EP0663925A1

EP0663925A1 - Affinity purification methods involving amino acid mimetics as elution reagents

Info

Publication number: EP0663925A1
Application number: EP93923139A
Authority: EP
Inventors: Brian Seed
Original assignee: General Hospital Corp
Current assignee: General Hospital Corp
Priority date: 1992-10-02
Filing date: 1993-09-27
Publication date: 1995-07-26
Also published as: EP0663925A4; WO1994007912A1; JPH08503698A; AU5292693A; CA2146162A1; MX9306174A

Abstract

Disclosed is a method of isolating a protein from a sample, involving (i) providing a first molecule which is capable of forming an affinity complex with the protein; (ii) contacting the sample with the first molecule under conditions which allow affinity complex formation; (iii) isolating the complex; (iv) treating the complex with a second molecule, the second molecule mimicking an amino acid residue of either the protein or the first molecule which is critical to the complex formation, so that the second molecule disrupts the complex; causing the release of the protein from the complex; and (v) isolating the protein. According to one embodiment, the amino acid mimetic imidazole is used as a very gentle elution reagent to disrupt a protein A-antibody fusion protein complex, a technique which has general application for the isolation of antibodies or recombinant antibody fusion proteins.

Description

AFFINITY PURIFICATION METHODS INVOLVING AMINO ACID MIMETICS AS ELUTION REAGENTS Background of the Invention The invention relates to protein isolation and purification techniques.

There currently exists a variety of methods, materials, and approaches for the separation of a particular protein from the other components of a biological sample. One general approach exploits the non-specific affinity of a protein for a substrate. For example, proteins may be separated based upon their molecular charge using ion exchange chromatography, whereby protein mixtures are applied to an oppositely charged, chromatographic matrix, and the various proteins bind to the matrix by reversible, electrostatic interactions. The adsorbed proteins are eluted, in order of least to most strongly bound, by increasing the ionic strength or by varying the pH of the elution buffer. Another general approach makes use of a protein's physical characteristics as a means of separation. For example, a protein may be separated based upon its size, using gel filtration. By this method, protein mixtures are applied to a gel-filtration column containing a chromatographic matrix of defined pore size. Proteins are eluted, generally with an aqueous buffer, collected as individual chromatographic fractions and analyzed.

Finally, a third general approach makes use of the specific affinity of a protein for a purifying reagent. A protein, for example, may be purified using an antibody specific for that protein or conversely an antibody may be purified using its specific antigen. Typically, the antibody or antigen is bound to a column substrate and a solution which includes the particular antigen or antibody applied to the column, allowing immunocomplex formation. Bound immunocomplex partners are then eluted by destabilizing the antigen-antibody complex, e.g., by exposure to buffers of very high ionic strength or high or low pH. Alternatively, immunocomplex formation may be exploited to purify the antigen or antibody by immunoprecipitation. Antigen-antibody complexes may be precipitated following aggregation, or alternatively, one of the binding partners may be covalently linked to a solid particle (such as Sepharose or agarose) and immunoaffinity complexes isolated by centrifugation. In either method, the protein of interest is then released from the complex, again, e.g., by exposure to buffers of high ionic strength or high or low pH.

Of particular interest to molecular biologists are isolation and purification methods for antibodies or recombinant antibody fusion proteins. Structurally, an individual antibody molecule includes two identical heavy (H) chains and two identical light (L) chains; each light chain is disulfide bonded to a heavy chain, and the heavy chains are disulfide bonded to each other to form the basic dimeric structure of the molecule. Within each chain, units made up of about 110 amino acids fold up to form compact domains, themselves held together by a single internal disulfide bond. The L chain has two domains, and the H chains have four or five domains.

