is envisaged that any number of assemblies may be connected.

3623084-1 A sample containing targets 1 and 2, for example, a water sample containing multiple different pathogenic and non-pathogenic organisms, may be passed through the system either by gravity flow or under pressure whereby on contact with the linkers the targets (one of more) bind to the linkers and are captured. Upon capture of the organisms the PC module is removed and the targets may be eluted in a single step from both CC1 and CC2 as shown in Figure 18. Alternatively, the CC1 and CC2 may be separated from each other and the targets separately eluted from each as shown in Figure 19. By elution of ^• each entity they can thus be purified or recovered from the sample. In some embodiments the entities subsequently may be identified by any means known in the art.

In the above examples, the assemblies of the present invention may be directly or indirectly connected with each other, for example the PC and CC of Figure 18 may be screwed or clipped together by any suitable means. Alternatively the assemblies may be indirectly connected, for example, by way of an adapter or conduit linking the assemblies.

As exemplified herein captured biological entities, for example pathogens such as Cryptosporidium oocysts, may be eluted directly onto a membrane (e.g., a polycarbonate membrane) for further processing as shown in Figure 21 (e.g., two- and three-color fluorescence in situ hybridization).

In some embodiments the eluted entity may be identified by any means known in the art for example, by molecular methods or immunological methods such as ELISA. In particular embodiments the identification may be performed in a hand-held device.

Typically, the sample will be a liquid. However in some embodiments the sample will be a gas. In particular, entities that for example are produced in the fine chemical, flavour and petrochemical industries may be insoluble in the aqueous phase although may be present in the gas phase. Other substances produced in the environmental biotechnology area, sometimes result in gases (in the form of odours).

Accordingly, it is envisaged that the complexes of silica-containing materials and linker peptides as described herein, for example where the capture moiety is an enzyme and the entity is thus its substrate may be useful in gas-phase- catalysis as illustrated in Figure 22. In one embodiment it is envisaged that a complex of a silica-containing compound and linker peptides comprising an enzyme capture moiety could be used for the removal of volatile odorous compounds from the gas-phase as illustrated in Figure 23.

3623084-1 Elution of biological or chemical entities

Captured biological entities may be eluted from the peptide linker and/or the silica containing compound to which the peptide linker is bound. Typically, an aqueous elution buffer is used.

The elution buffer typically has a pH about3.0, or about 3.5, or about 4.0, or about 4.5, or about 5.0, or about 5.5, or about 6.0, or about 6.5, or about 7.0, or about 7.5, or about 8.0, or about 8.5, or about 9.0, or about 9.5, or about 10.0. In particular embodiments the buffer has a pH of about 3.9.

The buffer may comprise an agent selected from betaine, imidazole, MgCl ₂ , NaCl, ammonium sulphate, cetyl trimethylammonium bromide (CTAB), polyethyleneimine (PEI), KC1, guanidine hydrochloride (CNaHs'HCl), ammonium thiocyanate (NH ₄ SCN), sodium deoxycolate, KI, L-histidine monohydrochloride or L-arginine monohydrochloride.

The concentration of the agent may be at least about 25mM, at least about 50mM, at least about lOOmM, at least about 200mM, at least about 250mM, at least about 500mM, at least about 750mM, or at least about 1M, at least about 1.5M, at least about 2M, at least about 2.5M or at least about 3M.

In a particular embodiment the agent may be NaCl. The NaCl concentration may be at least about 250mM, at least about 500mM, at least about 750mM or at least about 1M, at least about 1.5M, at least about 2M, at least about 2.5M or at least about 3M.

In some embodiments addition of NaCl (e.g., 1 M NaCl) to the buffers (for example a betaine buffer) facilitates the elution of a peptide linker from the silica- containing material, for example a betaine buffer with 1M NaCl may be used to elute an LPG-IgG complex from a silica-containing material.

In some embodiments, the linker peptide may be eluted from the silica-containing material. With reference to Example 15, buffers suitable for elution of the linker peptide from the silica-containing material typically contain betaine, imidazole, MgCl ₂ , ammonium sulphate, cetyl trimethylammonium bromide (CTAB), polyethyleneimine (PEI) and L- histidine monohydrochloride. These buffers typically have a pH in the range of 3.0 and lO.O.These buffers are particularly useful for the release of excess zeolite-bound peptide linker which may not have formed a complex with the biological entity to be captured.

In other embodiments the peptide linker and biological entity may be eluted from the silica containing material. Buffers useful for this application include buffers of L- arginine monohydrochloride.

3623084-1 In a further embodiment the peptide linker and biological or, chemical entity may be . eluted from the silica-containing material by way of a peptide linker comprising an enzyme cleavage sequence. Elution is achieved by contacting the peptide linker with at least one enzyme capable of cleaving at the cleavage site under conditions suitable for cleavage of the cleavage sequence. Examples of suitable cleavage sequences include (but are not limited to) the recognition sequences of trypsin, chymotrypisin, thrombin, Factor Xa, and tobacco etch virus (TEV) protease.

Kits

In one aspect there is provided a kit for the capture of a biological or chemical entity. The kit typically comprises a complex of a silica-containing material with a peptide linker capable of interacting, directly or indirectly, with the biological or chemical entity.

In another aspect there is provided a kit for the capture of a biological entity, the kit comprising at least one assembly wherein said assembly comprises a chamber having at least two apertures defining a flow path therethrough and a complex of a silica-containing material and a peptide linker capable of interacting, directly or indirectly with the biological entity wherein the complex is disposed within the chambfer and in the flow path.

The kit may further comprise at least one elution buffer for the elution of the biological entity from said complex.

The kit may also include printed instructions for using the kit to capture a biological entity, elute the entity and/or qualitatively or quantitatively determine the level of the entity in a sample.

Typically, the kits of the present invention will also comprise one or more other containers, containing for example, wash reagents, elution buffers, and/or other reagents as required for the performance of the methods of the invention, for example the kit may contain reagents for the identification of a biological or chemical entity such as one or more antibodies.

In the context of the present invention, a kit may include any kit in which reagents are contained in separate containers, and may include small glass containers, plastic containers or strips of plastic or paper. Such containers may allow the efficient transfer of reagents from one compartment to another compartment whilst avoiding cross- contamination of the samples and reagents, and the addition of agents or solutions of each container from one compartment to another in a quantitative or qualitative fashion.

3623084-1 For application to capture, detection and/or identification of different entities, a single kit of the invention may be applicable. Alternatively different kits may be required, for example kits containing reagents specific for each entity. Methods and kits of the present invention find application in any circumstance in which it is desirable to capture, detect or identify any biological or chemical entity.

The invention will now be described in more detail, by way of illustration only, with respect to the following examples. The examples are intended to serve to illustrate this invention and should not be construed as limiting the generality of the disclosure of the description throughout this specification.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention without departing from the spirit or scope of the invention as broadly described. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

Examples

General Methods

Bacterial strains, culture conditions and plasmids construction

The Escherichia coli strain used in the Examples was E. coli Tuner® (DE3) (Novogen). The plasmids used were pET22b, pET26b and pETDuet-1 (Novogen). Escherichia coli was grown at 37°C in Luria-Bertani medium (LB) with 50 μg/ml carbenicillin (final concentration). Carbenicillin was substituted with 30 μg/ml kanamycin in constructions carrying pET26b. DNA fragments encoding the different recombinant proteins were amplified by PCR using the primer pairs in Tables 1 and 2. Table 1 Oligonucleotides used in this study

