IMMOBILIZATION

Field of the Invention

[0001 J This invention relates to novel amino acid sequences that can function as linkers or anchors to facilitate immobilization of a protein ligand or another macromolecule onto a solid support.

Background of the Invention

[0002] Protein A is a membrane protein encoded by a bacterium (Staphylococcus aureus). Protein A consists of five homologues domains arranged as E-D-A-B-C. Protein A is unique in its ability to bind to most antibodies (including IgG, IgM, IgE, IgA) from various species including humans. Thus, Protein A has been widely used as a protein ligand immobilized on a solid support (eg., resin) to form an affinity column for antibody purification For research and therapeutic applications.

[0003] To fully exploit the antibody-binding property of Protein A for both pharmaceutical and biotechnological applications, methods to improve the immobilization efficiency of Protein A on a solid resin have been under investigation. Immobilization of a protein onto a solid support may be mediated through the ε-amine of the Lys side-chain. Although convenient, this method is non-specific, inefficient and may lead to loss of protein function. US Patent Nos. 5,084,559 and 5.260,373 describe methods of immobilizing Protein A in which a single cysteine residue, or a single arginine residue, is added to the N-termmus of Protein A, or a domain, or multiple domains thereof, to facilitate their immobilization.

10004] Despite the foregoing, there remains a need to develop improved methods

Cor protein immobilization and enhanced ligand stability.

[0005] This invention focused on identifying short amino acid sequence motifs that may function as site-specific linker or anchor for a protein or another biomolecule. By flanking a Lys residue with polar, neutral amino acids such as Ser and Thr, we have identified a series of novel short peptide sequences that exhibit superior efficiency in coupling the peptide to a solid support. [0006 J These motifs can function as linkers to couple a protein, DNA molecules such as aptamers. or another biomolecule to the surface of a solid support in a site- specific and oriented manner. Immobilized ligands may be used in affinity purification of proteins and other biomolecules for research or therapeutic uses. Depending on the type of molecules immobilized and surface employed, they can also be developed into research or diagnostic tools.

Summary of the Invention

[0007] In one embodiment the present invention provides for an anchor peptide for linking a ligand molecule to a solid support, said anchor peptide comprising an amino acid motif having at least one neutral polar residue and at least one basic polar residue.

[0008] In another embodiment the present invention provides for a fused product comprising a ligand molecule and an anchor peptide, said anchor peptide comprising an amino acid motif having at least one neutral polar residue and at least one basic polar residue.

10009] In another embodiment the present invention provides for a solid support having an immobilized fused product, the fused product comprising a ligand molecule and an anchor peptide, said anchor peptide comprising an amino acid motif having at least one neutral polar residue and at least one basic polar residue.

[0010] In another embodiment the present invention provides for a method of immobilizing a ligand molecule to a solid support comprising: (a) forming a fused product comprising a ligand molecule and an anchor peptide, said anchor peptide comprising an amino acid motif having at least one neutral polar residue and at least one basic polar residue; and (b) contacting the fused product with a solid support under conditions to immobilize the fused product to the solid support.

[001 1] In another embodiment the present invention provides for a method for purifying a molecule of interest in a sample suspected to include the molecule of interest, characterized in that the method comprises: (a) contacting the sample with a fused product immobilized to a solid support, the fused product comprising a ligand molecule lhal binds to the molecule of interest and an anchor peptide, said anchor peptide comprising an amino acid motif having at least one neutral polar residue and at least one basic polar residue, said contacting being under conditions wherein a complex is formed between the molecule of interest and the ligand molecule if said molecule of interest is present in the sample; and (b) recovering the molecule of interest from the complex thereby purifying the molecule of interest from the sample.

10012] In another embodiment the present invention provides for a method for detecting a molecule of interest in a sample suspected to include the molecule of interest, characterized in that the method comprises: (a) contacting the sample to a fused product immobilized to a solid support, the fused product comprising a ligand molecule that binds to the molecule of interest and an anchor peptide, said anchor peptide comprising an amino acid motif having at least one neutral polar residue and at least one basic polar residue, said contacting being under conditions wherein a complex is formed between the molecule of interest and the ligand molecule if said molecule of interest is present in the sample; (b) recovering the molecule of interest from the complex; and (c) detecting the recovered molecule of interest.

[0013] In another embodiment the present invention provides for a vector comprising at least one copy of a gene of a ligand molecule and at least one gene capable of coding for an anchor peptide, said anchor peptide comprising an amino acid motif having at least one neutral polar residue and at least one basic polar residue,

10014] In one embodiment of the present invention the at least one neutral polar residue is serine (S) or threonine (T), and wherein said at least one basic polar residue is lysine ( ).

10015] In one embodiment of the present invention the amino acid motif comprises the formula XX, wherein one X is a K and the other X is S or T.

[0016] In one embodiment of the present invention the anchor peptide further comprises at least one neutral non-polar residue. [0017] In one embodiment of the present invention the at least one neutral polar residue is S or T, the at least one basic polar residue is K and the at least one neutral non- polar residue is alanine (A).