The first two N-terminal domains of the H chains interact with the two L chain domains, producing the "Fab domain", a portion of the molecule which directs specific antigen recognition and binding. At the other end of the molecule, the extreme C-terminal domains of the H chains (termed the C_H2 and C_H3 domains) interact to produce the "Fc domain", a portion of the molecule which directs a number of immunoglobulin functions including binding to cells, fixing complement, and traversing the placenta. And, finally, lying between the Fab and Fc domains is a small number of amino acids which make up the hinge region, a flexible domain which facilitates the free movement of the antigen binding portion of the molecule.

Summary of the Invention It is an object of the invention to provide a cheap and general method for eluting proteins adsorbed to affinity chromatography columns.

As discussed generally above, affinity chromatography is a highly effective method for purifying proteins which exploits specific interactions between the proteins to be purified and a solid phase immobilized ligand. Typically the solid phase ligand has some unique chemical character which results in the selective adsorption of the protein of interest. Contaminant proteins either do not bind the solid phase or can be removed by washing the solid phase with appropriate solutions. Historically, elution of an affinity column has been accomplished by either of two methods: (i) washing the matrix with a solution of specific ligand resembling the immobilized ligand, or (ii) washing the matrix with solutions of very high ionic strength, or very high (>11) or very low (<3) pH. In general, method (i) is more attractive than method (ii) because solutions of very high ionic strength, while usually not deleterious to proteins, are also usually not effective at desorbing proteins which bind to the affinity matrix tightly, and many proteins are labile to buffers sufficiently acid or basic to elute the protein of interest. However method (i) is not applicable if a specific eluting ligand can not be found or if the use of a specific eluting ligand is infeasible for practical reasons, e.g. if the specific ligand is unstable or expensive. Elution of proteins from immobilized ligands in which the ligand itself is a protein can rarely be achieved by the use of an eluting solution containing a specific ligand. This is because the specific ligand usually must be a protein or peptide fragment, and elution is then infeasible for the practical reasons mentioned above.

The instant invention describes a new method for eluting affinity matrices based on the use of inexpensive low molecular weight compounds which mimic the side chains of specific amino acids. To desorb a protein bound to a nonproteinaceous affinity ligand, the eluting compounds are chosen to mimic the side chains of specific amino acids participating in recognition of the ligand. To desorb a protein bound to a proteinaceous affinity ligand, the compounds may mimic the side chains of either the protein to be purified or the immobilized ligand protein.

Suitable side chain mimetic compounds are chosen for their water solubility and compatibility with the stability of the protein to be isolated. Because protein-protein interactions are frequently stabilized by hydrophobic interactions, and particularly by aromatic hydrophobic interactions, the most favored elution reagents will be chosen from the group comprising side chain mimetics of histidine, tyrosine, tryptophan, and phenylalanine. In particular examples, imidazole is an appropriate choice for destabilizing interactions promoted by binding to histidine residues. Hydroxylated aromatic compounds such as phenols and phenolic compounds whose solubility in water is promoted by other substituents, such as aminophenols or hydroxybenzenesulfonates, are appropriate mimetics for tyrosine side chains. Substituted indoles, such as indole-3-sulfate or indole-3-acetic acid, or benzimidazole salts are appropriate mimetics for tryptophan. Benzoate salts, phenylsulfonates, and water soluble heterocycles, such as nicotinic acid salts or nicotinamide, are appropriate mimetics for phenylalanine. Useful amino acid mimetics may also be chosen from those molecules which disrupt charged residue interactions. Examples of compounds useful for interfering with such charged residue interactions include guanidine salts to mimic arginine, alkylamines to mimic lysine, and alkanoic acids to mimic glutamate or aspartate.

Finally, other compounds which are useful as elution reagents in the invention include those which interfere with aliphatic hydrophobic residue interactions, for example, aliphatic alcohols, aliphatic ethers of water soluble polyalkylene glycols, sulfate esters of aliphatic alcohols and aliphatic amines. Compounds with dual hydrophobic action, such as phenols substituted with aliphatic groups, may also prove useful in mimicking side chain residues.