Primer Sequence ¹ ' ^■ SEQ ID NO

GFP1-F 5 '-CCGGTAG AAAAAG ATCTT AAAGG AG A AG AA-3 ' 5

GFP1-R 5'-GCGCTCAGTTGGAATTCTACGAATGCTATT-3' 6

GFP2-F 5'-CCGGTAGAAATCATGAGTAAAGGAGAAG-3' ! 7

GFP2-R S'-GAATTCTACGACTGCAGTTTGTATAGTTC-S' 8

TPLF1 δ'-αΑΤθαΑΤΑΤαθΟΑΑΤΤΟΑΤΑΤΟΟΑΤΟΟΑ ΟΟΟΟ · 9

TPLR1 5'-GCGGCCGCAAGAATTCGGCTGCAAGCTC-3' 10

LinXynAd2-lF 5'-CGGTAGAAGAAGATCTTCCGTCCCTGAAAGATGTTT-3' 1 1

LinXynAd2-lR 5'-ACTCAGAATTCTACTAACTTAGGATCCGACTACCGC-3' i 12 ekAFl 5'-CAATATGAGGCATAAAGGATCCTGCTCAATCTAAGCTCGCCGCT-3 13

MekARl 5'-AGCCTCAACTAATGAGCAAGCTTTCGTCATCAAGCCATTTCAAAG-3 14

3623084-1 MekBFl 5'-TAAACGAGGTAACCTGAGGATCCCAGCTATTACACCGA^GAAAATC-3 15

MekBRl 5'-TGGGTTATTGGCTGGCAAG£TTTTATCAATACGCGGGAGATGACAG-3 16

Linker IF 5'-GACACCAGAAATGCATAlGCAGACTCAGGC-3' 17

Linkerl R 5'-GGTTTTCCGGATCCTCGAGGCTGGTC-3' 18 Linker2F 5'-GAC ACC AGAAATGCCTCTGC AGACTCA-3 ' 19

Linker2R 5'-GCCCGGTTTrCAAGCTTCTAGAGGCTGGTCGC-3' ¹ 20

Linker3F 5 '-G AACTATAC AAACTG AATTCTC AGGCG ACC A-3 ' 21

DuetDown 1 5'-GATTATGCGGCCGTGTACAA-3' ¹ 22

LinklxFl 5'-GCAAACATCGTCCGCATATGCAAACCCAGGCGACCA-3' 23 Link2xFl 5'-GCAAACACCGTCCGC IArGCAAACCCAGGCGACCA-3' i 24

Link3xFl S'-GCAAACATCGTCCGCATATGCAAACCCAGGCGACCAGCCGCGAA-S' 25

PG-R 5'-GAGCTCGAATTCGGATCCGATTATTATTCC-3' j 26

"Engineered restriction sites are underlined. To generate pLinkerlpET22b (encoding the Linker sequence VKTQATSREEPPRLPSKHRPG) ₄ VKTQATS, the amplified DNA fragment was digested with Ndel I BamWl and ligated into similarly-cut pET22b. The expression plasmid pLinker-GFP (encoding the Linker sequence

VKTQATSREEPPRLPSKHRPG) ₄ VKTQATS followed by GFP) was generated from the amplified DNA sequence digested with BgRl I EcoRl and ligated into BamHl I EcoRl digested pLinkerlpET22b. In the expression plasmid pGFP-Linker, the recombinant GFP gene product was fused to the N-terminus of the Linker sequence. The amplified DNA fragment was digested with BspHl I Pstl and ligated into Ncol / Pstl-cut pETDuet-1 to create pGFPpETDuet-1. A Linker DNA fragment encoding the Linker VKTQATSREEPPRLPSKHRPG) ₄ VKTQATS was amplified with the Linker2F and Linker2R primer set, digested with Pstl I Hindlll and ligated into similarly-cut pGFPpETDuet-1 to generate pGFP-Linker. ,

The TPL (lipase) gene was excised directly from pTPL with BamHl I Hindlll and ligated into similarly digested pLinkerlpET22b to generate expression plasmid pLinker- TPL (encoding the Linker sequence followed by the TPL lipase) _k To obtain expression plasmid pTPL-Linker (encoding the TPL lipase followed by the Linker sequence), the DNA fragment amplified with the TPLFl and TPLRl primer pair was digested with Ndel I EcoRI and ligated into similarly digested pET22b to create pTPLpET22b. A Linker DNA fragment (encoding the sequence VKTQATSREEPPRLPSKHRPG) ₄ VKTQATS) was amplified with the Linker3F and DuetDown primer set, digested with EcoRI / Hindlll and ligated into similarly digested pTPLpET22b to generate pTPL-Linker.

Expression plasmids pLinker-MekA (encoding the Linker sequence followed by the Mek monooxygenase) and pLinker-MekB (encoding the Linker sequence followed by 3623084-1 the MekB esterase) were generated by digesting the amplified DNA fragments with BamHl I Hindlll and ligation into similarly-cut pLinkerlpET22b. To obtain expression plasmid pLinker-XynAd2 (encoding the Linker sequence followed by the XynA xylanase), the amplified DNA fragment was digested with Bglll I EcoKl and ligated into BamHl I EcoRI-cut pLinkerlpET22b. The expression plasmids are shown schematically in Table 2.

Table 2 Expression plasmids.

Plasmid Primers PCR template Construction Linker Position Function

1

I

Production of recombinant proteins

Cultures of E. coli harbouring a plasmid encoding a recombinant protein were incubated at 37°C with shaking (250 rpm) until the Α ₆₀ ο was between 0.6 and 0.8. The incubation temperature was reduced to 20°C and protein synthesis was induced by the addition of 0.2 mM IPTG. Cells were harvested by centrifugation for 15 min at 10,000 xg and 4°C and were stored at -20°C. The recombinant proteins were extracted from bacterial cells using the Bacterial Protein Extraction Reagent (B-PER II, Pierce, USA), as recommended by the manufacturer.

Standard binding assay

The binding of protein (purified or soluble crude extracts) to zeolite was determined as follows. Synthetic zeolite CBV 100 (5 mg, Zeolyst International, USA) was washed three times with zeolite washing buffer (10 mM Tris-HCI, pH 7.5, 100 mM NaCl

3623084-1 and 1% Triton-XlOO). Soluble protein in a final volume of 100 μΐι was mixed with zeolite and incubated by rotation at room temperature for 1 h. After centrifiigation at 14,000 xg for 20 sec, the unbound fraction was removed. The zeolite pellet was washed three times with 100 μΐ of 100 mM Tris-HCl buffer, pH 8.0 and vortexing. The bound protein was eluted after addition of 100 μΐ of SDS PAGE-loading buffer and incubation at 99°C for 10 min (with short mixing every 2 min). Fractions containing the protein were identified by SDS-PAGE and staining with Coomassie brilliant blue. The SDS PAGE-loading buffer step was omitted for direct detection of remaining enzyme activity in the zeolite-bound fraction and the pellet was resuspended in buffer or appropriate enzyme substrate.

Construction of a Linker-Protein G (LPG) recombinant protein

The Protein G sequence used was based on that reported by Goward et al., Biochem. J. 267:171-177, 1990 which codes for a truncated; recombinant form of Streptococcus strain G148 Protein G (Protein G'). A DNA sequence encoding the Linker (VKTQ ATSREEPPRLP SKHRPG) ₄ VKTQ ATS) and a DNA sequence encoding a truncated Protein G' DNA sequences were joined with the linker sequence positioned at the N-terminus of Protein G. This construction is referred to as wiljd type LPG (WTJLPG, SEQ ID NO: 3). The codon usage of the final combined WT_LPG sequence was optimised for expression in E. coll Rare codons and negative cis-acting motifs (such as internal RBS sites, TATA-boxes, Chi-recombination sites, etc) that can hamper expression in E. coli were removed during codon optimisation. Codon usage was adapted to the bias of E. coli resulting in a CAI (Codon Adaptation Index) value of 0.91. Codon optimisation and gene synthesis of the final combined WT_LPG sequence was performed by GENEART AG (Regensburg, Germany). The final optimised LPG sequence (OPT_LPG, SEQ ID NO: 4) was designed with flanking restriction sites (Ndel and ΒαηϊΆΥ) I for easy ligation into plasmid pET22b (Novagen). The codon-optimised LPG gene (OPTJLPG) was ligated into plasmid pET22b to give expression plasmid pOPT_LPGpET22b.The protein expressed from the codon-optimised LPG sequence (OPT LPG) is referred to herein as Linker- Protein G (LPG). i

Production and purification of recombinant LPG

For the production of recombinant LPG, 300 ml Luria Bertani (LB) medium supplemented with 50 μg ml carbenicillin was inoculated with 3 ml of an overnight culture of E. coli Tuner® (DE3) cells (Novagen) harbouring pOPT_LPGpET22b. The culture was

3623084-1 incubated at 37°C with shaking (250 rpm) until the Aeoo was approximately 0.7-1.0. The incubation temperature was reduced to 20°C and protein synthesis was induced by the addition of 0.2 raM IPTG. After 3 to 4 hours induction, cells were harvested by centrifugation for 15 min at 10,000 xg and 4°C and were stored at -20°C.

Cells cultured as described above were resuspended in ice-cold lysis buffer (25 mM Tris-HCl, pH 8.0, lOOmM NaCl, 1.25mM EDTA and 0.05% Tween 20), supplemented with 4 mM of the serine protease inhibitor Pefabloc (Roche) and 1.5 mg lysozyme (Sigma). The cells were ruptured by three passages through a French pressure cell. After cell rupture, 50 U of DNase I (Invitrogen) and 4 mM Pefabloc were added. The sample was incubated with rotation at room temperature for 20 min. The debris was removed by centrifugation for 30 min at 20,000 xg and 4°C. The supernatant obtained was filtered through a 0.45 μπι and then through a 0.2 μιη sterile filter and stored at 4°C.

