A SYSTEM FOR THE CAPTURE OF A BIOLOGICAL OR

CHEMICAL ENTITY II

Technical Field

The present invention relates to methods of capturing biological and chemical entities indirectly or directly bound to a peptide linker. In particular, the present invention relates to the use of silica-containing materials to capture biological and chemical entities indirectly or directly bound to a peptide linker.

Background

Enzymes are used as biocatalysts for organic synthesis because they generally promote reactions in high yields with regio- and stereo-selectivities under mild reaction conditions.

Enzymes may be used either in solution or immobilised on a solid or insoluble support. Significant progress has made on a range of immobilisation supports. However the immobilization of proteins has been a particularly challenging task, mainly due to the heterogeneous nature of proteins and the marginal stability of the active tertiary structure over the denatured (and inactive) random coil structure. Immobilizing proteins onto solid or insoluble supports often relies on nonspecific adsorption or the reaction with amine • and/or carboxyl groups of the proteins with appropriate reactive groups on the support. In either case, the proteins are immobilized to the solid or insoluble support in a random orientation which typically causes some loss of the protein's biological activity.

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 silica-containing inorganic materials with a highly ordered structure that can be synthesized as nanocrystals. They offer interesting characteristics as support materials to immobilise proteins such as high mechanical and chemical resistance coupled with a high surface area.

The present invention is predicated on the inventor's findings that a complex of a protein and a peptide linker can bind silica-containing materials including zeolites.

Summary of the Invention

In a first aspect there is provided a method for capture of a biological or chemical entity comprising: (a) providing a peptide linker optionally comprising a capture moiety, wherein the peptide linker is bound directly or indirectly to the entity wherein the linker is capable of forming a complex with a silica-containing material;

(b) contacting said peptide linker bound to the entity with a silica-containing material under conditions permitting interaction of the peptide linker with the silica- containing material.

In a second aspect there is provided a method for capture of a biological or chemical entity comprising contacting a complex of a peptide linker optionally comprising a capture moiety, wherein the peptide linker is bound directly or indirectly to the entity with a silica-containing material, under conditions permitting interaction of the silica- containing material with the complex.

The interaction of the silica-containing material with the complex results in the formation of a substantially insoluble composite comprising the silica-Containing material and the complex.

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

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 bea 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 such as a strip or dipstick.

The peptide linker may comprise the sequence (VKTQATSREEPPRLPS HRPG) _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 VKTQATSREEPPRLPSKHRPG. For Example, the peptide linker may comprise the sequence (VKTQATSREEPPRLPSKHRPG) ₃ VKTQATS or (VKTQATSREEPPRLPS HRPG) ₄ VKTQATS. The peptide linker may further comprise a capture moiety such as 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, protein G, protein L and any combination thereof. The capture moiety may be a complex of protein A, protein G, protein A G or protein L with an antibody or antigen-binding fragment thereof.

The silica-containing material may comprises 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 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 .

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, 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. 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 released from the peptide linker, or the entity and the peptide linker are 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, gCl ₂ , NaCl, ammonium sulphate, cetyl trimethylammonium bromide (CTAB), polyethyleneimine (PEI), KC1, guanidine hydrochloride (CN ₃ H ₅ »HC1), ammonium thiocyanate (NHUSCN), sodium deoxycolate, KI, L-histidine monohydrochloride and L-arginine monohydrochloride. The concentration of the agent may be at least about 250mM, at least about 500m , 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 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.

The parasite may be selected from Cryptosporidium, Cyclospora, Entamoeba, Giardia, Microsporidium, Toxoplasma or Trichinella.

The pathogenic organism may be selected from a bacterium, a virus, a fungus or an apicomplexan. For example, the bacterium may be pathogenic bacterium selected from the group of genera comprising: Actinobacillus, Actinomyces, Bacillus, Clostridium, Campylobacter, 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 fourth aspect there is provided an assembly for 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 silica-containing material capable of interacting, directly or indirectly, with a peptide linker optionally comprising a capture moiety; and

wherein the silica-containing material is disposed within the at least one chamber and in the flow path.

In a fifth aspect there is provided a kit for capture of a biological or chemical entity comprising a peptide wherein the peptide linker optionally comprises a capture moiety, the kit comprising a silica-containing material.

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 (PEI), C1, guanidine hydrochloride (CN ₃ H ₅ 'HC1), ammonium thiocyanate (NHiSCN), 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 sixth aspect there is provided an expression vector comprising a nucleotide sequence encoding a peptide linker and a biological entity.

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 is a schematic diagram of a standard extraction of recombinant soluble proteins from bacterial cells using the Bacterial Protein Extraction Reagent (B-PER). Following extraction, soluble proteins were used for the standard binding assay as described in the General Methods. Z; zeolite.

Fig. 2 illustrates the binding of recombinant GFP (Green Fluorescent Protein) and GFP-Linker to zeolite. (A) GFP-Linker construction with the linker peptide at the C- terminus. (B) Fluorescence micrographs of zeolite-bound GFP and GFP-Linker.

Fig. 3 illustrates the binding affinity of purified LPG (linker Protein-G) 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 Mg); U, unbound LPG fraction; B, zeolite-bound LPG fraction.

Fig. 4 illustrates the binding affinity of purified LPG (linker Protein-G) to commercial synthetic zeolites (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, zeolite-bound LPG fraction.

Fig. 5 illustrates the binding affinity of purified LPG (linker Protein-G) 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.

Fig. 6 illustrates the results of a truncated LPG binding assay. Purified truncated LPG (linker Protein-G) 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 Coomassie brilliant blue. PG', truncated Protein G without linker sequence (Sigma-Aldrich P4689); 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 lOO μΐ) fractions were not loaded onto the gels.

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 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. 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 wash fractions (3 xlOO μΐ) were not loaded onto the SDS-PAGE gels. 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. 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 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. 11 illustrates the elution of zeolite-bound LPG and IgG by a Class 3 buffer (1 L-Arginine) 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 g 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) Zeolite-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 Zeolite binding and xylanase activity assays of Linker-XynAd2 enzyme. Zeolite binding and xylanase assay was performed as described in the General Methods. U, xylanase activity in unbound fraction; Wl-3, xylanase activity in wash fractions 1 -3 ; A 1 -A5 , xylanase activity of re-used bound fractions 1 -5.

Fig. 15 illustrates partial purification of recombinant Linker-MekB esterase from E. coli crude extract. (A) binding assay of Linker-MekB to synthetic zeolite. (B) Esterase activity of binding assay fractions. S, starting E. coli soluble proteins crude extract; U, unbound fraction; Wl-2, wash fractions 1 and 2; B, zeolite-bound fraction.

Fig. 16 shows confocal microscope images of the binding of GFP and magnetic silica nanoparticles. Left panel shows GFP fluorescence; middle panel shows the corresponding DIC image and the right panel shows the GFP fluorescence image overlayed on the DIC image. .