[0018] In one embodiment of the present invention the amino acid motif comprises at least 2 amino acid residues.

10019] In one embodiment of the present invention the amino acid motif comprises the formula XX-UU-XX (SEQ ID NO: 3), wherein each X is K, S or T and wherein U is any amino acid.

100201 In one embodiment of the present invention the amino acid motif comprises the formula XX-UU-XX (SEQ ID NO: 4), wherein each X is A, K, S or T and wherein U is any amino acid.

[0021 J In one embodiment of the present invention the amino acid motif comprises the formula XX-GA-XX (SEQ ID NO: 5), wherein each X is K, S or T.

[0022] In one embodiment of the present invention the amino acid motif comprises the sequence AT (SEQ ID NO: 1) or ASK (SEQ ID NO: 2).

[0023] In one embodiment of the present invention the amino acid motif comprises the sequence ATKASK (SEQ ID NO: 6).

[0024] In one embodiment of the present invention the amino acid motif comprises the sequence ATKGATK (SEQ ID NO: 7).

10025] In one embodiment of the present invention the ligand molecule includes an immunoglobulin-binding protein, an enzyme, a target protein binding partner or a nucleic acid molecule.

[0026] in one embodiment of the present invention the ligand molecule is protein

A or a domain of protein A. [0027] In one embodiment of the present invention the anchor peptide is labelled with a delectable label.

[0028] In one embodiment of the present invention said anchor peptide is labelled with a cytotoxic molecule, a fluorescent molecule or radioactive molecule.

[0029] These and other aspects of the invention will become apparent from the detailed description by reference to the following Figures.

Brief Description of the Figures

[0030] Figure 1 is a table of a peptide library constructed by chemically synthesizing short peptides containing 14-15 amino acid residues with different motifs in accordance to Example 1.

10031 1 Figure 2 shows the efficiency of immobilization of different peptides (the sequences of which are provided in Table 1) onto an epoxy-activated 96- well plate as evaluated by an ELISA assay.

[0032] Figure 3 A illustrates the anchor peptide ATKAS (SEQ ID NO; 6) motif linked to a single Z domain. Figure 3 B illustrates eight fusion products (SEQ ID NOS: 1 1 - 18) according to one embodiment of the present invention. The fusion products labelled 901 and 700 contain the ATKASK (SEQ ID NO: 6) motif.

[0033] Figure 4 illustrates a dot staining analysis of the immobilization efficiency of six of the eight fusion products illustrated in Figure 3 as well as a control bovine serum albumin (BSA) sample on an epoxy resin. On strip A, 901 and 902 fused products were dotted; on strip B, 903 and 904 fused products were dotted; on strip C, 123 fused product was dotted; on strip C, 201 fused product was dotted; and on strip £. control BSA was dotted. On, orgininal sample without dilution.

[0034] Figure 5 is a graph showing a BCA protein analysis of the fused products referred to in Figure 4 using a protein assay kit (Pierce Co,). The protein concentrations in those samples were measured. The immobilization efficiency of a protein ligand was illustrated as percentages of a protein ligand concentration before and after immobilization process.

[0035] Figure 6 panels A to D show the IgG binding capacities of eight affinity columns composed of the protein A ligand (Z) fused to various N-Hnkers illustrated in ig 3. Λ. fusion products 901 and 902, B. fusion products 903 and 904, C, fusion products 285 and 700, and D. fusion products 123 and 201.

Detailed Description of the Invention

[0036] Definitions

100371 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, unless indicated otherwise, except within the claims, the use o "or" includes "and" and vice versa. Non-limiting terms are not to be construed as limiting unless expressly stated or the context clearly indicates otherwise (for example "including", "having" and "comprising" typically indicate "including without limitation"). Singular forms including in the claims such as "a", "an" and "the" include the plural reference unless expressly stated otherwise.

[0038] The term "functionally equivalent derivative" is used herein to refer to derivatjzed versions which retain the function of anchor to immobilize the selected protein without substantial loss of protein function.

(003 1 The term "ligand" as used herein refers to a molecule or group of molecules capable of binding to one or more specific sites of a receptor, another molecule or target analytc. There can exist more than one ligand for a given target. The ligands may differ from one another in their binding affinities for the target molecule.

[0040] Overview of the Invention

[0041 ] The present invention relates in general to an anchor peptide. The anchor peptide may be used for linking a ligand molecule to a solid support. The anchor peptide may comprise an amino acid motif having at least one neutral polar residue and at least one basic polar residue. In one embodiment of the present invention the anchor peptide may further comprise at least one neutral non-polar residue. The neutral polar residue may be a threonine (T) or a serine (S) residue, the basic polar residue may be a lysine (K) residue, and the neutral non-polar residue may be an alanine (A) residue.

[0042] In another aspect, the present invention provides for an anchor peptide for linking a ligand molecule to a solid support, said anchor peptide comprising an amino acid moti f having at least one neutral polar residue, at least one basic polar residue and at least one neutral non-polar residue.