The extract was loaded onto a 5 ml HiTrap Q anion exchanger column (GE Healthcare) previously equilibrated with 25mM Tris-HCl, pH 8.0, supplemented with lOOmM NaCl. The column was washed extensively with the same buffer. Under these conditions the LPG was found in the lOOmM NaCl fraction. The LPG fraction was applied to a 5 ml HiTrap SP cation exchanger column (GE ^; Healthcare) previously equilibrated with buffer containing lOOmM NaCl. The column was washed extensively with the same buffer and the LPG was eluted with 150-200mM NaCl. Fractions containing the LPG were identified on a NuPAGE 4-12% Bis-Tris Gel (1.5 mm, 10 well, Invitrogen) by SDS-PAGE and staining with Coomassie brilliant blue, pooled and concentrated using an Amicon Ultra- 15 centrifugal filter (10 kDa cut-off, Millipore). Samples were stored in 25mM Tris-HCl, pH 8.0, lOOmM NaCl at -20°C after sterile filtration through a 0.1 μηι filter. The final yield of pure LPG protein from 300 ml of medium was approximately 20 mg.

Minimal number of repeats required for binding

Several deletion constructions of pOPT_LPGpET22b were prepared in Order to determine the minimum number of repeat sequences (VKTQATSREEPPRLPS HRPG) required for functional zeolite binding LPG (Table 3). DNA fragments encoding the different truncated recombinant proteins were amplified by PCR using the primer pairs described in Table 1. The amplified DNA fragments were digested with Ndel I BamHl and

3623084-1 ligated into similarly-cut pET22b. Production and purification of the different recombinant truncated LPG versions was as described above.

Table 3. Primers and expression plasmids used to produce the truncated LPG derivatives.

Recombinant protein Plasmld Primers Linker sequence repeats

PG pOPT_LPGpET22b N.A" 4

PG pLlnk3xpET22b Link3xF1 / PG-R 3

pLink2xpET22b Llnk2xF1 / PG-R 2

pl_ink1xpET22b Linkl xFI / PG-R 1

I PG' N.A N.A

N.A, not applicable.

"Purified recombinant truncated Protein G' (Sigma-Aldrich P4689)

Binding of IgG to zeolite-bound LPG

LPG (30 μg) was bound to zeolite as described in the standard binding assay but without the final SDS PAGE-loading buffer elution step. After washing, 20 g of rabbit anti-mouse IgG (Thermo Scientific) in a final volume of 100 μΐ was mixed with the "LPG- zeolite complex" and incubated by rotation at room temperature for 1 h. The unbound fraction was removed by centrifugation at 14,000 xg for 20 sec. The zeolite pellet was washed three times with 100 μΐ of 100 mM Tris-HCl buffer, pH 8<0 by vortexing. The bound "LPG-IgG complex" was eluted after addition of TOO μΐ of SDS PAGE-loading j

buffer and incubation at 99°C for 10 min (with short mixing every 2 min). Fractions containing the LPG and IgG were identified by SDS-PAGE and staining with Coomassie brilliant blue. A control sample to determine the possible non-specific binding of the antibody was prepared by incubating 20 μg of rabbit anti-mouse IgG with zeolite in the absence of LPG as described in the standard binding assay. Example 1: Binding of Cryptosporidium to zeolite-bound LPG-IgG complex

Zeolite-bound LPG-IgG complex was used for the capture of Cryptosporidium. Zeolite (5 mg) was loaded with 30 μg each of LPG and CRY 104 Cryptosporidium monoclonal antibody (BTF Pty Ltd), specific to the walls of Cryptosporidium oocysts, by the procedures described above. The zeolite-bound "LPG-CRY104 complex" was incubated with a sample of Cryptosporidium oocysts diluted in 500 μΐ of PBS (phosphate

3623084-1 buffered saline) buffer + 0.05% Tween 20, pH 7.4. A control zeolite sample was incubated

I

only with CRY 104 (but not LPG) and after the washing steps it was incubated with Cryptosporidium oocysts. Cryptosporidium oocysts in both samples were incubated with monoclonal antibody CRY 104 (10 μg/ml) conjugated to Fluorescein isothiocyanate (FITC, BTF Pty Ltd). Oocysts were visualized by epifluorescence microscopy on an Axioskop 2 microscope (Carl Zeiss, Sydney, Australia) and by confocal microscopy on an Olympus Fluoview FV 300 confocal laser-scanning microscope.

Example 2: Elution of zeolite-bound LPG

Several buffers were formulated for the elution of the zeolite-bound LPG, IgG and/or "LPG-IgG complex". The samples were prepared as described under "Binding of IgG to zeolite-bound LPG" but with 20 μg each LPG and IgG and without the final SDS PAGE-loading buffer elution step. After the final washing step, the zeolite pellet containing the bound LPG-IgG complex was resuspended in 100 μΐ of elution buffer and incubated for 5 min at room temperature with strong vortexing every 2.5 min. After centrifugation at 14,000 xg for 20 sec, the eluted fraction (elution 1) was removed. This elution step was repeated one more time (elution 2). Finally the zeolite pellet was resuspended in of 100 μΐ of SDS PAGE-loading buffer (bound fraction) and incubated at 99°C for 10 min (with short mixing every 2 min). Fractions were identified by SDS- PAGE and staining with Coomassie brilliant blue.

Zeolite (5 mg) was washed three times with zeolite washing buffer and then resuspended in 200 μΐ of 100 mM Tris-HCl buffer, pH 8.0 containing 50 μg of purified LPG. The mixture was incubated with rotation at room temperature for 30 min. After centrifugation at 14,000 xg for 20 sec, the zeolite pellet was washed by vortexing three times with 200 μΐ of 100 mM Tris-HCl buffer, pH 8.0. After the washing step, 5 μg of CRY 104 Cryptosporidium monoclonal antibody in a final volume of 200 μΐ was mixed with the "LPG-zeolite complex" and incubated by rotation at room temperature for 30 min. After centrifugation, the zeolite pellet was washed by vortexing three times with 200 μΐ of 100 mM Tris-HCl buffer, pH 8.0. The zeolite-bound "LPG-CRY104 complex" was incubated with a sample of Cryptosporidium oocysts diluted in 200 μΐ of PBS buffer + 0.05% Tween 20 (pH 7.4) and incubated by rotation at room temperature for 30 min. After centrifugation, the zeolite pellet was washed by vortexing three tjmes with 200 μΐ of 100 mM Tris-HCl buffer, pH 8.0. The pellet was resuspended in 100 μΐ of 1 M L-arginine

3623084-1 monohydrochloride ( H 3.9) and incubated for 5 min. Then the zeolite pellet was washed by vortexing three times with 100 μΐ of 1 M L-arginine monoriydrochloride. The final pellet was resuspended in 100 μΐ of PBS buffer + 0.05% Tween 20, pH 7.4. Samples from different binding steps were removed and incubated with i QDot605 nanocrystals (Invitrogen) followed by confocal microscopy on an Olympus Fluoview FV 300 confocal laser-scanning microscope.

Example 3: Binding affinity of purified recombinant LPG to natural and synthetic i

zeolite

The affinity of the purified recombinant LPG towards different types of commercial natural and synthetic zeolites was carried out as described in Example 3: Standard binding assay. Two types of natural zeolites were tested (Fig.1). Zeolite Australia (Zeolite Australia Pty Ltd, NSW, Australia) is a zeolite comprising a 54% clinoptilolite composition while Castle Mountain (Castle Mountain Enterprises Pty Ltd, NSW, Australia) zeolite is comprising 85% clinoptilolite. The cation-exchange capacity

I

(CEC) for Zeolite Australia and Castle Mountain zeolites are reported as 1.19 and 1.47 meq/g, respectively. The Zeolite Australia zeolite (7 Mohs) is much harder than the Castle Mountain zeolite (5 Mohs). The purified LPG showed high affinity to both types of natural zeolites (<10 μπι to 2.0 mm). However, this affinity decreased with increasing zeolite particle size, especially above 0.5 mm.

Thirteen synthetic zeolites belonging to 8 different zeolite families were tested as binding substrates for the purified LPG (Fig. 2). Similarly, LPG displayed low affinity (40- 50%) to Valfor 100 as well as Molecular Sieve 5 A (Linde Type A, LTA), and Molecular Sieve 13X (Faujasite-X, FAU-X). LPG displayed no affinity to CBV400 (Faujasite-Y, FAU-Y), CP914C (Ferrierite, FER), CP814E (Beta Polymorph A, BEA) and CBV2314 (ZSM-5, MFI). Of the other zeolites tested, the highest binding affinity (100% binding) was displayed against CBVIOO and CBV300 (FAU-Y), CBVIOA, and CBV21A (Mordenite, MOR). Table 5 summarizes some properties of the synthetic zeolites tested.