Fig. 1 shows confocal microscope images of the binding of GFP-Linker and magnetic silica nanoparticles. Left panel shows GFP fluorescence; middle panel shows the corresponding DIC image and the right panel shows the GFP fluorescence image overlayed on the DIC image. Fig. 18 illustrates binding of LPG and CRY 104 IgG to magnetic silica nanoparticles. Two magnetic silica nanoparticles samples were incubated individually with LPG. One tube was treated as described in the standard binding assay and the second tube was handled according to the binding of IgG to zeolite-bound LPG section in the General Methods. Fractions were resolved by SDS-PAGE and visualised by staining with Coomassie brilliant blue. Wash 2 and 3 (100 μΐ each) fractions were not loaded onto the gel. S, starting protein; U, unbound protein fraction; Wl, wash 1 protein fraction; El, first arginine elution fraction; E2, second arginine elution fraction; E3, third arginine elution fraction; B, zeolite-bound fraction.LPG, Linker-Protein G; HC, IgG heavy chain; LC, IgG light chain.

Fig. 19 shows phase contrast microscope images of virgin and zeolite-loaded fabric fibres. Bar = 50 μπι.

Fig. 20 shows confocal microscope images of virgin and zeolite-loaded fabric after incubation with GFP and GFP-Linker. Left-black panels show GFP fluorescence; right panels, corresponding DIC images. Bar = 100 μηι.

Fig. 21 is a close up of confocal microscope image of zeolite-loaded fabric treated with GFP-Linker. Left panel shows GFP fluorescence; middle panel shows corresponding DIC image and right panel shows GFP fluorescence image overlayed on the DIC image. Bar = 50 μηι.

Fig. 22 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. 23 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).

Fig. 24 illustrates the results of crude extract Linker-MekB binding to synthetic zeolite CBV100. Fractions were resolved by SDS-PAGE and visualised by staining with Coomassie brilliant blue.

Fig. 25 illustrates the results of crude extract Linker-MekB binding to magnetic silica particles. Fractions were resolved by SDS-PAGE and visualised by staining with Coomassie brilliant blue.

Fig. 26 illustrates the results of premixed LPG and CRY104 binding zeolite CBV100. Fractions were resolved by SDS-PAGE and visualised by staining with Coomassie brilliant blue. HC; CRY014 heavy chain, LC; CRY014 light chain, LPG; Linker-Protein G.

Fig. 27 shows binding affinity of (A) purified LPG; (B) commercial Protein G'; and (C) basic Dictyoglomus XynBto commercial synthetic zeolite, silica and Castle Mountain natural clinoptilolite (106-250 μπι fraction). Each substrate tested was at a concentration of 5 mg. Unbound and wash fractions (3 xlOO μΐ) were not loaded onto the SDS-PAGE gels. S, starting protein (30 μg); U, unbound protein fraction; B, substrate- bound protein fraction; FAU, faujasite; MOR, mordenite; CLI, clinoptilolite; SIL, silica.

Fig. 28 shows binding affinity of purified basic Dictyoglomus XynB to synthetic zeolite CBV100. Binding was performed over pH range 4 to 9. Zeolite tested was at a concentration of 5 mg. Wash fractions (3 xlOO μΐ) were not loaded onto the SDS-PAGE gels. S, starting protein (30 μg); U, unbound protein fraction; B, zeolite-bound protein fraction.

Fig. 29 illustrates truncated derivatives of LPG carrying the synthetic (GGGGS)n sequence.

Fig. 30 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-P GE and visualised by staining with Coomassie brilliant blue.

Fig. 31 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.

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 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 acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, 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.

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. 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 acknowledgment or admission or any form of suggestion that 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. Detailed Description

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

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

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 co//allowing the bacterium to bind to zeolites. It has been demonstrated also 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 the sequence of SEQ ID NO: 1 (VKTQATSREEPPRLPSKHRPG) or a fragment, variant or derivative thereof. The peptide linker typically comprises more than one occurrence of the peptide sequence, for example (VKTQATSREEPPRLPSKHRPG),, in the case of SEQ ID NO: 1, where n can be any number. Typically n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14or 15.

In some embodiments the linker may comprise or further comprise a fragment of SEQ ID NO: 1. The term "fragment" as it relates to SEQ ID NO: 1 refers to an amino acid sequence that comprises a portion of the amino acid sequence of SEQ ID NO: 1. A fragment of SEQ ID NO: 1 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: 1. Such deletions can occur at the amino-terminus or carboxy-terminus of SEQ ID NO: 1, 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: 1. The term "derivative" as it relates to SEQ ID NO: 1 and fragments thereof, refers to a sequence comprising having 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:l, or (V TQATSREEPPRLPSKHRPG)n where n is 2, 3, 4, 5, 6, 7, 8, 9,10, 1 1, 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 repeat 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 (VKTQATSREEPPRLPSKHRPG) _n and a capture moiety.

In some embodiments, the peptide linker may further comprise a capture moiety, typically bound or attached to a carboxy or amino terminus of the peptide linker. The skilled person will understand that the term "capture moiety" refers to any moiety that binds to a biological or chemical entity. The capttire moiety may selectively or specifically bind to one or more biological 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. The antibody-binding protein may be protein A, protein G, protein A/G or protein L.

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, protein A, protein A/G or protein L and an antibody.

Where the capture moiety is a protein, the protein may be expressed as a fusion protein with the peptide linker. The peptide linker may be fused to the amino-terminus or the carboxy-terminus or both. In some embodiments the linker peptide may be expressed within a protein sequence, for example in a solvent accessible loop region. The peptide linker also may be used to join capture moieties derived from two chains of a multi-chain protein or two separate capture moieties with different specificities or selectivities.

In some embodiments the capture moiety may be a non-polypeptide moiety such as a toxin, drug, radioisotope, metabolite, contaminant, enzyme substrate or organic compound.

The wild type nucleotide sequence encoding linker-protein G is 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 (VKTQATSREEPPRLPSKHRPG) 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 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.

For Example, a capture moiety may be covalently or non-covalently bound to a peptide linker. 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(Bissulfosuccinimidyl suberate). Other crosslinking reagents include, for example carbodiimide crosslinkers such as EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide)).

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, virus, bacterium, yeast, fungus archaeon or algae.

In another embodiment, the biological entity may be a eukaryotic cell, a 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, for example, pathogenic and nonpathogenic bacteria.

In other embodiments the biological entity may be an organelle for example mitochondria, nuclei, chloroplasts, endoplasmic reticulum, golgi, acrosomes,

ί autophagosomes, centrioles, cilia, glycosomes, glyoxysomes, lysosomes, melanosomes, myofibrils, thylakoid, 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 a protein such as a glycoprotein, lipoprotein, phosphoprotein, 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 mono-saccharide, an oligosaccharide, a poly-saccharide, a mineral, a catalyst, a volatile compound, an aromatic 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 waste water or sewerage.

In some embodiments 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-products 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. 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 material for example, 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, 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.

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 .

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 _2j 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 microparticles or nanoparticles.

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

Silica microparticles may have a mean diameter of about Ιμπι to about 10 μπι, about ΙΟμι ^η to about 20 μιη, about 20μπι to about 50 μιη, or about 50μπι to about 100 μπι, about ΙΟΟμ ^η ι to about 150 μπι, about 150μπι to about 200 μιη, about 200μιη to about 250 μπι, about 250μπι to about 300 μιη, about 300μηι to about 350 μπι, about 350μπι to about 400 μπι, about 400μπι to about 450 μηι, about 450μπι to about 500 μη , about 500μηι to about 550 μιη, about 550μιη to about 600 μπι, about 600μιη to about 650 μπι, about 650μιη to about 700 μπι, about 700μπι to about 750 μπι, about 750μπι to about 800 μπι, about 800μπι to about 850 μπι, about 850μπι to about 900 μιη, about 900μιη to about 950 μπι, about 950μιη to about 1000 μπι.