Accordingly, in general, the invention features a method of isolating a protein from a sample, involving (i) providing a first molecule which is capable of forming an affinity complex with the protein; (ii) contacting the sample with the first molecule under conditions which allow affinity complex formation; (iii) isolating the complex; (iv) treating the complex with a second molecule, the second molecule mimicking an amino acid residue of either the protein or the first molecule which is critical to the complex formation, so that the second molecule disrupts the complex, causing the release of the protein from the complex; and (v) isolating the protein.

In a preferred embodiment, all or a part of the second molecule mimics an amino acid side chain. In another preferred embodiment, the first molecule is protein A and the protein to be isolated is an antibody or an antibody fusion protein which includes a protein A-binding domain. According to this embodiment, the second molecule preferably mimics a histidine residue and is, for example, imidazole.

In two other preferred embodiments, the first molecule is an antibody and the protein to be isolated is a recombinant protein; or the first molecule is an antigenic protein and the protein to be isolated is an antibody which specifically binds that antigenic protein.

By "mimicking an amino acid residue" is meant being of the same or similar chemical composition to either the amino acid residue itself or to a part of the amino acid residue which is critical to that residue's ability to interact with a proteinaceous or nonproteinaceous affinity ligand (i.e., the "first molecule") . Preferably, the portion of the amino acid mimicked is that residue's side chain. Because such a mimetic molecule is used as an elution reagent, the molecule should preferably also be water soluble and compatible with the stability of the protein to be purified. Although any amino acid mimetic is useful in the invention, those which disrupt hydrophobic protein- protein interactions are preferred; these include interactions involving the amino acids histidine, tryptophan, tyrosine, and phenylalanine. Preferable mimetics for these amino acids are described above.

By "antibody fusion protein" is meant a protein which includes at least a portion of an immunoglobulin Fc domain directly or indirectly covalently bonded to a non- immunoglobulin polypeptide.

By "amino acid side chain" is meant that moiety bound to the molecule's central carbon atom which determines the amino acid's identity. Preferable side chains according to the invention include those hydrophobic side chains which characterize histidine, tryptophan, tyrosine, and phenylalanine.

By "protein A-binding domain" is meant that portion of the immunoglobulin molecule which interacts with the Staphylococcus aureus cell wall component termed protein A. By crystallographic studies, this domain is most likely positioned at the C_H2/C_H3 cleft.

Applicant has recognized that affinity purification techniques may be modified such that amino acid mimetics are used as elution reagents in the final step of purification to release the protein of interest from the affinity complex. Because this approach is considerably gentler than more conventional elution techniques (e.g., elution steps based on drastic changes in solution pH) , applicant's method facilitates the isolation and purification of those proteins which are destroyed (e.g., irreversibly denatured) or reduced in activity by standard elution procedures. Moreover, because the amino acid mimetics represent convenient and inexpensive elution reagents, they may be utilized as alternative elution reagents even for the purification of more stable proteins.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.

Brief Description of the Drawings FIGURE 1 shows a schematic of the immunoglobulin fusion protein CD62 Rg. FIGURE 2A shows flow cytometric results of CD62 Rg binding to the surface of neutrophils. FIGURE 2B shows flow cytometric results of CD62 Rg binding to the surface of H3630 cells. FIGURE 2C shows flow cytometric results of CD62 Rg binding to the surface HSB2 cells. FIGURE 2D shows flow cytometric results of CD62 Rg binding to the surface of K562 cells.

Detailed Description There now follows a description of a protein 5 isolation and purification procedure according to the invention, and a description of its use in the isolation of one particular immunoglobulin fusion protein. Unusually gentle elution of the recombinant protein facilitates purification without appreciable loss of 0 native binding reactivity. This example is provided for the purpose of illustrating, not limiting, the invention. Affinity Purification of IσGl by Elution with the Histidine Mimetic Imidazole

Human IgGl was purified by affinity chromatography 5 followed by elution with the histidine mimetic imidazole as follows.