Under the binding assay conditions exemplified herein LPG displayed approximately 40-60% binding affinity to FAU and EMT. As exemplified herein, two Mordenites (CBVIOA and CBV21A) and two FAU-Y (CBVIOO and CBV300) zeolites provide greater binding of the LPG component (100% LPG bound) than FAU and EMT. Also two LTA (Valfor 100 and Molecular Sieve 5 A) and one FAU-X (Molecular Sieve

3623084-1 13X) type of zeolite showed LPG affinity comparable to that previously reported. Furthermore, the affinity of the linker peptide towards two different types of natural clinoptilolite zeolite has been demonstrated. Table 5. Properties of the commercial synthetic zeolites tested and LPG binding results.

Zeolites SIO2/AI2O3 Nominal Cation NazO weight Unit cell size Surface area Binding ³

(mole ratio) form <%) (A) ^! (mz/g) U B

Fau)asite (FAU)

(FAU-Y)

CBV 100 5.1 Sodium 13.0 24.65 900 . mm

CBV 300 5.1 Ammonium '2.8 24.68 925

CBV 400 5.1 Hydrogen 2.8 24.50 730

(FAU-X)

Molecular Sieve 13X 5.1 Sodium 14.6 24.94 700 mm mm

Mordenite (M0R)

CBV 10A 13.0 Sodium 65.0 N.A ⁶ 425 mm

CBV 21 A 20.0 Ammonium 0.08 N.A 500 mm

H-MOR-14 14.0 N.A N.A N.A 471 mm

Fen erlte (FER)

CP 914c 20.0 Ammonium 0.05 N.A 400

Beta Polymorph A (BEA)

CP 81 E 25.0 Ammonium 0.05 N.A 680

H-BEA-25 25.0 Sodium N.A N.A 563 mm

ZSM-5 (MFI)

CBV 2314 23.0 Ammonium 0.05 N.A 425

H-MFI-90 N.A N.A N.A N.A 426

Linden Type A (LTA)

Valfor 100 1.1 Sodium 15.0* N.A 71 ^c mm mm aSDS-PAGE of LPG binding to material. U; unbound fraction, B; bound fraction. Wash fractions not shown.

bData not available.

cHui and Chao 2008.

Example 4: Binding affinity of purified recombinant LPG to silica

The affinity of LPG was tested against different type of silica substrates (Fig. 7). Twelve different types of commercial silica materials were tested. LPG displayed high binding affinity to all silica samples, except for Silica LC 60A (50% binding affinity) and Silica Gel 60 (30% binding affinity). LPG displayed between 90-100% binding affinity to all other commercial silica samples tested. Table 6 summarizes isome properties of the silica samples tested. LPG also displayed weak affinity to 3 different glass powders, which usually have a silica composition between 31 and 70% (Fig. 7). Only 40% of the LPG was found in the bound fraction of glass beads (74% silica), whereas 20% was found i

in the bound fractions of zirconia/glass beads (31% silica) and flint glass powders (45% silica). Thus, the peptide linker displays high affinity to different types of commercial silica. ·

3623084-1 . Table 6. Main properties of the commercial silica materials tested and LPG binding results.

Silica SIO2 Particle size Pore size Surface area Binding ⁹

( ) (μπι) (A) (m2/g) U B

Silica (precipitated) 99.0* N.A ^C non-porous* ⁸ 62 ^s

Silica gel Grade 646 99.4 250-500 107-189 275-375 — mm

Silica Davisil LC 60A 99.4 40-63 60 500-600 m» m

Silica Gel 60 Scharlau 99.4 60-200 60 500 m» m

Rhodoline HP 34M 94.0 2.6 N.A 157 .

Tixosil 38 94.0 15 N.A 190 . ^' «■»

Tixosil 38A 94.0 60 N.A 230 m ^' m

Tixosil 68 94.0 250 N.A 160

Perkasil S 660 98.0 17 N.A 200. . . ^■

Perkasil KS 300-PD 98.0 0.02 N.A 125 ^' - mw.

Elfadent SM 514 98.0 12 N.A 125 mm

Durafill 200 ^f 82.0 6.0 N.A 80

aSDS-PAGE of LPG binding to material. U; unbound fraction, B; bound fraction. Wash fractions not shown. bQaykara and Guven 1998.

cData not available.

^Possibly some pores of less than 4 nm diameter.

eFurlong 1982.

'Sodium Magnesium Aluminium Silicate (8.5% AI2O3, MgO 2%).

Example 5: Minimal number of repeats of the linker required for binding

Binding assays with LPG (4 linker sequence repeats) and truncated derivatives indicated that the minimum number of repeats required for complete binding to zeolite or silica was 3 (Figure 4 and Table 7).

There was no clear difference in the binding efficiency to both substrates with either 3 or 4 repeats. The truncated derivative with only 2 repeats was still able to bind to zeolite and silica but displayed less than half the affinity displayed by the two other derivatives. There was no difference in the binding affinity between the derivative carrying one linker sequence and the truncated derivative without linker sequence. In both cases only a non-specific binding of less than 5% was observed.

I

Table 7. Summary of the zeolite and silica binding results of the different truncated derivatives.

Recombinant protein Linker sequence repeats Binding assay ³

Zeolite Silica

PG 100 100

PQ 100 100

PG 40 30

PG <5 <5

PG' <5 <5

Percentage of purified protein found in the bound fraction. Binding assays were perfomed as described in the standard binding assay section. Zeolite (CBV100) and precipitated silica (B.D.H) were at 5 mg each. Purified truncated proteins were at a final concentration of 30 £ g.

3623084-1 I Example 6: Binding of IgG to zeolite-bound LPG

Binding assays were performed using rabbit anti-mouse IgG in order to test the potential of using zeolite-bound LPG as a generic antibody anchoring system. A control experiment was carried out by incubating IgG with zeolite in the iabsence of LPG. As shown in Figure 5A, the rabbit anti-mouse IgG displayed no affinity towards the zeolite matrix and accordingly, all the protein was localized in the unbound fraction. However, when the IgG was incubated with a LPG-bound zeolite sample (Figure 5B), all of the added IgG was found in the zeolite-bound fraction.

These results clearly indicated that zeolite-bound LPG indeed could be used as a generic antibody anchoring system. Protein G binds to different mammalian immunoglobulins with high affinity. The affinity and binding specificity depends on the source and antibody subclass. The incorporation of the linker sequence onto other antibody-binding proteins (ABP) e.g., Protein A, Protein A/G and Protein L, would extend the application of this technology and increase the antibody specificity range available. Thus, the use of a LPG-bound zeolite and/or silica (and by extension, Linker-Protein A, Linker- Protein A/G and Linker-Protein L) as an antibody anchoring or binding system allows the facile exchange of the antibody (e.g., at least one of an IgG, IgM, IgD and an, IgA) and accordingly, the fine tuning of the final antibody specificity , desired.

Example 7: Binding of Cryptosporidium to zeolite-bound LPG-IgG complex

Zeolite-bound LPG-IgG complex was used to capture Cryptosporidium. Cryptosporidium oocysts were surrounded and attached to zeolite, particles only when incubated with zeolite-bound "LPG-CRY104" complex (Figure 6B and D). However, when the oocysts were incubated with the control zeolite sample (no LPG), the oocysts appeared isolated and not bound to the zeolite particles (Figure 6A and C). These results indicate that the selective binding of Cryptosporidium oocysts to zeolite particles was mediated through the "LPG-CRY 104" complex. Ϊ

Example 8: Formulation of elution buffers

Several compounds were tested for their ability to elute (release) the bound proteins (LPG and/or IgG) from the zeolite matrix (Figures 7-1 1). From the results obtained, the solutions tested could be classified into 3 different classes. Class 1 contained those compounds/solutions that had no elution effect on the bound proteins, e.g., polyethylene

3623084-1 ι glycol (PEG). After addition of class 1 buffers, all the proteins , remained bound to the

i

zeolite matrix (Figure 7).

Class 2 buffers comprise compounds/solutions that mainly eluted the LPG, e.g., betaine, imidazole, MgCl ₂ , ammonium sulphate, cetyl trimethylammonium bromide (CTAB), polyethyleneimine (PEI) and L-histidine monohydrochlo|ride. After the addition of class 2 buffers, most of the "LPG-IgG complex" remained bound to the zeolite matrix (Figure 12).