The silica may be granular. Silica granules typically have a mean diameter of about 1000 μηι or greater. One example of granular silica is desiccated 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 at 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), 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 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 ) that is solid at room temperature and pressure. The skilled person will understand that any glass may be useful in the present invention.

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 0 ₃ )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 comprise metals, some zeolites are magnetic and some magnetic materials can be coated with zeolites or silica. 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 /Al ₂ 0 ₃ ratios. Dye-exclusion assays of proteins binding to zeolites have indicated that the affinity of the linker-peptide zeolite is a consequence 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 linker-peptide would have a very narrow and selective affinity for particular zeolites (Nygaard et al 2002, Adv. Mater. 14:1853-1856). However, in contrast, 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 embodiment, the silica-containing material comprises a zeolite comprising 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 _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% Si0 ₂₎ or at least about 99% Si0 ₂ . The silica-containing material may comprise a zeolite.

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.

Methods

The person skilled in the art will appreciate that a peptide linker according to the present invention forms a complex with a silica-containing material and can be used to capture any biological or chemical entity, for example via a capture moiety. Accordingly, a peptide linker bound directly or indirectly to a biological or chemical entity will be useful in any method or process where it is desirable to, for example, remove, purify, recover, reuse or reduce the amount of a biological or chemical entity in a sample.

With reference to Example .8 there is provided a method of recovering and/or reusing enzymes in industrial processes. It will be understood by a person skilled in the art that a peptide linker directly or indirectly bound to a protein or enzyme of interest may be used to facilitate the capture of that protein or enzyme and thereby allow reuse of the enzyme. For example, there is exemplified herein a peptide linker expressed as a fusion protein with a xylanase enzyme. However, the skilled person will understand that a peptide linker of the present invention may be expressed as a fusion protein with any known protein or enzyme. Examples of enzymes that may be expressed as a fusion with a peptide linker of the present invention include but are not limited to amylases, arabinoxylanases, amyloglucosidases, beta-glucanases, cellulases, glucoamylases, glucanases, lactases, lyases, mannanases, pectinases, polysaccharide lyases, puUulanases, xanthanases, xylanases, and other glycoside hydrolases. Further examples of enzymes that may be expressed as a fusion with a peptide linker of the present invention include but are not limited to acetolactate decarboxylases (ALDC), proteases, restriction enzymes, D A ligases, DNA polymerases, trypsin, rennin, lipases, papain, glucose isomerases, ligninases, catalases, , glycosyl transferases and carbohydrate esterases..

The peptide linker comprising the enzyme or protein then may be used in any process, such as the cleavage of xylan in Example 8. At the completion of the reaction or at any time during the reaction the enzyme may be captured by the addition of a silica- containing material. On contact with the silica-containing material, the linker peptide will bind to the material thereby forming a complex of the silica-containing material and the linker peptide/enzyme fusion protein. As the silica-containing material is typically a particulate or granular form it can easily be removed from an aqueous solution, for example by centrifugation, filtration, magnetic separation or by allowing the silica- containing material to settle from solution. Once the silica-containing material has been separated from the aqueous solution the bound linker peptide/enzyme fusion may be eluted from the silica-containing material as described above and thus reused. Alternatively, the silica-containing material bound to the linker peptide/enzyme fusion may be used, for example in a further process.

In one aspect a peptide linker bound directly or indirectly to a biological or chemical entity is useful for purification and or recovery of the biological or chemical entity, such as a protein, or for example microorganisms or eukaryotic cells expressing a peptide linker on the cell surface. With reference to Example 9 there is provided herein a purification tag for recombinant proteins. In this aspect, the purification tag comprises a peptide linker of the present invention. In Example 9 a peptide linker-MekB enzyme is expressed as a fusion protein with a peptide linker. The fusion protein was expressed in a bacterium and a crude extract of the bacterium was contacted with a silica-containing compound, in Example 9 a synthetic zeolite was used to purify the linker-MekB from the crude extract.

Accordingly a skilled person will understand that the peptide linker of the present invention may be expressed as a fusion protein with any protein of interest to facilitate purification of that protein. In such applications the peptide linker may further comprise a cleavage sequence to facilitate removal of the linker from the protein component after the purification. Examples of cleavage sequences include but are not limited to the recognition sequences for trypsin, chymotrypsin, thrombin, Factor Xa, and tobacco etch virus (TEV) protease. In some embodiments the silica particulars may be magnetic to facilitate the purification of linker peptides bound to those particles.

The skilled person will also understand that the linker peptide may be fused to a protein of interest at either or both of the amino or carboxy termini of said protein.

The biological entity may be recovered from the silica-containing material by elution, for example using an elution buffer described below or using an engineered enzyme cleavage point within the peptide sequence.

In industrial processes, the captured biological entity, for example an enzyme may be eluted from the complex for re-use.

With reference to Example 1 1 there is provided a method of attaching a biological entity to a synthetic fibre via a peptide linker. In particular the method relates to zeolite- loaded fabrics and proteins directly or indirectly associated with a peptide linker of the present invention. While Example 11 illustrates the use of a synthetic fibre, it is envisaged that any natural or synthetic fibre or fibrous material may be used. Examples of natural fibres include, cotton, coir, hemp, silk, flax, jute, sisal, wood, wool or other animal hair. Examples of synthetic fibres include, fibreglass, carbon fibre, kevlar, aramid, derclon, microfiber, modacrylic, nylon, olefin, polyester, polyethylene, spandex, vinalon and zylon.

Examples of fibrous materials include any fabric comprising a natural or synthetic fibre. In particular paper is envisaged to be useful in the present invention. As shown in Example 11 proteins associated with the peptide linker specifically bind to the zeolite attached to a fabric. Accordingly preloading fabrics with zeolite and/or silica provides a means to further modify the characteristics of the fabric, for example by including proteins or enzymes associated with a peptide linker which are suitable for the neutralization of odours, chemicals or toxins, or for example, proteins with anti-microbial properties or luminescent or fluorescent properties. In other embodiments peptide linkers of the present invention may interact directly or indirectly with fire retardant chemicals or toxin- neutralising moieties such peptide linkers may be preloaded on to zeolite-containing fabric to produce for example, fire retardant clothing, safety clothing, odour reducing clothing, temperature reactive clothing or fabrics for use in monitoring exposure to biological or chemical entities such as chemicals or toxins.

In another aspect, a peptide linker bound directly or indirectly to a biological entity may used to reduce the rate of a reaction mediated by a biological entity or stop a reaction mediated by a biological entity, such as an enzyme. The reaction rate may be reduced or the reaction stopped by contacting the peptide linker bound directly or indirectly to a biological or chemical entity (for example an enzyme) with a silica-containing material under conditions that allow the binding of the peptide linker to the silica-containing material. Alternatively, or in addition, the reaction rate may be reduced or the reaction stopped by contacting the peptide linker bound directly or indirectly to a cofactor essential for the reaction. The sample in which the reaction is occurring may be passed over the silica-containing material that may be present in a column, on a surface or other assembly. Alternatively the silica-containing material may be added to the sample. Binding of the peptide linker-bound entity thereby separates the peptide linker-bound entity from the environment in which it is active. In embodiments where the silica-containing material is added to the sample, the peptide linker-bound enzyme may be removed from the reaction mixture by any means known in the 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 peptide linker of the invention.