I ^' 0043 ^' l The present method is not particularly restricted with respect to the ligand molecule that may be immobilized, and is therefore useful for immobilizing any protein of interest. Of particular interest is use of the method to immobilize imrnunoglobulin- binding proteins, including but not limited to, Protein A, Protein G, Protein A/G and Protein L. Other ligand molecules of interest may include nucleic acid ligands, including aptamers,

10044] The Anchor Peptide

[0045] The anchor peptide may be suitable to immobilize the selected ligand molecule onto a solid support while substantially retaining the function of the selected ligand molecule, i.e. retaining at least about 50% of ligand molecule function, such as immunoglobulin binding, in its immobilized state. In one embodiment of the present invention, the anchor or linker may comprise a motif having at least 2 amino acid residues. The amino acid motif of the anchor may comprise equally three groups of amino acids- neutral non-polar, neutral polar, basic polar residues. An anchor sequence may also contain only the neutral-polar and basic residues in different combinations and arrangements.

100461 in one embodiment of the present invention the anchor peptide may comprise an amino acid sequence ATK (SEQ ID NO. \) or ASK (SEQ ID NO. 2). [0047] In one embodiment the present invention provides for a novel peptide including an amino acid formula XX-UU-XX (SEQ ID NO. 3), wherein each X may be K, S or T and U may be any amino acid. In one embodiment of the present invention the amino acid motif comprises the formula XX-UU-XX (SEQ ID NO: 4), wherein each X is A, , S or T and wherein U is any amino acid. In another embodiment the formula may be XX-GA-XX (SEQ ID NO. 5) wherein each X may be K, S or T.

[0048 J In one embodiment of the invention, the anchor peptide may comprise an amino acid sequence ATKASK (SEQ ID NO. 6), or a functionally equivalent derivative thereof including at least 50% sequence homology.

[0049] In one embodiment the present invention provides for a novel peptide including the amino acid sequence SEQ ID NO. 6.

[0050] In one embodiment of the invention, the anchor peptide may comprise an amino acid sequence ATKGATK (SEQ ID NO. 7), or a functionally equivalent derivative thereof including at least 50% sequence homology.

[0051 ] In one embodiment the present invention provides for a novel peptide including the amino acid sequence SEQ ID NO. 7.

[0052] The anchor peptides of the present invention may also be provided as a fused product comprising the anchor peptide and a ligand molecule. The anchor peptide may be an N- or C-terraraal extension of a ligand protein.

10053] The anchor peptide or the fused product comprising the anchor peptide and a ligand protein may be synthesized by any known method in the art of peptide synthesis including solid phase synthesis (Merrifield (1964) J. Am. Chem. Assoc. 65:2149, J. Amer. Chem. Soc 85:2149 (1963); and Int. J. Peptide Protein Res. 35:161- 214 (1990)) or synthesis in homogenous solution (Methods of Organic Chemistry, E. Wansch (Ed.) Vol. 15, pts. I and II, Thieme, Stuttgart (1987) to generate synthetic peptides. [0054] The anchor peptide or the fused product comprising the anchor peptide linked to a ligand protein may be made by recombinant methods known in the art. In this regard, the anchor peptide may generally be bound or fused to a terminus of the selected ligand protein such that the function of the protein may substantially be retained. Thus, depending on the ligand protein, the anchor may be bound to the N-terminus or the C- terminus of the selected ligand protein. In certain aspects, the anchor peptide may be bound to both termini, or may additionally be bound to an amino acid residue which may not be a terminal residue. In another aspect, the anchor peptide may be bound to a solid support prior to the coupling of the protein ligand by chemical or recombinant means or through the use of an ens^mc.

[0055] The anchor peptides of the invention may be labelled with a label to facilitate their detection in an assay as is understood by one of skill in the art. Such labels may include but are not limited to radioactive label, fluourescent label or cytotoxic label. The anchor peptides of the invention may be provided with a carrier such as for example coupled to bovine serum albumin (BSA) or keyhole limpet haemocyanin. The anchor peptides may be preceded by a Biotin-GGYG (SEQ ID NO: 55) N-terminal sequence that may facilitate peptide concentration determination by OD280 (of Tyr or Y) measurement. The N-terminal biotin moiety may provide a ligand for ELISA assay (see Figure 2) using avidin-HRP. <

[00561 Once the ligand molecule is bound or fused to the anchor peptide of the present invention, the fused product may be immobilized onto a solid support. Suitable solid supports include epoxy-based supports, e.g. beads or resins, or solid supports having the surface chemistry of an epoxy group, or materials that are functionalized on the surface to allow coupling of a protein via the anchor sequence.

[0057] To immobilize the fused product to the solid support, the solid support may be treated in an aqueous solution. The aqueous solution may include a binding buffer, such as 0.1 M sodium phosphate/0.5 M Na2S04, pH, e.g. in the range of about 6.5 to about 7.2. In one aspect the pH is 6.8. Suitable salts for use in this regard may include salts that may be compatible with the solid support and fused product, non-toxic with regard to the intended use of the immobilized protein and otherwise suitable for use. The solid support may be mixed with the fused product, for example at room temperature, for a sufficient period of time for immobilization to occur.

