[go: up one dir, main page]

EP1040122A1 - Polypeptides de liaison de proteines - Google Patents

Polypeptides de liaison de proteines

Info

Publication number
EP1040122A1
EP1040122A1 EP98962582A EP98962582A EP1040122A1 EP 1040122 A1 EP1040122 A1 EP 1040122A1 EP 98962582 A EP98962582 A EP 98962582A EP 98962582 A EP98962582 A EP 98962582A EP 1040122 A1 EP1040122 A1 EP 1040122A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
protein
protein binding
binding polypeptide
polypeptide according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98962582A
Other languages
German (de)
English (en)
Inventor
Harmesh Singh Ajoula
David John Clarke
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Anmat Technology Ltd
Original Assignee
Anmat Technology Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Anmat Technology Ltd filed Critical Anmat Technology Ltd
Publication of EP1040122A1 publication Critical patent/EP1040122A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C07K14/003—Peptide-nucleic acids (PNAs)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to protein binding polypeptides and applications for such polypeptides.
  • the only small molecules known to bind to ligand binding and receptor proteins were those natural molecules of biological origin which are known to bind to the usually highly specific functional sites of such proteins, or synthetic analogues of those natural molecules.
  • Common examples of these small molecules are the haptens, polypeptides and epitopes binding to the variable or complimentarity determining region (CDR) of antibodies and other immunoglobulins, biotin binding to avidin or streptavidin, glucose binding to concanavalin A and the tachykinins binding to tachykinin receptors.
  • CDR complementarity determining region
  • binding proteins Whilst other protein molecules are known to bind to other sites on such ligand binding and receptor proteins, such as Protein A or G binding to immunoglobulins, or antibodies raised to bind to epitopes on the surface of many proteins, these binding proteins are of a particularly large size which limits their usefulness in separation, detection and treatment.
  • antibody purification by immobilised Protein A is a frequently-used separation technique both in the laboratory and for pilot scale manufacture.
  • Protein A which is isolated from the cell walls of the pathogenic bacterium Staphylococcus aureus
  • Protein G which is isolated from the cell wall of a ⁇ haemolytic Streptococcus G strain
  • S R Narayaman Journal of Chromatography 658, 1994, pages 237-258.
  • paralogs are short polypeptides that simulate the binding site for the antigen on a molecule antibody.
  • Such polypeptides have high specificity for the antigen, and are reviewed in the article by Narayaman (supra), but require knowledge of an antibody amino acid sequence and are therefore not appropriate for general use.
  • Peptides which bind specifically to a protein have been identified using Phage or chemical libraries screened against known specificities or binding receptors (Eur. J. Biochem. 1974,43 71-375. Anal Biochem. 1979, 97, 302-308. Biotech. Bioeng 1995, 47, 288-297.). Again such peptides are specific ligands of those proteins.
  • the inventors have surprisingly found short polypeptide sequences which have the ability to bind both antibodies and several other proteins, typically those with ligand binding and receptor functions, but not to the majority of enzymes tested.
  • the protein binding polypeptides can be distinguished according to this invention in three important and previously unknown and unexpected ways: their particularly small size (between 2 and 50 and typically 4 to 30 amino acids); their binding at a different site to those sites known to define the function of the proteins bound; and their binding substantially to two or more unrelated proteins which may or may not have ligand binding functions.
  • Typical proteins bound by the polypeptides are immunoglobulins (eg antibodies) and related receptors (eg antibody receptors on B cells, and T cell receptors on T cells of the cellular immune system); immunoglobulin binding proteins (eg Protein A, Protein G), lectins (eg concanavalin A), vitamin binding proteins (eg avidin, streptavidin).
  • the polypeptides of the invention are advantageous in the fields of separation, detection and treatment, particularly in applications involving the binding of the polypeptides to proteins with protein binding and receptor functions.
  • a first aspect of the invention provides a protein binding polypeptide not directly derived from a natural protein-binding protein, the protein binding polypeptide comprising 2-50 amino acids preferably 4 to 30.
  • the polypeptide preferably comprises less than 17 amino acids. In a preferred embodiment, the polypeptide comprises 13 amino acids.
  • polypeptide protein interaction in the present invention may be elucidated by detailed structural analysis. It is possible that the polypeptides of the present invention may interact with themselves via aggregation or with other proteins. The binding and dissociation may in many, but not all cases, be governed by aggregation phenomena. Indeed the polypeptides in the invention may influence aggregation properties of other proteins including ⁇ amyloid polypeptides.