In another aspect, peptide linkers are envisaged to be useful for removing a biological entity, such as a protein or microorganism from a sample. For example, a peptide linker may be bound directly or indirectly to a protein or microorganism. The complex peptide linker-bound microorganism (for example) will be contacted with a silica- containing material, for example either by passing the sample over the silica-Containing material or by adding the silica-containing material to the sample under conditions that allow interaction of the peptide linker-bound microorganism with the silica-containing material. The peptide linker-bound microorganism may be recovered from the silica- containing material by elution, for example using an elution buffer described below.

In one embodiment the biological entity may be a microorganism, such as a prokaryotic or eukaryotic cell or virus that expresses a peptide linker on its cell surface or is bound via a capture moiety. A biological entity that may be bound via a capture moiety may be, for example, 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, Toxoplasm and Trichinella.

Examples of pathogenic bacteria include those bacteria from the genera Actinobacillus, Actinomyces, Bacillus, Borrelia, Bordatella, Brucella, Burkholderia, Campylobacter, Chlamydia, Clostridium, Coxiella, Enterococcus, Esherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Legionella, Leptospira Listeria, 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, Cryptosporidium, Eimeria, Isospora, 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), Flayiviradae (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), Togavindae (e.g. Rubella virus), Parvovitridae (e.g. Human bocavirus and Parvovirus B19), 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, blood or urine. The suspension may be a cell culture for example used in production of therapeutics expressed from eukaryotic or prokaryotic cells e.g. antibiotics and antibodies. The sample may be an enzyme catalysed chemical reaction.

Assemblies

Biological or chemical entities may be captured according to the methods of the invention using an assembly comprising a solid support having disposed thereon a silica- containing material. The silica-containing material may be present in a liquid or disposed on a solid support. The skilled person will appreciate the solid support may comprise any suitable substrate for example, particles, beads, powders, coatings, fibres such as paper or fabric, a surface of a vessel or chamber or a substantially planar substrate such as a strip or dipstick.

In one embodiment, biological or chemical entities may be captured according to the methods of the invention using an assembly typically comprising a chamber containing a silica-containing material capable of interacting with the 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 silica-containing material capable of interacting with the peptide linker is disposed within the chamber and in the flow path such that when a fluid containing the biological or chemical entity is passed through the chamber, the biological or chemical entity contacts the linker peptide.

The chamber may further comprise 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.

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 of 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 at least two assemblies.

Elution of biological or chemical entities

Captured biological or chemical 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 , NaCI, ammonium sulphate, cetyltrimethylammonium bromide (CTAB), polyethyleneimine (PEI), KC1, guanidine hydrochloride (CNsHs'HCl), ammonium thiocyanate (NH ₄ SCN), sodium deoxycolate, KI, L-histidinemonohydrochloride or L-arginine monohydrochloride.

The concentration of the buffering 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, 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, 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 6 buffers suitable for elution of the linker peptide from the silica-containing material typically contain betaine, imidazole, MgCl ₂ , NaCl ammonium sulphate, cetyltrimethylammonium bromide (CTAB), polyethyleneimine (PEI) and L-histidinemonohydrochloride. The buffers typically have a pH in the range of 3.0 and 10.0. The buffers are particularly useful for the release of excess zeolite-bound peptidelinker that may not have formed a complex with the biological entity to be captured.

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

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 said cleavage. Examples of suitable cleavage sequences include (but are not limited to) the recognition sequences for 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 silica-containing material capable of interacting with a peptide linker. The kit may further comprise a peptide linker.

In another aspect there is provided a kit for the capture of a biological or chemical 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 silica-containing material capable of interacting with a peptide linker wherein the a silica-containing material is disposed within the chamber and in the flow path.

The peptide linker may comprise a capture moiety.

The kit may further comprise at least one elution buffer for the ^~ elution of the biological or chemical entity from said silica-containing material and/or from said peptide linker.

The kit can also include printed instructions for using the kit to capture a biological or chemical 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.

For applications where the capture and/or detection of entities is desired, a single kit of the invention may be applicable, or alternatively different kits, for example containing reagents specific for each entity, may be required. Methods and kits of the present invention find application in any circumstance in which it is desirable to capture and/or detect any 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 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" SEQ ID NO:

GFP1-F 5'-CCGGTAGAAAAAGATCTTAAAGGAGAAGAA-3' 5

GFPl-R 5'-GCGCTCAGTTGGAATTCTACGAATGCTATT-3' 6

GFP2-F 5'-CCGGTAGAAATC_ATG_AGTAAAGGAGAAG-3' 7

GFP2-R 5'-G AATTCT ACG ACTGC AGTTTGT ATAGTTC-3 ' 8

TPLF1 5'-CATGGATATCGGAATTCATATGGATCCAGCGG-3' 9

TPLR1 5'-GCGGCCGCAAGAATTCGGCTGCAAGCTC-3' 10

LinXynAd2-lF S'-CGGTAGAAGAAGATCTTCCGTCCCTGAAAGATGTTT-S' 11

LinXynAd2-lR 5'-ACTCAGAATTCTACTAACTTAGGATCCGACTACCGC-3' 12

MekAFl 5'-CAATATGAGGCATAAAG^AICCTGCTCAATCTAAGCTCGCCGCT-3 13

MekARl 5'-AGCCTCAACTAATGAGCAAGCTTTCGTCATCAAGCCATTTCAAAG-3 14

MekBFl 5*-TAAACGAGGTAACCTGAGGATCCCAGCTATTACACCGAAGAAAATC-3 15 ekBRl S'-TGGGTTATTGGCTGGCAAGCTTTTATCAATACGCGGGAGATGACAG 16

Linker IF 5'-GAC ACCAGAAATGC^TATGC AGACTC AGGC-3 ' 17

Linker 1R 5 ^, -GGTTTTCCGGATCCTCGAGGCτGGTC-3 ^, 18

Linker2F 5'-GAC ACC AGAAATGCCTCTGC AGACTC A-3 ' 19

Linker2R 5'-GCCCGGTTTTCAAGCTTCTAGAGGCTGGTCGC-3' 20

Linker3F 5'-GAACTATACAAACTGAATTCTCAGGCGACCA-3' 21

DuetDownl 5'-GATTATGCGGCCGTGTACAA-3' 22

LinklxFl 5'-GCAAACATCGTCCGCATATGCAAACCCAGGCGACCA-3' 23

Link2xFl 5'-GCAAACACCGTCCGCATATGCAAACCCAGGCGACCA-3' 24 Link3xFl 5'-GCAAACATCGTCCGCATATGCAAACCCAGGCGACCAGCCGCGAA-3' 25

PG-R 5'-GAGCTCGAATTCGGAT£CGATTATTATTCC-3' 26

"Engineered restriction sites are underlined.