[0058] In embodiment, the present invention relates to a matrix for affinity separation. One end of the anchor peptides of the present invention may be bound to the matrix, and the other end of the anchor peptide may be bound to the ligand molecule. The matrix of the present invention, when compared to a matrix having the ligand directly bound to the matrix, may exhibit an increased immobilization capacity during two or more separations.

[0059] Examples of the material for the solid support is based on polymers having hydrophilic surface, such as polymers having hydroxyl group (-OH), carboxyl group (- COOH), aminocarbonyl group (-CONH¾ possibly in N-substituted forms), amino group (-NH ₂ . possibly in substituted form), oligo or polyethyleneoxy group on their external surface and if present, also on internal surface. In one embodiment, the polymer may be a synthetic polymer (e.g., polymethacrylate, polyacrylamide, or styrene-divinylbenzene copolymer). Such a synthetic polymer may be easily produced by a known method (see J. MATER. CHEM 1991 , 1(3), 371-374, for example). Alternatively, a commercially available product such as TOYOPEA L™ may also be used. In another embodiment, the polymer is a polysaccharide (e.g., dextran, starch, cellulose, pullulan, or agarose). Such a polysaccharide may be easily produced by a known method (see Japanese Patent No. 4081 143, for example). Alternatively, a commercially available product such as Sepharose (manufactured by GE Healthcare Biosciences) may also be used. In still another embodiment, an inorganic support (e.g., silica or zirconium oxide) may be used.

(0060J The solid support may be in the shape of particles. The particles may be porous or nonporous. The solid support in the shape of particles may be used as a packed bed, or may be used in a suspended form. The suspended form may be an expanded bed or a pure suspension, in which the particles can move freely. When using a packed bed, or an expanded bed. separation process used in known affinity chromatographic methods is used. When using a pure suspension, a batch method is used. [0061] The solid support in the shape of particles according to this embodiment may preferably have a particle size (volume average particle size) of about 20 to about 200 micrometers, and more preferably about 30 to about 100 micrometers. If the solid support has a particle size of less than about 20 micrometers, the pressure of column packed with this support may increase to an impractical level at a high flow rate. If the solid support has a particle size of more than about 200 micrometers, the binding capacity may deteriorate. Note that the term "particle size ^" used herein refers to a volume average particle size determined using a laser diffraction/scattering particle size distribution analyzer.

100621 The solid support according to this embodiment may preferably be porous, and having a specific surface area larger than about 50-1000 m ² /g-, and more preferably about 80-400 m ² /g. If the affinity chromatography packing material has a specific surface area of less than about 50 m ² /g, the binding capacity may deteriorate. If the affinity chromatography packing material has a specific surface area of more than about 1000 m ² /g, the packing material may be destroyed at a high flow rate due to a decrease in strength, so that the column pressure may increase. Note that the term "specific surface arca ^,: used herein refers to a value of the surface area of pores (pore size: 10 to 5000 nm) determined using a mercury porosimeter by the dry weight of the particles.

[0063] The solid support according to this embodiment may preferably have a volume average pore size of about 50 to about 400 nm, and more preferably about 100 to about 300 nm. If the affinity solid support has a volume average pore size of less than about 50 nm, the binding capacity may significantly deteriorate at a high flow rate. If the affinity chromatography packing material has a volume average pore size of more than about 400 nm, the binding capacity may deteriorate irrespective of the flow rate. Note that the term "volume average pore size" used herein refers to the volume average pore size of pores (pore size: 10 to 5000 nm) determined using a mercury porosimeter.

[0064] Specific examples of porous particles used as the solid support according to this embodiment may include porous organic polymer particles including a copolymer of 20 to 50 wt% of a crosslinkable vinyl monomer, 3 to 80 wt% of an epoxy group- containing vinyl monomer, and 20 to 80 wt% of a diol group-containing vinyl monomer, and having a particle size of 20 to 80 micrometers, a specific surface area of 50 to 150 m ² /g, and a volume average pore size of 100 to 400 run.

[00651 In yet another form embodiment, the solid support may be in another form such as a surface, a monolith, a chip, capillaries, or a filter.

[0066] The ligand molecule may be attached to the support via conventional coupling techniques utilizing, e.g. amino and/or carboxy groups present in the ligand. 1 isepoxidcs, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS) etc. are well-known coupling reagents. Between the support and the ligand, a molecule known as a spacer may be introduced, which will improve the availability of the ligand and facilitate the chemical coupling of the ligand to the support. Alternatively, the ligand may be attached to the support by non-covalent bonding, such as physical adsorption or biospecific adsorption.

100671 In one embodiment of the present invention, the molecule ligands may be peptide ligands that may be used in the purification of immunoglobulins of interest from a composition comprising at least one immunoglobulin of interest and at least one other substance from which the immunoglobulin is to be separated.