Class 2 buffers appear to selectively elute the bound LPG, as shown in both elution fractions (El and E2, Figure 9). Under the experimental conditionij used, LPG is present at 7.5 molar excess to IgG (assuming a 1 :1 binding stoichiometry). This result implies that class 2 buffers selectively eluted the excess zeolite-bound LPG. Zeolite-bound LPG refers to the LPG (excess) bound to the zeolite but has no IgG bound to it. This LPG has to be tightly bound to the zeolite since during the binding assay it was washed by vortexing three times with buffer before being incubated with IgG. Upon binding of the IgG to the zeolite- bound LPG the efficacy of this class of elution buffers might be hindered by the unavailability or lack of access to the IgG-bound LPG. In order to confirm that the action of class 2 elution buffers was selectively targeted to only zeolite-tyound LPG, the binding experiment was repeated as before using L-histidine monohydrochloride and three different LPG:IgG molar ratios, 1 :1, 4:1 and 10:1. The results are summarized in Figure 9.

At a 1 : 1 molar ratio, no LPG was found in both elution fractions, while all proteins (LPG and IgG) remained with the zeolite-bound fraction. When a 4- and 10-molar excess of LPG was used, most of the excess LPG was found in the elution 1 fraction, with residual excess amounts found in the second elution fraction. However, the protein distribution in the zeolite-bound fraction was similar to that observed with the 1 :1 molar ratio. Thus, this result clearly confirmed that class 2 elution buffers can only release the excess zeolite- bound LPG but not the zeolite-bound "LPG-IgG" complex.

The most efficient elution buffer, class 3 (e.g., L-arginine monohydrochloride), eluted the LPG and "LPG-IgG complex" (Figure 10). Furthermore, addition of 1 M NaCl to L-arginine monohydrochloride and betaine also resulted in the i class 3 type of elution pattern (Figure 10). After addition of class 3 buffers, most of the LPG and IgG is found in the first elution fraction. After a second elution, only residual LPG and IgG remained bound to the zeolite. A third elution step could be used to improve the final yield of eluted proteins. !

3623084-1 The elution efficiency of 1 M L-arginine monohydrochloride was also tested at pH values between 3.0 and 8.0 (Fig. 15). Under these conditions, the same elution effect was observed between pH 3.9 and 8.0. However, at pH 3.0 there was a decreased amount of eluted and/or bound protein released. This result appeared to be due to degradation of the LPG at 1 M L-arginine monohydrochloride pH 3.0.

In addition to the above buffers, the following compounds/solutions were also effective in the elution of zeolite-bound LPG; 3 M KC1, 1M guanidine hydrochloride (CN3H5 ^» HC1), 1 M ammonium thiocyanate (NH4SCN) andl% sodium deoxycolate (data not shown). KI (at 2.5 M) was also effective in the elution of zeolite-bound LPG and "LPG-IgG complex" (data not shown).

Example 9: Arginine elution buffer.

Arginine elution buffer was tested using QDot605 nanocrystals. We have observed previously that these quantum dots were able to bind to synthetic zeolites and silicas. However, binding of the LPG protein to the zeolite or silica resulted in the exclusion of the QDot605 nanocrystals from the matrix surface (unpublished results). Thus, binding or exclusion of QDot605's was used to investigate the mode of action of the arginine elution buffer.

^• Synthetic zeolite CBVIOO appeared as small particles of approximately 1 μιη under the confocal microscope and displayed no autofluorescence (Figure 12A). Addition of QDot605 nanocrystals to the zeolite sample resulted in a strong fluorescent signal due to the binding of the nanocrystals to the zeolite surface (Figure 12B). However, when the LPG was previously bound to the zeolite, there was no fluorescent signal upon addition of QDot605 (Figure 12C). Similarly, no fluorescence was observed when the CRY 104 antibody and the Cryptosporidium oocysts were bound (through the LPG) to the zeolite particles (Figure 12D). In both cases (Figure 1 1C and D), QDot605 nanocrystals were found surrounding but excluded from the surface of the zeolite particles.

Incubation of the "zeolite-LPG-^RY O -Cryptosporidium" complex with the class 3 arginine elution buffer and subsequent incubation witlji QDot605 restored the fluorescent signal, indicating that the QDot605 was able to bind again to the surface of the zeolite particles (Figure 13). These results indicated that the action of the arginine elution buffer was directed to the LPG-zeolite complex, and the primary target is the LPG. Furthermore, the arginine elution buffer appeared to release the complete "LPG-CRY104- Cryptosporidium" complex. This result was confirmed by the ability of anti-mouse IgG 3623084-1 (ab') ₂ fragment (conjugated to AlexaFluor 444) secondary antibody to bind to the CRY 104-Cryptosporidium bound antibody after the arginine elution step (data not shown).

Example 10: Single and Multiple Capture - Proof of Application

Natural clinoptilolite zeolite was size-sieved to 106 - 250 μπι particle size. The zeolite was washed thoroughly with Milli Q water and dried. A stainless steel mesh disc (38 μιη pore size) and a porous polyethylene frit (approx. mean pore size 50 μιη) filter were placed in a 10 cm Pierce disposable polystyrene column (Pierce, Rockford, IL, USA). Columns were packed with 1 g of washed natural zeolite (106-250 μπι size) and attached to a peristaltic pump. The columns packed with zeolite were washed with PBS buffer (pH 7.4) at a rate of 5 ml/min. Then a sample of purified recombinant LPG (300 μg) was diluted to 5 ml with PBS buffer and run through the zeolite column at a rate of 1 ml/min. Flow-through was fed back through the column three times to allow for complete LPG binding. The column was washed extensively with PBS buffer and loaded with 100 μg of either CRY 104 (anti-Cryptosporidium monoclonal antibody, BTF Pty Ltd), G203 (anti- Giardia monoclonal antibody, BTF Pty Ltd) or a mixture (50 μ each) of CRY 104 and G203 antibodies. The antibodies were diluted to 5 ml with PBS buffer and run through the column at a rate of 1 ml/min and the flow-through was fed back through the column three times to allow for antibody binding. Unbound antibody was removed after washing with PBS buffer. The ColorSeed Kit (BTF Pty Ltd) containing a suspension of 100 Cryptosporidium oocysts and 100 Giardia cysts, which had been modified by attachment of Texas Red to the cell wall, was used for the capture experiments, Fluorescently labelled (oo)cysts were loaded onto the column and run at a rate of 1 ml/min and the flow-through was fed back through the column three times. The column was washed with PBS to remove unbound (oo)cysts and the flow-through was collected and transferred to a 13 mm polycarbonate membrane filter of 0.8 μηι pore size (Millipore Australia Pty Ltd) mounted on a vacuum manifold. (Oo)cysts recovered on the membranes were counted using an epifluorescence microscope (Axioskop 2, Carl Zeiss Australia). A control column also was prepared as described above but without antibody. Also, a quality control mud (QC- MUD) sample spiked with ColorSeed Cryptosporidium oocysts and Giardia cysts was used for capture experiments. QC-MUD was prepared from a large volume of backwash water and 100 μΐ of the final concentrated QC-MUD was equivalent to 5 litre of original untreated water.

3623084-1 Table 8 Comparison of total number of Cryptosporidium and Giardia (oo)cysts captured with natural zeolite columns containing LPG, CRY 104 and G203 IgG.

Capture efficiency (%)"

Cryptosporidium Giardia

Without QCMUD

Without antibody 0 0

CRY 104 92 U

G203 0 91

CRY104 + G203 94 94

With QCMUD

CRY104 + G203 92 ^• 87

"EachColorSeed tube sample contained a suspension of 100 (± 2.2) Cryptosporidium and 100 (± 2.2) Giardia labelled (oo)cysts.

All of the (oo)cysts were found in flow-through and wash fractions when the

ColorSeed Cryptosporidium-Giardia suspension was loaded onto the control column i

(zeolite with LPG but without antibody). When the zeolite column was loaded with only the mti-Cryptosporidium monoclonal antibody CRY 104, 92 Cryptosporidium oocysts remained bound to the column, along with only 14 Giardia cysts. However, when the column was loaded with only anti-Giardia monoclonal antibody G203, 91 Giardia cysts remained bound to column but no Cryptosporidium oocysts were observed to be bound.

When a mixture of both antibodies was loaded onto the zeolite capture column, a capture efficiency of 94% for both Cryptosporidium and Giardia (oo)cysts was achieved. In the presence of QC-MUD, a capture efficiency of 92% and 87% for Cryptosporidium and

• j

Giardia (oo)cysts was achieved, respectively. These results indicated that the natural zeolite-LPG complex can be used as a universal anchoring point for antibodies with different specificities. Captured organism specificity was mediated by the antibody or combination of antibodies used.