To generate pLinkerlpET22b (encoding the Linker sequence VKTQATSREEPPRLPSKHRPG^VKTQTAS, the amplified DNA fragment was digested with Ndel I BamHl and ligated into similarly-cut pET22b. The expression plasmid pLinker-GFP (encoding the Linker sequence

VKTQATSREEPPRLPSKHRPG) ₄ VKTQTAS followed by GFP was generated from the amplified DNA sequence digested with BgHl I EcoRl and ligated into BamUl I EcoRIcut 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 BspUl I Pstl and ligated into Ncol I iM-cut pETDuet-1 to create pGFPpETDuet-1. A Linker DNA fragment encoding the Linker

VKTQATSREEPPRLPSKHRPG) ₄ VKTQTAS 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-cut pLinkerlpET22b to generate expression plasmid pLinker-TPL (encoding the Linker sequence followed by the TPL lipase). To obtain expression plasmid pTPL-Linker (encoding the TPL lipase followed by the Linker sequence), the DNA fragment amplified with the TPLF1 and TPLR1 primer pair was digested with Ndel I EcoRl and ligated into similarly-cut pET22b to create pTPLpET22b. A Linker DNA fragment was amplified with the Linker3F and DuetDown primer set, digested with EcoRl / Hindlll and ligated into similarly-cut pTPLpET22b to generate pTPL-Linker.

Expression plasmids pLinker-MekA (encoding the Linker sequence followed by the MekA monooxygenase) and pLinker-MekB (encoding the Linker sequence followed by 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 BgRl I EcoRl and ligated into BamHl I EcoRI-cut pLinkerlpET22b. The expression plasmids are shown schematically in Table 2. Table 2 Expression plasmids used in this study.

Plasmid Primers PCR template Construction Linker Position Function pGFP" N.A I GFP N.A Green fluorescent protein pLinker-GFP 1- 1- pGFP N« Green fluorescent protein

2- 2- pGFP-Linker pSN6 ^c

Linker2F / Unker2R pGFP Γ C Green fluorescent protein pTPL' N.A N.A Lipase pLinker-TPL N.A N Lipase

TPLF1 /TPLR1 pTPL

pTPL-Unker C Lipase

Linkei3F / DuetDown! pGFP-Unker

pUnker-XynAd2 LinXynAd2-1F / LinXynAd2-1R pSUN18 ⁹ Xylanase pUnker-MekA ekAFI / MekARI pJOE5332.10" Monooxygenase pLinker-MekB MekBFI / MekBRI pJOE5332.tO Esterase

"Commercial plasmid (Novagen).

''N.A, not applicable.

cNygaar<3 et el. (2002) Adv. Mater. 14:1853-1856. Plasmid kindly provided by Stanley Brown. University of Copenhagen.

*N, Linker is at N-termlnus of fusion protein.

"C, Linker Is at C-termfnus of fusion protein.

'pET26b plasmid carrying the TPL lipase gene. Plasmid kindly provided by Noosha Ehya, Macquarie University.

"Sunna et al. (2000) Microbiology 1 6:2947-2955.

"Onaca et al. (2007) J. Bacteriol. 189:3759-3767. Plasmid kindly provided by Josef Altenbuchner, Universitat Stuttgart.

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 Agoo 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 (Figure 1) 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-HCl, pH 7.5, 100 mM NaCl and 1% Triton-XlOO). Soluble protein in a final volume of 100 μΐ was mixed with zeolite and incubated by rotation at room temperature for 1 h. After centrifugation 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 PAGErloading 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.

Enzyme assays

Lipase activity was determined using p-nitrophenyl ester as substrate. The standard assay reaction mixture contained 1.0 mM p-nitrophenyllaurate (CI 2), 100 mM Tris-HCl buffer (pH 8.0) and enzyme; the final volume was 200 μΐ. The reaction mixture was incubated at 50°C for 5 min.

Xylanase activity was determined by the dinitrosalicylic acid method. The standard assay reaction mixture consisted of 1% (w/v) xylan supplemented with 120 mM universal buffer, pH 6.0, and enzyme to give a final volume of 0.1 ml. Samples were incubated at the specific temperature (60 - 80°C) for 10 min.

a-Naphthyl acetate-hydrolyzing esterase activity was assayed according to Miller and Karn (1980, J. Biochem. Biophys. Methods, 3:345-354). The reaction mixture included 100 μΐ enzyme solution, 50 mM phosphate buffer, pH 7.0, 200 μΐ of Fast Blue RR salt (0.5 mg/ml) in 100 mM Tris-HCl buffer pH 7.4, and 50 μΐ of α-naphthyl acetate (10 mg ml) in a total reaction volume of 350 μΐ.

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

The Protein G sequence used was based on that reported by Go ward et ah, Biochem. J. 267:171-177, 1990 which encodes for a truncated recombinant form of Streptococcus strain G148 Protein G (Protein G'). Linker and 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 wild type LPG (WT LPG, SEQ ID NO: 3). The codon usage of the final combined WTJLPG sequence was optimised for expression in E. coli. Rare codons, 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 BamHl) 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 linker-protein G expressed from wild type nucleic acid is provided in SEQ ID NO: 2. The linker-protein G protein expressed from the codon optimised linker-protein G nucleic acid sequence (OPT LPG) is referred to herein as Linker-ProteinG (LPG). LPG comprises the (VKTQATSREEPPRLPSKHRPG) 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.

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 incubated at 37°C with shaking (250 rpm) until the A ₆₀ o 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 mM 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, 1.25 mM 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 μηι 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 25 mM Tris-HCl, pH 8.0, supplemented with 100 mM NaCl. The column was washed extensively with the same buffer. Under these conditions the LPG was found in the 100 mM NaCl fraction. The LPG fraction was applied to a 5 ml HiTrap SP cation exchanger column (GE Healthcare) previously equilibrated with buffer containing 100 mM NaCl. The column was washed extensively with the same buffer and the LPG was eluted with 150-200 mM 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 25 mM Tris-HCl, pH 8.0, 100 mM 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 (VKTQATSREEPPRLPSKHRPG) 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 BamRl and 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 d to produce the truncated LPG derivatives.

Recombinant protein Plasmld Primers Linker sequence repeats

Unter

BfeM PG ~1 pOPT_LPGpET22b N.A* 4

pLink3xpET22b Llnk3xF1 / PG-R

pLink2xpET22b Llnk2xF1 / PG-R

I PG I pUnk1xpETT22b Link1xF1 / PG-R 1

I PG' |" N.A N.A 0

aN.A, not applicable.

. Purified recombinant truncated Protein G' (Sigma-Aldrlch 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 μg 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 100 μΐ of SDS PAGE-loading 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: 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 mMTris-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 times with 200 μΐ of 100 mMTris-HCl buffer, pH 8.0. The pellet was resuspended in 100 μΐ of 1 M L-arginine monohydrochloride (pH 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 monohydrochloride (pH 3.9). 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 QDot605 nanocrystals (Invitrogen) followed by confocal microscopy on an Olympus Fluoview FV 300 confocal laser-scanning microscope. Example 2: Effect of peptide linker on recombinant protein/enzyme function

In order to test the effect of the peptide linker, the peptide linker was introduced into plasmids containing the genes for several enzymes/proteins at either the N- or C- terminus and assessed for the activity/biological function of the resulting recombinant fusion protein. Table 4 summarises the results obtained with GFP and 4 enzymes.

The presence of the peptide linker at the N-terminus of the recombinant GFP protein resulted in no fluorescence whereas the C-terminal linker fusion resulted in a functional GFP protein displaying both fluorescence and high binding affinity to zeolite CBV100 (Figure2).