IO068J The fused product comprising the anchor peptides of the present invention bound to peptide ligands, may be immobilized on to a solid support or may be used in solution. The solid support may then be contacted with a composition containing an immunoglobulin, the immunoglobulin then binding to the peptide ligand.

[0069] When using the fused products immobilized on a solid support, the composition containing the immunoglobulin may be contacted to the fused products-solid support either before or after the solid support material is packed into a column. For example, the fused products-solid support material may be incubated with the composition containing the inununoglobulin and then this mixture may be packed into a column for further purification steps, Alternatively, the fused product-solid support may be first packed into the column followed by the addition of the composition containing the immunoglobulin of interest. The fused products-solid support material may first be packed in the column followed by the introduction of the composition containing the immunoglobulin of interest.

[0070] The recovery of the immunoglobulin from the fused products-solid support may involve any technique conventionally used in affinity chromatography to remove compounds from solid supports, including changes in pH or ionic strength. In one preferred embodiment of this invention, the immunoglobulin is eluted using a buffer at or below pH 4.

[0071 ] In an additional embodiment of the invention, the fused products may be used in assays for the detection of molecules of interest such as antibodies in a solution or composition suspected of containing the same. The fused products may be coupled to a detectable group or label. The contacting and separating steps may be carried out in solution or with immobilized fused products. Methods of detecting labelled fused products include those for the detection of labelled immunoglobulin. When carried out in solution, fused products bound immunoglobulin may be separated and/or detected from the solution using a variety of techniques including, but not limited to, fluorescence- activated cell sorting, membrane filtration and mass spectrometry.

[00721 One embodiment of this invention includes methods of using a fused product o f the present invention in methods for determining the presence or concentration of a molecule of interest in a sample. The ligand component of the fused product being a lingand of the molecule of interest. The method may include:

[0073] (a) contacting said sample to the fused product under conditions wherein a molecule of interest ligand complex is formed if said molecule of interest is present in the sample; and

[0074] (b) detecting of the molecule of interest by detecting the molecule of interest/li and complex, thereby determining the presence of the molecule of interest in the sample. Step (b) may also include measuring the amount of the molecule of interest in the sample by quantitatively detecting the complex. [0075] One embodiment of this invention includes methods of using a fused product of the present invention in an affinity column for the determination of presence and/or concentration of molecules of interest in a sample suspected of containing the same. The methods may include the following steps:

[0076] (a) immobilizing a fused product of the present invention to an affinity column, the ligand component of the fused product being a ligand of the molecule of interest;.

|0077 | (b) running the sample through the affinity column under conditions whereby the molecule of interest is captured by the fused product if said molecule of interest is present in the sample, thereby ;

[0078] (c) recovering the molecule of interest from the column with a recovering agent; and

10079] (d) measuring the quantity of molecule of interest captured by the column by methods such as direct fluorescence measurement, high performance liquid chromatography and mass spectrometry of the molecule of interest. Alternatively, prior to the recovering step, the molecule of interest/ligand complex formation within the column may be detected by the use of fluorescence, or fluorescence in combination with quenchers, or fluorescence polarization, and electro-affinity analysis.

100801 embodiments of the invention are described in the following specific example which is not to be construed as limiting.

EXAMPLES

Example 1 - Chemical synthesis of a peptide library

|0081 1 A peptide library was constructed by chemically synthesizing short peptides containing 14-15 amino acid residues with different motifs (Figure 1). These peptides were synthesized by the Fraoc chemistry based solid-phase peptide synthesis and labelled at the N-terminus with biotin. After synthesis, the peptides were dissolved in water and the identities (eg., molecular masses) were confirmed by MALDI-TOF mass analysis.

Peptides 1 to 24 of Figure 1 are designed in such a way that a lysine (K) is next to an Ser (S) or Thr (T) residue or flanked by different combinations of serine (S), threonine (T) and other types of amino acids. The motif length may be six as in XX-UU-XX (SEQ ID NO: 3) or XX-GA-XX (SEQ ID NO: 5) (peptides 9 to 16, wherein at least one K is in the X positions). The GA dipeptide may serve as a spacer between the XX motif, but GA can be replaced by other amino acid types as in the motif XX-UU-XX (SEQ ID NO: 3), where U represents any amino acid. The motif can be as short as containing two amino acid as in the motif XX. wherein one X is K, and the other X is S or T. The peptide core sequence may be preceded by a Biotin-GGYG (SEQ ID NO: 55) N-terminal sequence that may facilitate peptide concentration determination by OD280 (of Tyr or Y) measurement The N-terminal biotin moiety may provide a ligand for ELISA assay (see Figure 2) using avidin-HRP.