Human IgGl was loaded on protein A trisacryl beads (Pierce, Rockford, IL) , and washed with phosphate buffered saline. The beads were divided among several 0 small columns, and the columns were eluted with solutions containing imidazole at 1, 2, 3, 4, or 5M concentration, adjusted to a final pH of 6, 7, 8, or 9. The results are given in Table 1 as the percent of maximum elution obtained for any one pH.

TABLE 1

Mean + std. error Negligible amounts of IgGl were retained by the columns at the highest imidazole concentrations, at any pH. In general the pH did not play a significant role in mediating the elution power of imidazole, which was somewhat unexpected, given that the pK of imidazole is 7.1, and so approximately 90% of the molecules would be charged at pH 6, whereas approximately 90% would be uncharged at pH 8.

Using this method, immunoglobulin fusion proteins, as well as IgGl alone, were purified from protein A columns without loss of biological activity of the protein moiety fused to the immunoglobulin constant domain (in particular, see the purification of CD62 Rg below) . For several of the fusion proteins purified in this manner, it was known that acidic elution conditions destroyed the activity (i.e., the ligand binding activity) known to reside in the portion of the protein fused to the immunoglobulin domain. In general, the purification of these immunoglobulin fusion proteins involved an initial isolation of a crude preparation of a fusion protein (including the hinge, C_H2 and C_H3 domains of human IgGl joined to the extracellular domain of some surface antigen) which had been prepared by transfection of COS cells with the appropriate cDNA constructs. Media supernatants were collected from transfected cells which had been grown for a further 5 to 10 days, clarified by centrifugation, and adsorbed to protein A trisacryl or protein A agarose beads. The beads were collected, washed thoroughly with phosphate buffered saline containing 1% nonionic detergent (Nonidet P40 or Triton X-100) followed by buffer alone, then eluted with 4 M imidazole adjusted to pH 8 with acetic or hydrochloric acids. The eluted fusion proteins were dialyzed against buffer, or the imidazole was removed by two cycles of centrifugal ultrafiltration (Centricon 30, Amicon Corp., Beverly, MA) .

One particular example of such an antibody fusion protein purification now follows. Isolation of a Soluble CD62;Immunoglobulin Fusion Protein CD62 protein chimeras were prepared by genetic fusion of the first four N-terminal extracellular domains of CD62 to the hinge domain of human IgGl as follows. CD62 cDNA sequences encoding the lectin (L) , epidermal growth factor (EGF) , and first two complement regulatory protein repeat elements (CR) were amplified in polymerase chain reactions using synthetic oligonucleotides designed to allow fusion to the human IgGl artificial splice donor sequences described previously (Aruffo et al., Cell 61. 1303-1313, 1990) (Figure 1). The forward primer bore the sequence GGC GCC GAA GCT TCC ATG GCC AAC TGC CAA ATA GCC ATC TTG (SEQ ID NO:l), while the reverse primer bore the sequence GGC CAG ATC TCC CTG CAC AGC TTT ACA CAC TGG GGC TGG (SEQ ID NO:2); the sequence allowed the CD62 fragment to be inserted as a Hindlll to Bglll fragment into

Hindlll- and BamHI-digested vector. To amplify the DNA, 20 PCR cycles were conducted, consisting of 30 s at 94°C, 2 min at 45°C, and 3 in at 72°C, using the reaction buffer recommended by the enzyme vendors (US Biochemical, Cleveland, OH) , and Mlul-digested DNA prepared from a previously described endothelial cell expression library (Bevilacqua et al., Science 243_F 1160-1165, 1989). A schematic of the resultant fusion protein, termed CD62 Rg, is shown in Figure 1. The CD62 Rg expression plas id was transfected into COS cells using DEAE dextran as previously described (Seed and Aruffo, Proc. Natl. Acad. Sci. USA 84:3365- 3369, 1987); typically, ten 100mm semiconfluent plates of COS cells were transfected with each construct. Twelve hours following transfection, cells were trypsinized, seeded onto fresh 100 mm dishes, and allowed to grow for 7-10 days. On the fourth day, 5 ml of fresh media/10% calf serum was added per dish. Supernatants were harvested, centrifuged to remove nonadherent cells and debris, pooled, and stored at 4°C. Gel electrophoresis of such supernatants demonstrated that the expression plasmids encoded the recombinant globulins and that these globulins appeared in soluble form in the supernatants of the transfected COS cells. Initial attempts to purify the CD62 Rg fusion proteins by chromatography on protein A columns were hampered by the lability of the fusion proteins to the acidic buffers typically used to elute immunoglobulins. To circumvent this problem, applicants eluted instead with a solution of imidazole, reasoning that an excess of this molecule would disrupt the interaction (as postulated from crystal structure studies) between protein A and the histidine residue contacts at the C_H2/C_H3 cleft of the antibody fusion protein. 4M imidazole proved to be a mild and effective eluant, allowing retention of carbohydrate and tissue reactivity (see below) .