  • polypeptide is used herein in a broad sense to indicate that a particular molecule comprises a plurality of amino acids joined together by peptide bonds. It therefore includes within its scope substances, which may sometimes be referred to in the literature as peptides, polypeptides or proteins.
  • the polypeptide may be further truncated below 13 amino acids.
  • the amino acid sequence of the protein binding polypeptide includes at least two of non polar amino acids which are separated by n polar amino acids where n is 0 or 1 and the said sequence has the ability to bind to at least two or more unrelated other proteins and where the full length of the sequence bears less than 90% identity with any stretch of sequence present in the said protein
  • the protein binding polypeptides of the present invention include at least one Gin residue adjacent to at least one non-polar residue.
  • the protein binding polypeptides of the present invention may be non-branching and are capable of binding to a greater range of polypeptides Such as lectins, vitamin binding proteins and immunoglobulin binding proteins (including Protein A itself).
  • the polypeptide may bind to sites on a protein which do not compete with natural protein active sites.
  • the polypeptide preferably binds an antibody at a site or sites not competing-with the antigen binding site of the antibody.
  • the polypeptide of the invention has several advantages over the prior art protein-binding protein-molecules such as Proteins A and G.
  • polypeptide of the present invention is distinguished from the polypeptides described in J. Neuroimmunol (1993) supra. Unlike the peptides of the present invention, these peptides were membrane bound polypeptides binding specifically to IgG at hinge region only and failed to show binding to Fc or Fab fragments. There are further distinctions that the amyloid peptides required specific hydropathic profile, were longer in nature and showed reluctance to reversing the binding in the presence of strong denaturing conditions.
  • the polypeptide of the invention Due to its smaller size, the polypeptide of the invention has a higher capacity than conventional protein binding proteins that is to say it binds more protein per unit weight of polypeptide.
  • the polypeptide of the present invention has been found to bind strongly to enzyme labelled rabbit and goat IgG antibody, and native antibodies from a range of animal species (goat, human, dog, cat, horse), but not to the common enzymes used in labels in such immunological procedures.
  • Affinity chromatography which is based upon the ability of molecules in solution to bind specifically to immobilised ligands or receptors on solid phase is simple in concept. It is performed in a column containing a ligand derivatised matrix, molecules to be separated from crude preparations binding specifically and tightly to the matrix, whereas most of the contaminants, which lack specific binding sites, are washed away. The specifically absorbed molecules are then eluted with desorbing agents and collected. Affinity chromatography has become an important method for the purification of molecules for use as research probes, diagnostic tools and therapeutic agents.
  • the binding of proteins is typically not very efficient, serving only to resolve some of the desired proteins into "bands" or separated fractions eluting from separation media.
  • the polypeptide ligands according to this invention typically allow the contaminants to be washed away whilst retaining the desired proteins on or within the separation media.
  • the short length of the polypeptide sequence means that the polypeptide is more stable in the harsh conditions used in affinity chromatography columns and also has improved storage properties. It is an important aspect of this invention that such harsh conditions can be avoided which is of particular advantage to the proteins being separated.
  • bound proteins can be eluted from the polypeptides of the invention using reagents such as dilute acid solutions or by using buffers or electrolytes of higher ionic strength than used to bind the polypeptides.
  • antibodies bound onto one polypeptide in accordance with the invention in weak buffer eg 10 mM Tris HCI pH 7
  • loading and elution buffers of this type can be designed for particular proteins and polypeptide combinations according to this invention.
  • polypeptides of this invention may be made by recombinant DNA methods. Alternatively, the polypeptides of the invention may be made synthetically. This reduces the risk of pyrogenic substances, typically from the cell envelope of bacteria, contaminating a particular product when the polypeptides of the invention are used with affinity columns. Conventional protein binding proteins Protein A and Protein G are derived from the cell envelope of pathogenic bacteria, and may comprise such pyrogenic substances as contaminants. The pyrogenic substances produce fever in animals, which often makes traditional protein preparations unsuitable for medical use or requires careful and extensive production and quality analysis procedures.
  • the production of the short polypeptides of the invention by synthetic means has the additional advantage that the cost of producing them is significantly reduced in comparison to the conventional isolation of known protein binding polypeptides. This results in a significant reduction in the cost of affinity chromatography columns including the polypeptides of the invention compared with conventional columns.
  • polypeptides of the invention synthetically has the further advantage that the structure of the polypeptide can be altered by known techniques to improve its stability. For example, some or all labile bonds of the polypeptide can be chemically modified to prevent a ⁇ f ⁇ ck by proteolytic enzymes, thus making the polypeptide non-biodegradable and resistant to microbiological attack. This is very difficult to achieve with native Protein A or Protein G even through recombinant DNA technology.