Example 2 - Identification of peptide motifs that allow efficient ligand immobilization onto a solid support

] 00821 The synthetic peptides are each dissolved in dd¾0 and the pH value of the solution adjusted to pH 7-8 using a 0.1N NaOH solution. The resulting peptide solutions are added, respectively, to different wells of a 96-well plate containing epoxy groups on the surface and incubated at 4 °C overnight. The plate is washed in PBS and then blocked with 5% skim milk at room temperature for 1 hour. After blocking, a avidin-HRP solution (1 :40,000 dilution) was added to the plate and incubated at room temperature for 1 hour. The plate is washed again three times with PBS and incubated with tetramethylbcnzidine (TMB), a colorless substrate. After 20 minutes, the reaction is stopped by adding 2M H2S04. The OD at 405 nm was recorded for the plate using a Multi-label reader (Perkin-Em mer). As shown in Figure 2, a higher OD405 value corresponds to a greater amount of a peptide immobilized in a well. As illustrated in Figure 2, a number of peptides showed efficient ligand immobilization to the solid support. Peptides that exhibited the greatest immobilization efficiency contained the ΛΤΚ (SEQ ID NO: 1) and ASK (SEQ ID NO: 2) motifs (highlighted in Figure 1), Example 3 - Protein ligand immobilization on the epoxy resin

[0083] Using a molecular cloning method, a single Z domain was fused with various N-Hnker sequences as illustrated in Fig. 3. These fused protein ligands were expressed in a bacterial expression system. The target proteins were purified with the Ni- NT A affinity column (GE Healthcare) to highly homology. Protein ligand's purity was examined by SDS-PAGE analysis.

[0084] The purified protein ligands were prepared in the immobilization buffer

(0.1 M sodium phosphate/0.5 M Na2S04, pH 6.8). Five milligram of each protein ligand solution was mixed with 1 millilitre of the epoxy-activated resin (JSR). The protein ligand/resin mixture was incubated at about room temperature overnight with a rotating mixer, immobilization of the fused product in each case may be monitored by the dot staining method (FIG. 4) and by the BCA protein measuring kit (FIG. 5).

[0085] The results illustrated in FIGS. 4 and 5 confirmed that the Z protein ligand fused with a N-linker containing either "ATKASK" (SEQ ID NO: 6) (ligand 901) motif or "ATKGATK" (SEQ ID NO: 7) (ligand 902) motif was successfully immobilized on the beads, while the Z ligand fused with other N-linker sequences without the motifs "ATKASK" (SEQ ID NO: 6) or "ATKGATK" (SEQ ID NO: 7) were less efficient to be immobilized onto the beads under the same conditions.

Example 4 - IgG binding capacity of Immobilized Protein A containing an ATKASK fSEO ID NO: 6) or ATKGATK (SEP ID NO: 7) anchor

[0086] Eight affinity columns of 1 ml bed volume, packed with the epoxy beads

(JSR) immobilized with eight protein ligands with various N-Iinkers, were analyzed to determine their human IgG binding capacity (Fig. 5). The IgG solution at 1 mg/ml of PBS was added to each affinity column. The flow through samples were collected. OD280 values were monitored between the loading IgG solution and the flow through samples. The differences of the OD280 values between the initial IgG solution and the flow through samples are the indication of the affinity column's IgG binding capacity. The higher an affinity column's IgG binding capacity, the lower the percentage of the initial IgG OD280 reading and the flow through sample OD280 reading. Results are depicted in Figure 6. Figure 6 A and 6 C show that in both 901 and 902 columns as well as 700 columns, the percentage of eluted IgG (unbound IgG) was approximately at 25-30 % at the 20 mg IgG loading, while the rest of the columns showed the percentage above 50%. These three columns contain the protein ligands fused with the N-linker containing the motif of "ATKASK" (S.EQ ID NO: 6) (901 and 700) or the motif of "AT GAT " (SEQ ID NO: 7) (902). These data indicate that the N-linker containing the motif of "ATKASK" (SEQ ID NO: 6) would greatly facilitate a protein ligand to be immobilized on an epoxy bead and thus enhance the IgG binding capacity.

Methods and Materials for Examples 3 and 4

[0087] (1) Vector constructs for Protein A expression - The Z domain of protein A fused to a different anchor peptide was cloned into the pETMl l vector (European Molecular Biology Laboratory) or pET302 N-His (Invitrogen). The original Protein A gene was amplified by PCR method using a TAP-Tag plasmid (EMBL) containing Protein A gene as a template. The Z domain was created by the PCR assisted QuickChange Site-Directed Mutagenesis method (Stratagene Cloning System).

[0088] All protein A ligands with various N-linkers listed in FIG. 3B (201, 123,

901 , 902, 903, 904, 285, 700) were manufactured in the E. coli expression system, BL21 (DE3) pLysS competent cells, as described in the following paragraphs.

[0089] The eight expression vectors listed in Fig. 3B were constructed as follows:

[0090] 201 expression vector was constructed by PCR the single Z domain with the restriction enzyme sites: EcoRI/BamHI and cloned the single Z domain into the expression vector of pET302/N-FIis (Invitrogen).