In the case of the TPL lipase, the presence of the peptide linker at either N- or C- terminus had little effect on the lipase activity. However, only the C-terminal linker displayed binding affinity to the zeolite. All three N-linker constructions pLinker- XynAd2, pLinker-MekA and pLinker-MekB, retained their enzymatic activity and displayed high affinity to zeolite CBVIOO (Table 4). The results suggest that as with several affinity tags, e.g. His tag, glutathione 5-transferase (GST) or maltose binding protein (MBP), the effect of the peptide linker on the protein function and potential zeolite binding affinity must be determined empirically.

Table 4. Expression plasmids used to produce the recombinant proteins with and without the linker sequence. The ability to retain the protein/enzyme function after linker incorporation and the zeolite binding affinity of the expressed proteins is shown.

Plasmld Construct Linker Function retained Zeolite binding pGFP 1 GFP 1 N.A" Yes No

pLinker-GFP """sq 1 N" No N.O ^c

pGFP-Linker 1 C Yes Yes

pTPL 1 TPL 1 N.A Yes No

pUnker-TPL ^ TPL 1 N Yes No

pTPL-Unker 1 — j

TPL C Yes Yes

pLinker-XynA N Yes Yes

pLInker-MekA N Yes Yes

pLInker-MekB N Yes Yes

"N.A. not applicable.

Ή Unker Is at N-termlnus of fusion protein.

'N.D, not determined.

dC, Unker Is at C-termlnus of fusion protein. Example 3: Binding affinity of purified recombinant LPG to natural and synthetic zeolite

The affinity of the purified recombinant LPG towards different types of commercial natural and synthetic zeolites was carried out as described under the General Methods section. Two types of natural zeolites were tested (Figure 3). Zeolite Australia (Zeolite Australia Pty Ltd, NSW, Australia) is a zeolite with a 54% clinoptilolite composition while Castle Mountain (Castle Mountain Enterprises Pty Ltd, NSW, Australia) zeolite is composed of 85% clinoptilolite. The cation-exchange capacity (CEC) for Zeolite Australia and Castle Mountain zeolites are reported as 1.19 and 1.47 meq/g, respectively. The Zeolite Australia (7 Mohs) was 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. Larger particle size may result in lower surface area and significant reduction inaccessible silica available for LPG binding.

Thirteen commercial synthetic zeolites belonging to 8 different zeolite families were tested as binding substrates for the purified LPG (Figure 4). 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 summarises the 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), 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 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 Na20 weight Unit cell size Surface area Binding ³

(mole ratio) form (%) (A) (m2/g) U B

Faujaslte (FAU)

(FAU-Y)

CBV 100 5.1 Sodium 13.0 24.65 900 mm

CBV 300 5.1 Ammonium 2.8 24.68 925 mm

CB 400 5.1 Hydrogen 2.8 24.50 730

(FAU-X)

Molecular Sieve 13X 5.1 Sodium 14.6 24.94 700 mm

Mordenlte (MOR)

CBV 10A 13.0 Sodium 65.0 N.A ^b 425 mm

CBV 21A 20.0 Ammonium _k 0.08 N.A 500 mm

H-MOFt-1 14.0 N.A N.A N.A 471 «■»

Ferrlerlte (FER)

CP 914c 20.0 Ammonium 0.05 N.A 400

Beta Polymorph A (BEA)

CP 814E 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 ΝΛ N.A 426

Linden Type A (LTA)

Valfor lOO 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.

"Data not available.

cHul 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 (Figure 5). Twelve different types of commercial silica materials were tested. LPG displayed high binding affinity to all silica samples, except for Silica LC 60 A (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 the main 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% (Figure 5). Only 40% of the LPG was found in the bound fraction of glass beads (74% silica), whereas 20% was found in the bound fractions of zirconia/glass beads (31% silica) and flint glass powders (45% silica). Thus, we have demonstrated also that the zeolite-specific linker peptide displays very high affinity to different types of commercial silica. Table 6 Main properties of the commercial silica materials tested and LPG binding results.

Silica

Silica (precipitated)

Silica gel Grade 646

Silica Davisll LC 60A

Silica Gel 60 Schariau

Rhode-line HP 34M

Tixosil 38

Tixosil 38A

Tixosil 68

Perkasil S 660

Perkasil KS 300-PD

Elfadent S 514

Durafill 200'

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

cPata 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 6 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 a linker sequence. In both cases only a non-specific binding of less than 5% was observed.

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

Recombinant protein Linker sequence repeats Binding assay ³

Zeolite SHica

Linker

PG 4 100 100

PG 3 100 100

PG 2 40 30

PG 1 <5 <5

PG' 0 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 pg.

Example 6: 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 7-11). 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 ^• glycol (PEG). After addition of class 1 buffers, all the proteins remained bound to the zeolite matrix (Figure 7).

Class 2 buffer comprise compounds/solutions that mainly eluted the LPG, e.g. betaine, imidazole, MgCl ₂ , ammonium sulphate, cetyltrimethylammonium bromide (CTAB), polyethyleneimine (PEI) and L-histidine monohydrochloride. After the addition of class 2 buffers, most of the "LPG-IgG complex" remained bound to the zeolite matrix (Figure8).

Class 2 buffers appear to selectively elute the bound LPG, as shown in both elution fractions (El and E2, Figure 9). Under the experimental conditions used, LPG is present at 7.5 molar excess to IgG (assuming a 1 :1 binding stoichiometry). This result implies that class 2 solutions 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-bound LPG, the binding experiment was repeated as before using L-histidinemonohydrochloride 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 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.

The elution efficiency of 1 M L-arginine monohydrochloride was also tested at pH values between 3.0 and 8.0 (Figure 11). 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, 1 M guanidine hydrochloride (CN3Hs ^» HCl), 1 M ammonium thiocyanate (NH ₄ SCN) and 1% 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 7: Mode of action of arginine elution buffer.

The mode of action of the 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 μηι under the confocal microscope and displayed no autofluorescence (Figure 12 A). 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 bound previously 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 (Figures 12C and 12D), QDot605 nanocrystals were found surrounding but excluded from the surface of the zeolite particles.

Incubation of the "zeolite-LPG^RY104- ?tO-fpor/i/m»i" complex with the class 3 arginine elution buffer and subsequent incubation with 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 (ab') ₂ fragment (conjugated to AlexaFluor 444) secondary antibody to bind to the CRY 104-Ctyptosporidium bound antibody after the arginine elution step (data not shown).