This imidazole purification procedure was carried out as follows. Twelve hours following transfection, a fraction of the COS cells transfected with each construct were seeded onto flasks. Thirty-six hours post- transfection, the cells were washed with phosphate- buffered saline (PBS) and overlayed with cysteine- methionine-free media for 30 min. [³⁵S]-methionine and [³⁵S]-cysteine (TransLabel, ICN, Costa Mesa, CA) were added to a final concentration of 150 μCi/ml, and the cells were allowed to incorporate the label overnight. The supernatants were harvested and incubated with 200μl of protein A Trisacryl (Pierce, Rockford, IL) at 4°C for 12 hr. The beads were collected by centrifugation and washed with PBS/1% Nonidet P-40. For analysis, the beads were eluted with 200 μl of 1% sodium dodecyl sulfate. Ten microliters of each eluate was loaded on a 6% discontinuous polyacrylamide gel with or without prior exposure to mercaptoethanol. For preparative elution, columns were washed with 5 bed volumes of 4M imidazole (pH 8) (neutralized with acetic acid) . Eluted proteins were stored for short periods of time in imidazole at 4°C or 8°C, or exchanged into PBS by centrifugal ultrafiltration for longer term storage. CD62 Rg Tissue Reactivity

To test the purified protein's ability to react with cells and tissues in a manner characteristic of CD62, the following binding assays were performed on myeloid and tumor cell lines, i.e., cells normally bound by native CD62.

Typically, 10⁶ cells were incubated with undiluted Rg supernatants for 30 min on ice in the presence of 10% rabbit serum. Cells were washed once with PBS and exposed to fluorescein-conjugated goat antibodies to human IgG or IgM (Cappel, Malver, PA) at a concentration of 1 to 5 μ.g/ml for 30 min on ice, followed by fixation in PBS containing 4% formaldehyde. Fluorescence profiles were determined by standard techniques with a FACScan analyzer. Results are shown in Figure 2; solid lines indicate reactivity with CD62 Rg, and dotted lines indicate reactivity with control CD7 Rg protein.

Flow cytometry and fluorescence microscopy showed that CD62 Rg reacted with a cell surface ligand on freshly isolated human granulocytes, on the breast carcinoma cell lines H3630 and H3396, and on the myeloid cell lines HL60, THP-1, and U937. Cell surface reactivity was not found with the leukemic T cell lines HSB-2, Jurkat, or HPB-ALL, with K562 (erythroleukemia) cells, HeLa cells, COS cells, RD (rhabdomyosarcoma) cells, H3606 and H3620 melanoma cells, or the L tk~ and NIH 3T3 murine fibroblast cells lines (Figure 2) . Control immunoglobulin fusion proteins CD7 Rg and CD8 Rg, and native IgG, did not show appreciable reactivity under these conditions (Figure 2) . In many cases, the amount of CD62 Rg bound to permeabilized cells greatly exceeded the amount bound to unpermeabilized cells, suggesting that substantial internal stores were present. CD62 Rg Carbohydrate Reactivity Because glycolipids frequently express complex carbohydrate determinants in lineage-restricted developmental patterns, we investigated whether lipid extracts of HL60 cells (a promyelocytic leukemia line) would bind to CD62 Rg in either soluble or adsorbed form. The upper and lower phases of a Folch partition of HL60 cells was subjected to thin layer chromatography on silica gel plates, and the chromatograms were incubated with radiolabeled CD62 or control fusion proteins, washed, and subjected to fluorograph as follows. Cells (1 x 10⁸ to 5 x 10⁸) were extracted by homogenization with 20 vol of a 2:1 chloroform:methanol solution. The crude extract was filtered through lipid- free filter paper and subjected to repeated Folch partitions as described (Hakomori and Siddiqui, Meth. Enzymol. 22:345-367, 1974). Both upper and lower phases were evaporated and subsequently dissolved in 200μl of methanol. Lipids from culture supernatants were extracted (1:1 v/v) with butanol saturated with 1M NaCl. The butanol phase was dried by evaporation and the residue resuspended in methanol.