[0091 ] 123 expression vector was constructed by PCR the single Z domain with the restriction enzyme sites: NcoI/BamHI and cloned the single Z domain into the expression vector of pETMl 1 (EMBL). 10092] 90 L 902, 903, and 904 expression vectors were constructed by deleting V and introducing AT ASK (SEQ ID NO: 6) (901), ATKGAT (SEQ ID NO: 7) (902), AHKGAHK (SEQ ID NO: 56) (903) and GHTKGHTK (SEQ ID NO: 57)(904) into the 123 by using the QuickChange Site-Directed Mutagenesis method (Stratagene Cloning system).

[0093] 285 expression vector was constructed by deleting MSDYDIPTT (SEQ ID

NO: 58) in 123 by using the QuickChange Site-Directed Mutagenesis method (Stratagene Cloning system).

[0094] 700 expression vector was constructed by deleting MV and adding

ATKASK (SEQ ID NO: 6) in 285 by using the QuickChange Site-Directed Mutagenesis method (Stratagene Cloning system).

[0095] (2) Recombinant protein expression and purification - BL2l(DE3) pLysS competent cells allow high-efficiency protein expression of any gene that is under the control of- a T7 promoter and has a ribosome binding site. BL21(DE3)pLysS is lysogenic for λ-ΟΕ3, which contains the T7 bacteriophage gene I, encoding T7 RNA polymerase under the control of the lac UV5 promoter. BL21 (DE3)pLysS also contains a plasmid, pLysS, which carries the gene encoding T7 lysozyme. T7 lysozyme lowers the background expression level of target genes under the control of the T7 promoter but does not interfere with the level of expression achieved following induction by IPTG. Target DNA plasmids were used to transfect BL21 competent cells. 0-5 μΐ of plasmid DNA containing about 0.5 ug of DNA was used for transfection. The BL21 competent cells were stored at - 80 degree Celsius. An aliquot of BL21 cells (20 μΐ) was thawed and put on ice. 0.5 μ) of plasmid DNA was mixed with the competent cells and put on ice for 60 minutes. After incubation on ice, these cells containing plasmid DNA were "heat shocked" in 45 degree C water batch for 45 seconds and immediately put on ice for another 2 minutes. Then, 30 μΐ of LB medium was added to the bacterial cells and shaken at 37 degree C for 1 hour. These cells were then spread on agar /LB plates containing kanamycine (final concentration: 100 igf l). These plates were incubated at 37 degree C overnight. [0096] For expression of recombinant Protein A domains, the DNA plasmids were transformed into E. . coli BL21. Overnight cultures were inoculated into fresh medium and grown to an OD600nm of approximately 0.5. Isopropyl beta-D-1- thiogalactopyranoside was added to a concentration of 1 mM, and the culture was grown for a further 15 hours at 18 degree C. Cells were harvested by centrifugation at 7,000 rpm for 10 min in a Sorvall GS-3 rotor.

100971 The pellet was resuspended in phosphate-buffered saline (PBS) containing protease inhibitor (Roche Applied Science), lysozyme (200 μ^Ι), and DNase I (3 μ§/ ^η ι1). Cells were lysed by repeated freeze/thaw cycles. Cell debris was removed by centrifugation.

|0098] Recombinant proteins expressed in pETMl l vector contained an ammo- terminal His-Tag. His-Tag fusion proteins were purified using the Ni-NTA columns (Qiagen) and djalyzed against PBS.

10099] (3) Protein Analysis by Poly acry lam idc gel electrophoresis (SDS-

PAGE) - Electrophoresis was performed on polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS). Samples (10 μΐ) were mixed with 10 μΐ of sample buffer (62.5mM Tris-HCl, pH 6.8 _; 2% SDS, 5% mercaptoethanol, 10% glycerol and 0.0025% brophenol blue), heated at 90 C for 2 min, cooled and loaded on gels. Electrophoresis was performed at 75 mA in an apparatus purchased from Bio-Rad. Gels were stained in a solution of 0.5 mg/ml Coomassie blue in 5:5: 1 methanol/water/acidic acid and de-stained in water heated in a microwave.

[00100] (4) Peptide synthesis -Fmoc solid-phase peptide synthesis method was used to synthesize all peptides. Protected amino acid derivatives were purchased from AnaSpec (US); N _; N-diisopropylethylamine (DIEA), HBTU, HOBT, piperidine were purchased from liMD (Canada); and Tental gel resin was purchased from Intavis (Canada). Other reagents and solvents were purchased from Sigma (Canada). MALDI- TOF-mass spectra were recorded on a Micromass® bench-top MALDI-TOF mass analysis with reflection ion mode. 100101 j (5). Synthesis of porous beads - 8.2 g of glycidyl methacrylate (manufactured by Mitsubishi Rayon Co., Ltd.), 65.9 g of trimethylolpropane trimethacrylate (manufactured by Sartomer), and 90.6 g of glycerol monomethacrylate (manufactured by NOF Corporation) were dissolved in 245.8 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) and 62 g of acetophenone (manufactured by Wako ure. Chemical Industries, Ltd.), and 2 g of 2,2 ^: -azoisobutyronitrile (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the solution to prepare an organic monomer solution.