  • the structure of the polypeptide may be modified to enhance its binding properties.
  • residues such as cysteine may be introduced into the sequence via which residues the polypeptide can be attached to an affinity chromatography matrix.
  • the polypeptide is capable of binding an antibody or a protein enzyme conjugate.
  • Preferred protein-enzyme conjugates includes: Protein A - horseradish peroxidase (HRP), concanavalin A - HRP, and avidin - HRP. Such conjugates find application in immunoassays and related immunological procedures.
  • a second aspect of the invention provides a polypeptide including the following amino acid sequence:
  • a third aspect of this invention provides a polypeptide including the following amino acid sequence:
  • a fourth aspect of this invention provides a polypeptide including the following amino acid sequence: DMHDFFVGLM
  • a fifth aspect of this invention provides a polypeptide including the following amino acid sequence:
  • a sixth aspect of the invention provides a polypeptide including the following amino acid sequence:
  • a seventh aspect of the invention provides a polypeptide including the following amino acid sequence:
  • Functional equivalents include polypeptides comprising additions, deletions, and/or substitutions to the above sequence having the same or similar protein binding abilities as the above polypeptide.
  • the determination of functional equivalents of the above sequence is within the scope of the skilled worker.
  • a polypeptide having the sequence AIKG derived from the polypeptides of the second and third aspects of the invention binds protein.
  • Functional equivalents of the polypeptide of the invention may have improved protein binding abilities.
  • One or more amino acids of the polypeptide may be replaced by an amino acid having similar properties.
  • Amino acids having similar properties include Amino acids having aliphatic side chains: gly, ala, val, leu, ile, pro.
  • Aromatic amino acids phe, tyr, trp
  • Basic amino acids lys, arg, his, asn, gin.
  • Acidic amino acids asp, glu.
  • Non-polar amino acids are amino acids not including acidic and basic amino acids.
  • the polypeptide may also contain synthetic amino acids such as aminoadipic acid, aminobutyric acid, desmosine, sarcosine, norvaline, norleucine and ornithine, ⁇ -alanine,homocysteine,citrulline,cyclohexylalanine,chlorophenylalanine,cystine,dehydrproli ne,homocitrulline,homoserine,hydroxyproline, ⁇ hydroxyvaline, penicillamine, statine.
  • synthetic amino acids such as aminoadipic acid, aminobutyric acid, desmosine, sarcosine, norvaline, norleucine and ornithine, ⁇ -alanine,homocysteine,citrulline,cyclohexylalanine,chlorophenylalanine,cystine,dehydrproli ne,homocitrulline,homoserine,hydroxyproline, ⁇ hydroxyvaline
  • the polypeptide binds to a site or sites on a protein which do not compete with the natural protein active sites, that is to say the normal function of the active site is not substantially affected.
  • the polypeptide may bind to the antibody at a site or sites which do not compete with a normal antigen binding site of the antibody.
  • polymers of a polypeptide according to the invention may be prepared. These have the advantage that several protein binding sites may be provided on the same molecule at the same time.
  • the polymer may be a homopolymer or a copolymer of a polypeptide in accordance with the invention together with another suitable polypeptide.
  • a polypeptide of the invention is used to modify another protein, by incorporation of the sequence of the polypeptide into the amino acid sequence of the protein, so that that protein can bind to other proteins.
  • Proteins which may have the polypeptide of the invention added or substituted to their amino acid sequence include Protein A and Protein G. This improves the protein's ability to bind antibodies and other proteins, and increases its binding affinity to other molecules.
  • the polypeptide sequence of a polypeptide in accordance with the invention may be added to other proteins to enable novel protein conjugates to be made.
  • polypeptides of the invention may be used as affinity chromatography agents. Accordingly the polypeptides of the invention may be conjugated to one or more solid materials suitable for use in affinity chromatography such as, acrylic polymers cross-linked dextran, silica, glass, agarose, methacrylamide-methylbisacrylamide, cellulose, vinyl polymers, polyacrylamide or combinations thereof.
  • the polypeptides may be covalently or non-covalently attached to such substrates by any means known in the art.
  • sequence GQVLQGAIKG can be assembled on solid phase removing a portion for testing after each amino acid.
  • a Lys residue is incorporated with temporary side chain protection such as Fmoc which can be removed (prior to testing) with 20% piperidine in DMF without peptide cleavage from the resin.