I

Example 11: Capture of Cryptosporidium in clean samples

A natural clinoptilolite zeolite (Castle Mountain Enterprises Pty Ltd, NSW, Australia) column was used to capture Cryptosporidium oocysts from clean samples. Purified, unlabelled and flow cytometric cell-sorted Cryptosporidium oocysts were used in this and all further experiments. Cryptosporidium oocysts were freshly extracted from bovine faeces using a gradient centrifugation method and sorted by size exclusion using a

3623084-1 I FACSAria flow cytometer (BD Biosciences). In the first set of experiments two 1 ml AC disposable columns (Shen Zhen Biocomma Biotech Co. Ltd, China) were prepared containing natural zeolite with LPG (380 μg) and CRY 104 IgG (2ίθ μg). One column was loaded with all reagents by gravity flow while the other column was loaded manually using a disposable syringe to provide pressure and to reduce the overall loading time of the reagents. Two columns were prepared and all reagents (LPG and CRY 104 IgG) were loaded once using either gravity or a syringe pressure. _; A sample of sorted Cryptosporidium oocysts diluted in 5 ml PBS buffer + 0.05% Tween 20 was loaded onto the column with a single passage using gravity or a syringe. All reagents were passed through the column only once. Bound oocysts were eluted with 1 M arginine pH 3.9.

Table 9 illustrates that a single passage of all reagents and Cryptosporidium oocysts by gravity resulted in a capture efficiency of 99%. However, the total time required for the capture assay was 165 min. Single passage using a disposable \ syringe resulted in 9% reduction in the capture efficiency of Cryptosporidium oocysts but the overall time required for the capture assay was reduced to 30 min.

Table 9. Comparison of total number of Cryptosporidium oocysts captured and eluted from natural zeolite columns containing LPG (380 μg) and CRY 104 IgG (20 μg) using gravity and low pressure. (syringe).

Gravity Syringe

Unbound fraction 0 4

Wash fraction 1 ' 7

Elution fraction 1 12 1 12

Total oocysts 1 13 123

Capture (%) 99 91

Total time (min) 165 30

The efficiency of the 1 ml AC zeolite column to capture Cryptosporidium oocysts from a larger sample (100 ml) was also tested using the single passage and the manual syringe approach (Table 10). All reagents (LPG and CRY 104 IgG) were loaded once using a syringe. A sample of sorted Crypstosporidium oocysts diluted in 100 ml PBS buffer + 0.05% Tween 20 was loaded onto the column with a single passage using a syringe. Oocysts were eluted with 1 M arginine pH 3.9. Under these conditions the capture efficiency (90%) was similar to that obtained above.

3623084-1 Table 10. Total number of Cryptosporidium oocysts captured aitd eluted from zeolite column containing LPG (380 μ ) and CRY 104 IgG (20 μg).

Unbound fraction 9

Wash fraction 0

Elution fraction 81

Total oocysts 90 ι

Capture (%) 90

Example 12: Capture of Cryptosporidium in QC-MUD Spiked Samples

The efficiency of the 1 ml AC zeolite column to capture Cryptosporidium oocysts from a larger sample (100 ml) spiked with 100 μΐ QC-MUD was tested using the single passage and the manual syringe approach. All reagents (LPG and CRY 104 IgG) were loaded once using a syringe. A sample of sorted Crypstosporidium oocysts diluted in 100 ml PBS buffer + 0.05% Tween 20 and 100 μΐ QC-MUD was loaded onto the column with a single passage using a syringe. Oocysts were eluted with 1 M arginine pH 3.9.Under these conditions it was impossible to determine the capture efficiency of this column (Table 11). There was an increase in the column pressure and this was possibly the result of blockage of the frit by QC-MUD particles.

Table 11. Total number of Cryptosporidium oocysts captured and eluted from natural zeolite column containing LPG (380 μg) and CRY 104 IgG (20 μg).

Unbound fraction 14

Wash fraction 0

Elution fraction 19

Total oocysts 33

Capture (%) ?

Two modifications were incorporated into the zeolite column in order to overcome blockage of the frit by the QC-MUD particles. A 38 μη stainless steel mesh filter disc was placed on top of the frit and an upper Swinnex pre-filter unit containing a 38 μπι stainless steel mesh filter disc was incorporated between the syringe and the column. All other parameters were as described above. '

All reagents (LPG and CRY 104 IgG) were loaded once using a syringe. A 38 μπι stainless steel mesh filter was placed on top of the 50 μιη frit within the column. A sample of sorted Crypstosporidium oocysts diluted in 100 ml PBS buffer + 0.05% Tween 20 and

i

100 μΐ QC-MUD was loaded to the column with a single passage through an upper

3623084-1 Swinnex pre- filter unit housing a 38 μη stainless steel mesh filter using a syringe. Oocysts were eluted with 1 M arginine pH 3.9. Under these conditions the column pressure did not increase and there was no visible blockage of the frit. However the capture efficiency from a 100 ml sample spiked with QC-MUD was only 30% (Table 12).

Table 12. Total number of Cryptosporidium oocysts captured and eluted from natural zeolite column containing LPG (380 μg) and CRY104 IgG (20 μg).

Unbound fraction 84

Wash fraction 0

Elution fraction 36

Total oocysts 120

Capture (%) 30

Example 13: Capture of Cryptosporidium in QC-MUD Spiked Samples

Several modifications to the column and material set up were tested in order to improve the Cryptosporidium oocysts capture efficiency of the zeolite column in QC- MUD spiked samples. A stainless steel mesh disc (38 μπι pore size) and a porous polyethylene frit (mean pore size 77 μιη, SPC Technologies Ltd, UK) were placed in a 10 cm Pierce disposable polystyrene column (Pierce, Rockford, IL, USA). Columns were packed with 2.5 g of washed Castle Mountain natural clinoptilolite zeolite (106-250 μπι particle size) plus an extra 1 cm top layer of Castle Mountain zeolite (300-500 μιη particle size). An upper Swinnex pre-filter unit containing a 38 μπι stainless steel mesh filter disc was incorporated between the syringe and the column. By increasing the length of the column and the amount/final height of the zeolite (106-250 μιη) layejr, it was expected that the LPG and accordingly, the CRY 104 IgG would be distributed more evenly along the zeolite layer. Furthermore the increase in the height of the zeolite layer would increase the travel and contact time of Cryptosporidium oocysts with the CRY104 IgG. Addition of a top layer of coarser grade zeolite (300-500 μηι) after LPG and CRY104 IgG were loaded onto the column was expected to act as a natural pre-filter for QC-MUD particles. In addition to these modifications, three different concentrations of CRY 104 antibody were tested in the capture assays.

All reagents (LPG and CRY 104 IgG) were loaded once using a syringe. A 38 μηι stainless steel mesh filter was placed on top of the column's 77 μπι frit. After loading the CRY 104 IgG, a 1 cm top layer of 300-500 μπι zeolite was added. A sample of sorted

Crypstosporidium oocysts diluted in 100 ml PBS buffer + 0.05% Tween 20 and 100 μΐ

I

3623084-1 I QC-MUD was loaded onto the column with a single passage through an upper Swinnex pre-filter unit housing a 38 μπι stainless steel mesh filter using a syringe. Oocysts were eluted with 1 M arginine pH 3.9.The results obtained are summarized in Table 13. First, addition of a top layer of zeolite apparently resulted in a cleaner flow through the column. Secondly, increasing the concentration of the Cryptosporidium antibody CRY 104 from 20 to 60 μg improved the capture efficiency to 82%. Accordingly, the size (height) of the capture column needs to be considered when scaling up the sample volume to be treated. Higher columns may allow for a longer contact time between the zeolite matrix, reagents (LPG and CRY 104 IgG) and Cryptosporidium oocysts.

Table 13 Total number of Cryptosporidium oocysts captured and eluted from a natural zeolite column containing LPG (380 μg) and CRY104 IgG (20-60 μg).

20 40 60

Unbound fraction 71 44 17

Wash fraction 0 0 0

Elution fraction 27 63 78

Total oocysts 98 107 95

Capture (%) 27 59 82 Example 14: Capture of Cryptosporidium in QC-MUD Spiked Samples - 2

Optimization

A two-column system consisting of a top pre-filtration column containing only zeolite and a lower capture column containing LPG-CRY104-bound zeolite was made.