[00102] 8.5 g of polyvinyl alcohol ("PVA-217" manufactured by Kuraray Co., Ltd.), 0.43 g of sodium dodecyl sulfate ("Emal 10G" manufactured by ao Corporation), and 21.3 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) were added to 4240 g of distilled water. The mixture was stirred overnight to prepare an aqueous solution.

[00103] A separable flask (7 1) was charged with the resulting aqueous solution. The separable flask was equipped with a thermometer, a stirring blade, and a cooling tube, and placed on a hot water bath. The aqueous solution was stirred at 600 rpm in a nitrogen atmosphere. When the temperature of the aqueous solution had reached 85°C, the organic monomer solution was added to the aqueous solution using a dropping funnel. The mixture was stirred for 5 hours.

[00104] After cooling the reaction solution, the reaction solution was transferred to a polypropylene bottle (5 1). The reaction solution was allowed to stand until the beads floated. Unnecessary water was then sucked out from the bottom of the bottle. Acetone was then added to the reaction solution to precipitate the beads. After allowing the reaction solution to stand for 3 minutes, acetone was removed by decantation. After repeating this operation twice, water was added to precipitate the beads. After allowing the mixture to stand for 3 minutes, the mixture was subjected to decantation. After replacing the dispersion medium of the bead dispersion with acetone, the mixture was air- dried overnight. The mixture was then dried using a vacuum dryer to obtain 90 g of porous beads (hereinafter referred to as "PB"). The average particle size of the PB was 43 micrometers, and the specific surface area of the PB was 83 m ² /g.

[00105] (6) Immobilisation of protein ligands on the epoxy beads- Epoxy- activated beads obtained as described in the previous section, were used for immobilization. The epoxy beads were washed with the immobilization buffer before the immobilization. Five milligram of protein ligand fused to an anchor peptide was added to 1 ml of the epoxy resin. The protein/resin mixture was incubated at room temperature overnight with a rotating shaker. After overnight immobilization, the protein ligand fused to an anchor peptide (such as 901, 902, etc) resin was blocked with 1M thioglyceol in 0.1 M Na2S04, pH 8.3 for 4 hours at room temperature. After blocking, the resin was washed with 1 x PBS (+ 0.5% Tween 20). After washing, a protein A affinity resin was ready for further use.

100106] (7) Dot analysis - Referring to Figure 4, products 901 (Figure 4 A), 902 (Figure 4 A), 903 (Figure 4 B), 904 (Figure 4 B), 123 (Figure 4 C) and 201 (Figure 4 D) before immobilization (top row) and after immobilization (product left in solution, bottom row) on the epoxy beads were diluted 1 :1 in PBS respectively and dotted on a nitrocellulose filter paper. The filter strips were stained by Coomassie blue. In a control experiment, BSA (bovine serum albumin, Pierce Co.) was immobilized on the same epoxy beads and samples were taken before and after the immobilization reaction and dotted on a nitrocellulose filter paper, as illustrated in Figure 4 E (Top row: before immobilization; bottom row: after immobilization).

(8) CA protein analysis - Referring to Figure 5 a BCA protein analysis of the fused products referred to in Figure 4 was performed using a protein assay kit (Pierce). Briefly, BCA Protein Assay combines the well-known reduction of Cu2+ to Cul+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cul+) by bicinchoninic acid. The first step is the chelation of copper with protein in an alkaline environment to form a light blue complex. In this reaction, known as the biuret reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate. In the second step of the color development reaction, bicinchoninic acid (BCA) reacts with the reduced (cuprous) cation that was formed in step one. The intense purple-colored reaction product results from the chelation of two molecules of BCA with one cuprous ion. The BCA/copper complex is water-soluble and exhibits a strong linear absorbance at 562 nm with increasing protein concentrations.

100) 07] For the BCA assay, 200 μΙ of the reaction solution was added into each well on a 96-well plate. Then, 25 μΐ of BSA standard samples or unknown protein samples was added into each well. The 96-well plate containing samples was incubated at 37 °C, for 30 minutes. The purple color was measured at OD595nm using the microplate reader (Perkin-Elmer). A BSA. standard curve was constructed by using protein concentration as X axis and OD595 as Y axis. Unknown protein samples' concentrations were deduced based on the BSA standard curve.

[001081 The protein concentrations in those samples were measured. The immobilization efficiency of a protein ligand was illustrated as percentages of a protein li¾and concentration before and after immobilization process,

100109] (9) IgG binding capacity of a Protein A affinity column-Protein A affinity columns were evaluated for their IgG binding capacities. Human IgG solution at 1 mg/ml of PBS was loaded on 1 ml of bed volume Protein A affinity resin. The flow- through samples were collected. OD280 value was measured in both loading IgG solution and flow-through samples to calculate the IgG content in samples. The percentage of IgG content in the loading solution and in the flow-through samples indicated a Protein- A affinity column's IgG binding capacity (illustrated at Y axis of Figure 6),