Example 8: Reuse of enzymes attached to solid matrices

As described above, the peptide linker sequence was introduced at the N-terminus of a xylanase enzyme XynAd2. The expressed recombinant fusion protein, Linker- XynAd2, retained its enzymatic activity and also was able to bind selectively to zeolite (Table 4). In this application we propose the use of zeolite and/or silica (or materials containing silica) as a solid matrix to remove enzymes/proteins carrying the peptide linker from solutions such as fermentation supernatants or reactant solutions. This approach could be used to stop an enzymatic reaction or to reuse (recycle) the matrix-bound enzyme (protein). A proof of application experiment was designed as follows. Recombinant Linker-XynAd2 was produced and extracted as described under the General Methods section. The soluble Linker-XynAd2 enzyme fraction was incubated with synthetic and natural zeolite according to the standard binding assay. Following three washing steps to remove unbound proteins, the xylanase activity of the zeolite-bound enzyme was assayed as described under the General Methods section. After 10 min enzyme assay, the mixture was centrifuged to remove the supernatant containing the reducing sugars produced by the xylanase. The zeolite pellet was incubated again with 100 μΐ of xylan substrate for 10 min. This cycle was repeated for a total of 5 times. Figure 14 shows a qualitative comparison of the results obtained with the Linker-XynAd2 enzyme and previous results obtained with the purified XynAd2 recombinant enzyme. First, most of the xylanase remained bound to the zeolite matrix as shown by the production of reducing sugars from the xylan substrate. Secondly, it was possible to remove the zeolite-bound enzyme from the reaction and recycle the enzyme for at least 5 cycles (10 min each). Thirdly, the half-life of the zeolite- bound Linker-XynAd2 matched that previously reported for the recombinant XynAd2. These results confirmed that zeolite/silica matrices could be used for the removal or recycling of enzymes, protein, metabolites etc carrying the zeolite peptide linker.

Example 9: Peptide linker as a purification tag

The linker peptide can be used as a simple purification tag for recombinant proteins. The expression plasmid pLinker-MekB (encoding the Linker sequence followed by the MekB esterase gene) was used to produce a recombinant Linker-MekB enzyme in E. coli. Enzyme production and zeolite binding assay was as described under the General Methods section. The mekB gene from Pseudomonas veronii MEK700 encodes a highly active esterase-like enzyme with sequence similarity to homoserine acetyltransferases. Incubation of a crude extract of soluble proteins from the E. coli expressing Linker-MekB and synthetic zeolite is shown in Figure 15. All of the Linker-MekB was found in the zeolite-bound fraction (Figure 15A).Several host protein bands were found in the unbound, wash and bound fractions. The overall surface charge of zeolite is negative and thus we expect at least some basic E. coli proteins to bind to the zeolite particles. As with other protein purification systems (e.g., His-tag, anion-, cation-exchange chromatography), the binding of host proteins can be reduced by addition and/or variations of salts (e.g. NaCl), pH and other compounds.

As shown in Figure 15B, MekB esterase activity was not observed in the unbound or wash fractions but only in the final zeolite-bound fraction. Thus, Linker-MekB does not only display high specific binding affinity towards zeolite (Figure 15 A) but also retained its catalytic activity upon binding to the zeolite matrix (Figure 15B).

Example 10: Magnetic silica particles

The linker peptide can be used as a specific affinity tag to mediate the binding of proteins to magnetic silica particles. A recombinant GFP protein carrying a C-terminal peptide linker was produced using the pGFP-Linker expression plasmid as described in the General Methods section. A soluble protein crude extract from the E. coli containing pGFP-Linker expression product (GFP-Linker) was incubated with non-porous organosilica Si02/Fe ₃ 0 nanoparticles (100 - 200 nm). The nanoparticles and the GFP-Linker were incubated as described in the standard binding assay and the nanoparticles were separated from the solution using a magnetic separator. A control experiment was performed using a GFP protein without the Linker (plasmid pGFP expression product). The binding was followed by confocal microscopy on an Olympus Fluoview FV 300 confocal laser-scanning microscope. As shown in Figure 16, no fluorescent signal was observed when the control GFP without linker was incubated with the magnetic silica nanoparticles. This result indicated that there was no affinity between the control GFP and the silica nanoparticles.

However, when the crude extract containing the GFP-Linker recombinant protein was incubated with the magnetic silica nanoparticles, strong fluorescence of the nanoparticles (Figure 17) was observed after binding and several wash steps. This result clearly confirmed the affinity of the linker for the silica nanoparticles and its potential to mediate a simple purification of recombinant proteins. The use of magnetic silica nano- or microparticles allows for a quick and simple removal of proteins (carrying the linker) of interests from solutions, reactions or host crude extracts.

Capture of the peptide linker can also be performed using magnetic silica particles. First, the affinity of LPG (and subsequent IgG) to magnetic silica particles was tested following the standard binding assay with minor modifications. A 1 mg sample of magnetic silica particles was washed three times with zeolite washing buffer and incubated with 15 μg of LPG. The sample was incubated for 15 min followed by three wash steps to remove unbound LPG. The sample was incubated with 15 g of CRY104 Cryptosporidium monoclonal antibody for another 15 min and the particles washed three times. The proteins were eluted with three washes of 1 M arginine pH 3.9 supplemented with 0.05% Tween 20. Any protein remaining bound to the magnetic silica particles after the previous elution step was eluted after addition of 100 μΐ of SDS PAGE-loading buffer and incubation at 99°C for 10 min (with a short mixing every 2 min). Fractions containing the protein were identified by SDS-PAGE and staining with Coomassie brilliant blue. As shown in Figure 18, most of the LPG and CRY104-IgG antibody remained bound to the magnetic silica particles. The LPG found in the first unbound fraction may represent an excess of LPG added to the magnetic silica particles. Almost all of the bound "LPG- CRY104" was eluted from the magnetic silica particles after incubation with the arginine elution buffer. Thus, the LPG displayed high affinity to the non-porous organosilica Si0 ₂ /Fe304 nanoparticles studied here.

Based on the results above, the potential for using magnetic silica particles plus the bound "LPG-IgG" complex for the capture of organisms was tested with Cryptosporidium. A 1 mg sample of magnetic silica particles was loaded with 15 μg each of LPG and CRY104-IgG Cryptosporidium monoclonal antibody, as described above. The magnetic silica particle-bound "LPG-CRY104 complex" was incubated with a sample of Cryptosporidium oocysts diluted in 500 μΐ of PBS (phosphate buffered saline) buffer + 0.05% Tween 20, pH 7.4. A control magnetic silica particle sample was treated as above but the LPG was replaced by Protein G' (Protein G without linker). The oocysts were eluted with 1 M arginine after the washing steps. Cryptosporidium oocysts in both samples were incubated with monoclonal antibody CRY 104 (10 μg ml) conjugated to Fluorescein isothiocyanate (FITC) and visualized by epifluorescence microscopy on an Axioskop 2 microscope (Carl Zeiss, Sydney, Australia).

Cryptosporidium oocysts were not captured when the magnetic silica particles were loaded with Protein G' and CRY104-IgG (Table 17). This result is due to the inability of Protein G' without the linker peptide to bind to the magnetic silica particles. When the magnetic silica particles were loaded with LPG and CRY104-IgG, 98% of the Cryptosporidium oocysts were captured from solution by the particle-bound "LPG- CRY104-IgG" complex (Table 17).

Table 17 Total number of Cryptosporidium oocysts captured and eluted from magnetic silica nanoparticles.

Protein G' LPG

Unbound fraction 22 0

Wash fraction 105 2

Elution fraction 3 131

Total oocysts 130 133

Capture (%) 2 98 Example 11: Selective attachment of proteins to synthetic fibres via a peptide linker

This application involves the ability of proteins and enzymes carrying the specific linker peptide to specifically bind or attach to fabrics, which have previously been loaded with zeolite and/or silica. A virgin (control) and a zeolite-preloaded fabric (40 gsm) were used in all experiments. Two forms of the Green Fluorescent Protein (GFP) were used in the binding assays. GFP (without linker peptide) was expressed using pGFP, while GFP- Linker was expressed from the pGFP-linker plasmid as described in the General Methods section. Two pieces (approx. 1.5 x 0.6 cm) of each fabric were cut and washed three times with 500 μΐ of zeolite washing buffer. GFP or GFP-Linker was added to the fabric samples and allowed to mix by rotation for 1 h. After binding, the liquid supernatant was removed and the fabrics were washed three times with buffer. Small pieces of the fabrics were removed carefully and used for examination by phase contrast and confocal microscopy.