A sample of sorted Crypstosporidium oocysts diluted in ¹ 100 ml PBS buffer + 0.05% Tween 20 and 100 μΐ QC-MUD was loaded onto a pre-filtraltion column containing 2.5 g zeolite (106-250 μηι ) with a single passage through an upper Swinnex pre-filter unit housing a 38 μπι stainless steel mesh filter using a syringe. The binding column was previously loaded with LPG (380 μg) and CRY104 IgG (20 μg). A 38 μηι stainless steel mesh filter was placed on top of the 77 μπι frit within the column. All reagents (LPG and CRY 104 IgG) were loaded once using a syringe. Oocysts were eluted with 1 M arginine pH 3.9.The results obtained with a 100 ml sample spiked with 100 μΐ QC-MUD are shown in Table 14. Passing the sample spiked with QC-MUD through an upper pre-filtration column containing only zeolite before the binding column increased the capture efficiency from 27 to 56% with only 20 μg of CRY 104 IgG. The incorporation of a zeolite upper

3623084-1 pre-filtration column also resulted in a reduction of the turbidity of the sample spiked with QC-MUD entering the lower binding column. This effect is most probably attributable to the natural filtration property of zeolites. Using an upper Swinnex pre-filter unit with a 38 μηι stainless steel mesh disc is recommended to avoid clogging of the zeolite columns by larger QC-MUD particles.

Table 14. Crypstosporidium oocysts captured using a two-column system.

Unbound + wash fractions 47

Elution fraction 59

Total oocysts 106

Capture (%) 56

Example 15: Capture of Cryptosporidium in QC-MUD Spiked Samples - 3 ^rd

Optimization

Optimisation of the two-column system was carried out by determining the binding affinity of the loaded LPG and CRY 104 IgG to the zeolite matrix. In all previous experiments the binding of LPG and CRY 104 IgG to the zeolite matrix was performed at pH 7.4. However, there is evidence that the Protein G-IgG binding is more effective below pH 7.0. A stainless steel mesh disc (38 μπι pore size) and a porous polyethylene frit (mean pore size 77 μπι, SPC Technologies Ltd, UK) were placed in a 10 cm Pierce disposable polystyrene column, (product no. 29920). Columns were packed with 2.0 g of washed Castle Mountain natural clinoptilolite zeolite (106-250 μηι particle size). LPG (210 μg) was loaded onto the column after washing it with PBS buffer at the specified pH. The column was washed with the same buffer to remove unbound LPG protein. CRY 104 (20 μg) was loaded onto the column and it was washed with the same buffer to remove unbound CRY 104 antibody. Unbound LPG and CRY 104 at each loading step were measured using the Micro BCA Protein Assay Kit (Pierce, Rockford, IL, USA) using a BSA standard curve and a control column sample containing only zeolite (no LPG or CRY 104, only buffer was passed through this column). At the working pH of 7.4, 18% of the initial LPG was found in the unbound fraction whereas 82% remained bound to the zeolite column (Figure 15A). However, at this pH only 49% of the originally loaded CRY104 IgG remained bound to the zeolite-LPG matrix (Figurel5B).Reducing the pH of the working buffer to 6.5, resulted in 91% of the initial LPG being found bound to the zeolite (Figure 15A). At this pH there was a dramatic decrease in the amount of unbound 3623084-1 CRY104 IgG (only 4%), with 96% of the initial CRY104 found bound to the zeolite-LPG matrix (Figure 15B). Further reduction to pH 5.5 did not improve the binding of LPG or CRY 104 to the zeolite matrix.

Example 16: Capture of Cryptosporidium in QC-MUD Spiked Samples - 4 ^th

Optimization

The two-column capture system was modified based on the above and previous results. The upper pre-filtration column was packed with 2.5 g Castle Mountain zeolite (250-300 μηι particle size). The pH of the working buffer was changed to 6.5. The Cryptosporidium oocysts were diluted in 100 ml tap water and spiked with 100 μΐ QC- MUD. This sample was loaded using a syringe onto a pre-filtration column containing 2.5 g zeolite (250-300 μηι) with a single passage through an upper Swinnex pre-filter unit housing a 38 μπι stainless steel mesh filter. The binding column contained 2.0 g zeolite (105-250 μπι) and previously was loaded with LPG (250 μg) and CRY104 IgG (10-20 μg). A 38 μηι stainless steel mesh filter was placed on top of 77 μηι frit in the column. All reagents (LPG and CRY 104 IgG) were loaded once using a syringe. All steps (except elution) were performed at pH 6.5. Oocysts were eluted with 1 M arginine pH 3.9.

An optimisation of the overall capture process was achieved under these conditions (Table 15). Capture efficiencies of 74 and 89% were obtained with 10 and 20 μg CRY104, respectively.

CRY104 IgG (ug)

10 , 20

Unbound fraction 27 14

Wash fraction 0 0

Elution fraction 79 109

Total oocysts 106 123

Capture (%) ^' 74 89

Example 17: Effect of QC-MUD on capture efficiency

QC-MUD was added to laboratory tap water samples to simulate an environmental sample. The QC-MUD used here was prepared as a concentrate from a large volume of backwash water as described elsewhere and 100 μΐ of the final concentrated QC-MUD was equivalent to 5 litres of the original untreated water. The binding (capture) assay was performed as described above with samples of sorted Cryptosporidium oocysts diluted in 3623084-1 , ^.. 100 ml tap water supplemented with QC-MUD (0.1 - 0.4% vol/vol final). The samples were loaded onto a pre-filtration column containing 2.5 g zeolite (250-300 μιη) with a single passage through an upper Swinnex pre-filter unit housing a 38 μπι stainless steel mesh filter using a syringe. The binding column contained 2.0 g zeolite (105-250 μιη) and was loaded beforehand with LPG (250 μg) and CRY104 IgG (20 g). A 38 μπι stainless steel mesh filter was placed on top of the 77 μηι frit in the column.: All reagents (LPG and CRY104 IgG) were loaded once using a syringe. All steps (except elution) were performed at pH 6.5. Oocysts were eluted with 1 M arginine pH 3.9.

The sample of QC-MUD added to samples appeared to have little effect on the capture efficiency of the two-column capture system (Table 16). Using the optimised setup described above, capture efficiencies of more than 85% were achieved in the presence of up to 0.3% QC-MUD. At a higher load of QC-MUD (0.4%) the upper pre-filtration column appeared to be clogged, most probably resulting in fewer oocysts passing into the lower binding (capture) column. Accordingly, the total oocysts reported for 0.4% QC- MUD in Table 16 maybe an underestimation of the original number loaded onto the column. The concentrations of QC-MUD tested here exceeded those used in common laboratory control tests, in which 400-800 μΐ (0.004-0.008% vol/Vol final) QC-MUD are added to a 10 L of water sample.

Added QC-MUD (%)

0.1 0.2 0.3 ^' 0.4

Unbound fraction 14 19 13 27

Wash fraction 0 0 0 , 0

Elution fraction 109 106 89 53 .

Total oocysts 123 125 102 , 80

Capture (%) 89 85 87 66

Example 18: Synthetic linker design

Individual repeats from the peptide linker sequence used in the above Examples were replaced by a synthetic (GGGGS) _n linker sequence. Three derivatives were synthesized by GENEART (Regensburg, Germany) and fused to Protein G. The derivatives used are depicted in Fig. 24. In the derivatives LinldX-(GGGGS)4-PG and Link2X-(GGGGS)g-PG two of the four repeats bf the sequence VKTQATSREEPPRLPSBCHRPG were replaced with four or eight repeats of the sequence

3623084-1 GGGGS. In the derivative LinklX-(GGGGS)i ₂ -PG three of the four repeats of the sequence VKTQATSREEPPRLPSKHRPG were replaced with 12 repeats of the sequence GGGGS. The sequences of the derivatives, including the Protein G sequence are shown in SEQ ID Nos:27-29. The recombinant proteins were expressed in E. coli and used in zeolite binding assays as described in the General Methods section above.

The zeolite binding assay results obtained with partially purified Link2X- (GGGGS) ₄ -PG (Fig. 25) and purified LinklX-(GGGGS)i ₂ -PG (Fig< 26) indicated that the space occupied by the repeat sequence is an important factor for binding. When synthetic (GGGGS) ₄ sequence was introduced to the Link2X-PG the binding affinity to the zeolite was increased from 40% to 80%. When a synthetic (GGGGS)i ₂ sequence was introduced to LinklX-PG the binding affinity to zeolite increased dramatically from less than 5% to above 80%. These results appear to indicate that the distance occupied by 3 repeats of VKTQATSREEPPRLPSKHRPG optimises binding to the substrate, i

Example 19: Capture efficiency validation using industrial standards

The binding (capture) assay was performed as described in Example 16 above, but using the industrial standard ColorSeedKit (BTF Pty Ltd, Sydney, Australia). Each vial in the kit contained a suspension mix of 100 Cryptosporidium oocysts and 100 Giardia cysts that have been permanently labeled with the red fluorescent dye Texas Red. One vial of ColorSeed was diluted inlOO ml tap water supplemented with 0.1% (vol/vol) QC-MUD. The binding column contained 2.0 g zeolite (105-250 um) and was loaded beforehand with LPG (250 μg) and CRY104 IgG (40 μg). Under these conditions a tbtal capture efficiency of 97% of ColorSeed Cryptosporidium oocysts was achieved (see Table 17), thus validating the efficiency of the two-column capture system.