Aluminum-backed silica gel HPTLC plates (5 cm x 7.5 cm) (E. Merck, Darmstadt) were used for chromatography, and glycolipids were separated in chloroform/methanol/water (120/70/14) . After chromatography, plates were dried, fixed by immersion in 0.1% polisobutylmethacrylate in hexane (Magnani et al. , Meth. Enzymol. 83.:235-241, 1982), and incubated for 1 hr at 22°C in blocking solution (150mM NaCl, 3mM CaCl₂, 2% BSA) . ³⁵S-labeled Rg (1 x 10⁵ to 2 x 10⁵ cpm/ l) , i.e., either CD62 Rg or control fusion protein ELAM-1 Rg, was added and allowed to incubate with the plates overnight. The chromatograms were then washed twice for 30 min each in 150mM NaCl, 3mM CaCl₂, dried, sprayed with En³Hance, and subjected to fluorography. Glycolipids migrating either as a single band or, in different solvent systems, as a closely spaced doublet, were found to react strongly with CD62 Rg. No reactivity was detected in ganglioside fractions under these or more potently eluting conditions. Parallel evaluation of the chromatographic pattern of different purified glycolipids indicated that the HL60 lipids comigrated in three different solvent systems [specifically, chloroform/methanol/water (120/70/14) , chloroform/methanol/water (73/21/4) , and chloroform/methanol/acetone/acetic acid/water

(10/2/4/2/1) (Ishizuka et al., J. Biol. Chem. 253:898- 907, 1978)] with commercial preparations of bovine brain sulfatides (Sigma, St. Louis, MO; Matreya, Beliefonte, PA) , 3-sulfated galactosyl ceramides bearing heterogenous fatty acyl substitution on the 2-amino position of the sphingosine moiety.

Chromatography and analysis of the purified glycolipids under the same conditions (i.e., two micrograms (by dry mass) of each of the lipid standards: either brain gangliosides (Sigma, St. Louis, MO; Matreya, Beliefonte, PA) , sulfatides (Sigma, St. Louis, MO; Matreya, Beliefonte, PA) , trisialyl ganglioside GTlb (sigma, St. Louis, MO) , galactosyl ceramides with hydroxyl substitution (Sigma, St. Louis, MO) , or lysosulfatide (Sigma, St. Louis, MO; Matreya, Beliefonte. PA) reacted with CD62 Rg or control ELAM-1 Rg and developed with chloroform/methanol/water 73/21/4) confirmed that sulfatides reacted strongly with CD62, and that the more polar form was recognized preferentially under these conditions. Lysosulfatides, lacking the fatty acyl substitution, were not recognized, nor were galactosyl ceramides, lacking the sulfate residue, either with or without hydroxyl substitution on the fatty acid chain. Glycolipid bearing CD15 did not detectably react with CD62 Rg under conditions allowing detection of sulfatides. Neither CD7 Rg, CD8 Rg (Aruffo et al., Cell 61, 1303-1313, 1990), ELAM-1 Rg (Walz et al., Science 150:1132-1135, 1990), intact IgGl, or a COS cell preparation of a fragment of the IgGl corresponding to the Fc fragment present in CD62 Rg reacted with sulfatides.