The fibres of the virgin fabric appeared smooth and regular in shape under the phase contrast microscope (Figure 19). In the case of the zeolite-loaded fabric, the fibres appeared more irregular and the zeolite particles attached were clearly visible (Figure 19).

Under the confocal microscope, the difference between the fibres from the virgin and zeolite-loaded fabrics was clearly visible (Figure 20). At the GFP excitation wavelength, there was no fluorescence detected in the fabric samples (virgin or zeolite- loaded) that were incubated with the GFP lacking the specific linker peptide (Figure 20). However, a localised GFP fluorescence was observed on the zeolite-loaded fabric that was incubated with the GFP-Linker (Figure 21). A close-up on the zeolite-loaded fabric incubated with GFP-Linker clearly indicated that the localised GFP fluorescence corresponded to areas along the fibres that had been previously loaded with zeolite particles (Figure 21).

Only the GFP carrying the peptide linker (GFP-Linker) was able to bind specifically to the zeolite attached to the fabric.

Example 12: Introduction of a protease cleavage sequence

The PreScission Protease is a genetically engineered human rhino virus 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. 22). This recognition sequence was introduced between the Linker and the MekB sequence of Linker-MekB to create the fusion protein Linker-rTEV-MekB (Fig. 22).

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 23 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.

Example 13: Peptide linker as a purification tag II

Soluble protein crude extract of recombinant Linker-MekB was produced as described in the General Methods section above. A 5 mg sample of zeolite CBVIOO was washed three times with 30 mM MOPS buffer (pH 6.5) supplemented with 100 mM NaCl and 1% Triton X-100 and incubated with 100 μΐ of Linker-MekB crude extract as described in the standard binding assay. After binding, the zeolite was washed three times with the same buffer but without Triton X-100. The zeolite-bound Linker-MekB was eluted by resuspending the zeolite in 100 μΐ of 20 mM Tris-HCl buffer, pH 8.0, supplemented with 150 mM NaCl, 1 mM DTT, 0.5 mM EDTA and 0.01% Triton X-100. The zeolite was incubated by rotation at 4°C for 2 h. Any protein remaining bound to the zeolite after the elution step was eluted after addition of SDS PAGE-loading buffer andincubation at 99 °C for 10 min (with a short mixing every 2 min). Fractions containing the protein were identified by SDS-PAGE and staining with Coomassie brilliant blue.

As shown in Figure 24, there is an excess of Linker-MekB crude extract in the starting material. Accordingly, Linker-MekB was also found in the unbound fraction and in the first wash fraction. All excess of Linker-MekB was removed after the third wash step. After incubation with elution buffer most of the zeolite-bound Linker-MekB was eluted from the zeolite fraction. The eluted Linker-MekB fraction contained few host proteins and was at least over 85% pure. Some Linker-MekB and several host proteins remained bound to the zeolite after the elution step.

Example 14: Magnetic silica particles II

Soluble protein crude extract of recombinant Linker-MekB was produced as described in the General Methods section above. A 0.5 mg sample of magnetic silica particles was washed three times with zeolite washing buffer and incubatedwith 100 μΐ of Linker-MekB crude extract as described in the standard binding assay and the nanoparticles were separated from the solution using amagnetic separator. After binding the particles were washed three times. The bound proteins were eluted with three washes of 1 M arginine pH 3.9 supplemented with 0.05% Tween 20. Any protein remaining bound to the magnetic silica particles after the previous elution step was eluted after addition of SDS PAGE-loading buffer and incubation at 99°C for 10 min (with a short mixing every 2 min). Fractions containing the protein were identified by SDS-PAGE and staining with Coomassie brilliant blue.

As shown in Figure 25, some of the Linker-MekB is found in the unbound fraction. This is most probably due to an excess of Linker-MekB present in the starting material. Most of the Linker-MekB remained bound to the magnetic silica particles during the washing steps. The first elution released most of the Linker-MekB from the particles. Two extra elution steps were used to further release bound Linker-MekB. Several host proteins and some of the Linker-MekB remained bound to the magnetic silica particles after the final elution step. Thus, magnetic silica particles can be used for easy and fast purification of proteins carrying the peptide linker described here.

Example 15: Binding to zeolite of premixed of LPG and CRY 104 complex

Purified LPG and CRY 104 IgG where mixed at a ratio of 1 : 1 in PBS buffer (pH 6.5) for 15 min. The mix was then intubated 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 26, 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 16: Effect of protein charge on binding

A control-binding assay was performed with LPG and two other proteins without the linker sequence. A truncated Protein G' (Sigma-Aldrich) and a basic Dictyoglomus XynB xylanase (provided by Dr. Moreland Gibbs) were used as controls for the binding experiments. Binding assays with Dictyoglomus XynB at different pH values were also performed to assess the effect of the overall protein charge on adsorption to silica- containing materials. Dictyoglomus XynB, binding and washing buffers were adjusted to the final pH of the experiment. The following buffers at a final concentration of 100 mM each were used in the assay: Na-acetate buffer (pH 4 and 5); 2-(N-Morpholino) ethanesulfonic acid buffer (MES, pH 6); Tris buffer (pH 7 - 9).

LPG displayed high binding affinity to all the zeolites and silicas tested (Figure 27A). Protein G' without the linker sequence (Figure 27B) displayed no binding affinity towards FAU-Y synthetic zeolites (CVBIOO and CVB 300). However, with MOR zeolites (CBVIOA and CVB21A), approximately 40% of the Protein G' remained bound to these zeolites. Protein G' displayed no affinity towards the natural zeolite clinoptilolite and less than 5% remained bound to silica. On the other hand, the basic recombinant Dictyoglomus XynB was expected to have a positive overall charge under the assay pH and to bind to the negatively-charged zeolite and silica. However, under our binding assay conditions, Dictyoglomus XynB displayed no binding affinity towards all the zeolite-containing materials tested (Figure 27C).

Zeolite and silica provides an extensive negatively-charged surface for positively- ί

charged molecules. The results presented herein show that the presence of the zeolite- specific linker peptide mediates the binding between the protein and the silica-containing materials. The basic protein, XynB, lacking the linker peptide carries a very high overall positive charge at the lowest pH of the binding assay. XynB was unable to bind to zeolite (Figure 28) even when the binding assay was performed over a wide pH range (4 - 9).

Example 17: Synthetic linker design

Individual repeats from the peptide linker sequence used in the above Examples were replaced by a synthetic (GGGGS)„ linker sequence. Three derivatives were synthesized by GENEART (Regensburg, Germany) and fused to Protein G. The derivatives used are depicted in Fig. 29. In the derivatives Link2X-(GGGGS) ₄ -PG and Link2X-(GGGGS)s-PG two of the four repeats of the sequence VKTQATSREEPPRLPSKHRPG were replaced with four or eight repeats of the sequence 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. 30) and purified LinklX-(GGGGS)i ₂ -PG (Fig. 31) 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.