■ Natural zeolite

Cryptosporidium

Unbound + Wash 3

Elution 1 68

Elution 2 23

Elution 3 6

Total oocysts 100

Capture (%)* 97

3623084-1 Example 20: Design of prototype columns and the use thereof in capturing

Cryptosporidium

A prototype column was designed and is shown in Fig. 27. Systems comprising the column were tested for their efficiency in capturing Cryptosporidium from varying volumes of tap water.

20.1 Single capture column with 100 ml tap water and environmental Cryptosporidium

Environmental Cryptosporidium oocysts were diluted in 100 ml tap water. This sample was loaded using a syringe onto a single binding column containing zeolite (105- 250 μπι) previously loaded with LPG (250 μg) and CRY104 IgG (40 g). The column was fitted with 77 μηι frit. All reagents (LPG and CRY 104 IgG) were loaded once using a syringe. All steps (except elution) were performed at pH 6.5. Oocysts were eluted with 1 M arginine pH 3.9. Under these conditions the column system displayed 90% Cryptosporidium capture efficiency (Table 18).

Table 18. Environmental Cryptosporidium captured with the prototype column shown in Fig. 27.

Cryptosporidium

Unbound + Wash 12

Elution 1 72.

Elution 2 30

Elution 3 0

Total oocysts 114

Capture (%) 90

20.2 Double capture column with 1 L tap water and environmental Cryptosporidium

Environmental Cryptosporidium oocysts were diluted in 1 L tap water. This sample was loaded onto a double binding column system (Fig. 28) using a pump at a flow rate of 20 ml/min. Each column contained zeolite (105-250 μπι) previously loaded with LPG (250 μg) and CRY104 IgG (40μg). The columns were fitted each with a 77 μπι frit. All steps (except elution) were performed at pH 6.5. Oocysts were eluted from each column with 1 M arginine pH 3.9. The use of two-column capture system resulted in a total capture efficiency of 75% Cryptosporidium oocysts (Table 19). The same capture efficiency was obtained with this system when the 1 L sample was loaded onto the column

3623084-1 at 40 ml/min (data not shown).

Table 19. Environmental Cryptosporidium captured with the double column capture system shown in Fig 28.

Cryptosporidium

Unbound + Wash 32

Top column

Elution 1 60

Eiution 2 8

Lower column

Elution 1 23

Elution 2 7

Total oocysts 130

Capture (%) 75

20.3 Double capture column with 5. L tap water and environmental Cryptosporidium

Environmental Cryptosporidium oocysts were diluted in 5 L tap water. This sample was loaded onto a double binding column system (Fig. 28) using a pump at a flow rate of 40 ml/min as described above. As shown in Table 20, under these conditions the Cryptosporidium capture efficiency of the two-column capture system was 69%.

Table 20. Environmental Cryptosporidium captured with the double column capture system shown in Fig 28.

Cryptosporidium

Unbound + Wash 42

Top column

Elution 1 50

Elution 2 11

Lower column

Elution 1 34

Elution 2 0

Total oocysts 37

Capture (%) 69

3623084-1 20.4 Triple capture column with 5 L tap water + 100 μΐ QC-MUD and environmental Cryptosporidium

Environmental Cryptosporidium oocysts were diluted in 5 L tap water spiked with 100 μΐ QC-MUD. This sample was loaded onto a triple binding column system (Fig. 29) s using a pump at a flow rate of 20 ml/min. The top filtration column contained zeolite (250-300 g) without LPG or CRY 104. Each of the two lower capture columns contained zeolite (105-250 μπι) previously loaded with LPG (250 μg) and CRY104 IgG (40μg). All 3 columns were fitted each with a 77 μιη frit. All steps (except elution) were performed at pH 6.5. Oocysts were eluted from each capture column with 1 M arginine pH 3.9. Table

10 21 summarises the results obtained with the three-column system. The final Cryptosporidium capture efficiency of his system was 78%

Table 21. Environmental Cryptosporidium captured with the triple column capture system shown in Fig 29.

15

Cryptosporidium

Unbound + Wash 32

Top column

Elution 1 82

Elution 2 26

Lower column

Elution 1 5

Elution 2 0

Total oocysts 145

Capture (%) 78

20.5. Triple capture column with 5 L tap water + 200 μΐ QC-MUD and environmental Cryptosporidium

Environmental Cryptosporidium oocysts were diluted in 5 L tap water spiked with 200 μΐ QC-MUD. This sample was loaded onto a triple binding column system (Fig. 29) using a pump at a flow rate of 450 ml/min. The top luer slip cap was replaced by a hose connector cap to allow for higher flow rates. All columns were as described above. Table 22 summarises the results obtained with the three-column system and QC-MUD. The final Cryptosporidium capture efficiency of this system was 82%.

3623084-1 Table 22. Environmental Cryptosporidium captured with the triple column capture system shown in Fig 29.

20.6 Triple capture column with 10 L tap water + 200 μΐ QC-MUD and environmental Cryptosporidium

Environmental Cryptosporidium oocysts were diluted in 10 L tap water spiked with 200 μΐ QC-MUD. This sample was loaded onto a triple binding column system (Fig. 29) using a pump at a flow rate of 450 ml/min. The top luer slip cap was replaced by a hose connector cap to allow for higher flow rates. All columns were prepared as described above. Table 23 summarises the results obtained with the three-column system. The final Cryptosporidium capture efficiency of this system was 82%.

Table 23. Environmental Cryptosporidium captured with the triple column capture system shown in Fig 29.

Cryptosporidium

Unbound + Wash 21

Top column

Elution 1 33

Elution 2 12

Lower column

Elution 1 35

Elution 2 15

Total oocysts 116

Capture (%) 82

3623084-1 Exaraple 21: Binding to zeolite of premixed of LPG and CRY104 complex

Purified LPG and CRY 104 IgG were mixed at a ratio of 1 :1 in PBS buffer (pH 6.5) for 15 min. The mix was then incubated with 5 mg zeolite CBVIOO (previously washed as described in General Methods above) and the assay was performed as described in the standard binding assay. As shown in Figure 30, the LPG-Cryl04 mix was able to bind specifically to the zeolite substrate. Some of the antibody light chain was observed in the wash steps and was probably the result of some degradation of the antibody upon storage.

Example 22: Introduction of a protease cleavage sequence

The PreScission Protease is a genetically engineered human rhinovirus 3C protease (HRV 3C protease). This protease specifically cleaves between the Q and G residues of the recognition sequence of L E V L F Q G P F (Fig. 31). This recognition sequence was introduced between the Linker and the MekB sequence of Linkef-MekB to create the fusion protein Linker-rTEV-MekB (Fig. 31).

The recombinant Linker-rTEV-MekB production and zeolite binding assay was as described in the General Methods section above, but with the following variations. The cells were resuspended in 30 mM MOPS (3-[N-morpholino]propanesulfonic acid) buffer, pH 6.5, supplemented with 100 mM NaCl before French pressure cell treatment. The zeolite-washing buffer was replaced by 30 mM MOPS buffer (pH 6.5) supplemented with 100 mM NaCl and 1% Triton X-100. The same buffer without Triton X-100 was used for binding of Linker-rTEV-MekB to zeolite CBVIOO (5mg) and subsequent washing steps. The zeolite-bound Linker-rTEV-MekB was resuspended in protease buffer (50 mM Tris- HC1, pH 7.0, supplemented with 1 mM EDTA, 1 mM DTT and 150 mM NaCl) and incubated with PreScission Protease (GE Healthcare) for 30 min at 4°C. A second binding assay was performed as above but the zeolite-bound Linker-rTEV-MekB was resuspended in the same buffer without NaCl before the protease treatment. Unbound, bound and elution fractions were resolved by SDS-PAGE and visualised by staining with Coomassie brilliant blue. Figure 32 summarises the results obtained.

The Linker-rTEV-MekB recombinant protein was clearly visible in the starting material but was not present in the unbound and wash fractions, indicating that it remained bound to the zeolite. After incubation with PreScission Protease most of the MekB (without the linker) was found in the elution fraction along with the PreScission Protease. No major differences were observed between the protease buffer with or without NaCl. Several host proteins were found in the final zeolite-bound fraction after the protease treatment. 3623084-1