Other Embodiments Applicant has recognized that affinity purification techniques may be modified such that amino acid mimetics are used in the final step of purification as elution reagents to release the protein of interest from the affinity complex. Such an amino acid mimetic elution step may be employed in any standard affinity purification procedure, e.g., to release a protein from a column-bound complex or from a complex included in an immunoprecipitate.

Because a number of amino acid mimetics (e.g., those described above) may be readily combined into a cocktail, the general elution technique described herein may be utilized for the purification of any protein, including those proteins whose interaction contact points or amino acid sequences are unknown. In one particular example, most or all of the exemplary amino acid mimetics listed above may be combined (solubility allowing) into a general purpose elution reagent which would disrupt any of a number of affinity complex interactions based on hydrophobic, aliphatic hydrophobic, and/or charged interactions. Such a general purpose elution reagent obviates the need to determine the amino acid sequence of specific affinity contact points or even of the protein to be isolated.

Elucidation of such amino acid sequences may, however, be used to guide selection of those mimetics which most efficiently elute the protein of interest. For example, a generally useful affinity interaction point would be that region of any given protein which directs antibody complex formation; the identity of this region could be determined either from the art or experimentally (e.g., by successive deletion or point mutation analysis of the protein followed by an assay of the mutant protein's ability to bind antibody, e.g., by immunoprecipitation or antibody column binding) . Examination of this region's amino acid sequence directs the choice of mimetics which may be utilized as elution reagents. For any given region of interaction, more than one candidate amino acid contact point may exist. Accordingly, more than one mimetic may be tested, alone or in combination, to determine which mimetic best disrupts the complex and directs the most efficient protein elution.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Seed, Brian (ii) TITLE OF INVENTION: AFFINITY PURIFICATION METHODS INVOLVING AMINO ACID MIMETICS AS ELUTION REAGENTS

(iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson

(B) STREET: 225 Franklin Street

(C) CITY: Boston

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02110-2804

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb

(B) COMPUTER: IBM PS/2 Model 50Z or 55SX

(C) OPERATING SYSTEM: MS-DOS (Version 5.0)

(D) SOFTWARE: WordPerfect (Version 5.1)

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: 07/956,660

(B) FILING DATE: October 2, 1992

(C) CLASSIFICATION:

(Vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Clark, Paul T.

(B) REGISTRATION NUMBER: 30,162

(C) REFERENCE/DOCKET NUMBER: 00786/153001

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070

(B) TELEFAX: (617) 542-8906

(C) TELEX: 200154 (2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GGCGCCGAAG CTTCCATGGC CAACTGCCAA ATAGCCATCT TG

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

GGCCAGATCT CCCTGCACAG CTTTACACAC TGGGGCTGG

What is claimed is:

Claims

1. A method of isolating a protein from a sample, said method comprising providing a first molecule which is capable of forming an affinity complex with said protein; contacting said sample with said first molecule under conditions which allow affinity complex formation; isolating said complex; treating said complex with a second molecule, said second molecule mimicking an amino acid residue of either said protein or said first molecule which is critical to said complex formation, so that said second molecule disrupts said complex, causing the release of said protein from said complex; and isolating said protein.

2. The method of claim 1, wherein said second molecule mimics said amino acid residue's side chain.

3. The method of claim 1, wherein said first molecule is protein A and said protein is an antibody or an antibody fusion protein which includes a protein A- binding domain.

4. The method of claim 3, wherein said second molecule mimics a histidine residue.

5. The method of claim 4, wherein said second molecule is imidazole.

6. The method of claim 1, wherein said first molecule is an antibody.

7. The method of claim 6, wherein said protein is a recombinant protein.

8. The method of claim 1, wherein said first molecule is an antigenic protein and said protein is an antibody which specifically binds said antigenic protein.