  • Fmoc temporary side chain protection
  • polypeptides of the invention are used to purify antibodies, or similar immunoglobulins, such as T cell receptor, lectins, streptavidin, avidin, or fragments or derivatives thereof, their ligands, ligands, substrates, antigens or other analytes.
  • immunoglobulins such as T cell receptor, lectins, streptavidin, avidin, or fragments or derivatives thereof, their ligands, ligands, substrates, antigens or other analytes.
  • Antibody binding proteins such as protein A and G are also widely used in the implementation of diagnostics tests or assays, including those assays, tests and monitoring methods using biosensor devices (eg surface plasmon resonance, surface acoustic wave, or other such microelectronic, optoelectronic devices).
  • biosensor devices eg surface plasmon resonance, surface acoustic wave, or other such microelectronic, optoelectronic devices.
  • the polypeptides of the invention may similarly be used, where their unique ability to bind a broad range of commonly used diagnostic molecules very close to the active surface of such sensor devices is particularly advantageous.
  • the small size of the polypeptide of the invention enables further improvements to be made in such assays, tests and monitoring methods, particularly those where the distance between reacting components needs to be short, which methods are typically called proximity assays.
  • a fluorescent dye e.g. dansyl
  • the natural (e.g. tryptophan) fluorescence of the protein may be used or fluorescence may be introduced into the polypeptide.
  • the fluorescence of the polypeptide or protein may be coupled in such a way as to change the nature of the fluorescence (its intensity, wavelength of emission or excitation, or the time scale or polarisation of the fluorescence).
  • the polypeptide enables the distance between the two molecules to be much shorter than would be possible using a protein such as Protein A or G. While the polypeptide may not bind to the functional site of proteins, its binding to the protein can present a molecule, such as a fluorophore, sufficiently close to the functional site that the fluorophore responds to binding processes occurring at the functional site. For example, the well known processes of fluorescence quenching or resonance energy transfer may be used. It will be similarly apparent to those skilled in the art that molecules or other materials may be attached to the polypeptide so as to interfere with the normal operation of the functional site of the protein.
  • polypeptide covalently to its site of binding on the protein molecule by procedures well established in the art, which typically involve the use of heterobifunctional cross-linking agents, whose reaction to couple the polypeptide to the protein may include photochemical methods.
  • Similar methods used in separation and diagnostic tests and assays may be used in the context of microbiological, animal cell or viral diagnostics tests, assay and monitoring procedures.
  • Some of these cells can possess the proteins to which the polypeptides of the invention bind, or the proteins can be introduced to bind to the micro-organisms, cells or viruses, by methods well known to those skilled in the field.
  • polypeptides of the invention can be used to treat micro-organisms, cells or viruses by attaching a bioactive agent, drug or their carriers to the polypeptide by well established methods.
  • the polypeptides of the invention can also be introduced onto the surface of larger drug molecules, such as those produced by biotechnology processes, commonly known as biopharmaceuticals, cells, micro-organisms, viruses, macromolecules, polymers or other particles or materials, such as medical implants, that are introduced into biological samples or the body of an animal.
  • biotechnology processes commonly known as biopharmaceuticals, cells, micro-organisms, viruses, macromolecules, polymers or other particles or materials, such as medical implants.
  • a particular problem in these procedures is that the molecule, cell, particle or material so introduced is often treated as foreign by the animal body, such that various processes (e.g. immune responses) result in unfavourable reactions in the body.
  • proteins such as antibodies, which label or opsonise the foreign matter introduced, which provokes the unfavourable response.
  • the foreign matter bearing the polypeptides bind proteins present in the host which are not recognised as foreign by the host, and furthermore may bind them in such a way that they do not present their normal labelling or opsonisation function.
  • polypeptide of the invention is able to bind to more than one protein at once it is possible to target one protein to another via interaction with the polypeptide.
  • an antibody bound to the polypeptide of the invention may be targeted to specific site and another protein could then be targeted to the same site , and vice versa, by interaction with the polypeptide.
  • the binding of the polypeptide to proteins may influence the specific functional properties of those proteins and this can be exploited to control the function of protein.
  • the aggregation of Alzheimer polypeptides may be controlled by binding to polypeptides of the present invention leading to treatment by minimising fibril formation.
  • polypeptides according to this invention may indicate the presence on ligand binding and receptor proteins of a previously unknown common or similar site or structural motif, which is substantially absent at least from one other major class of proteins with catalytic functions - the enzymes.
  • Fig. 1 is an HPLC trace of purified peptide TRNGQVLQGAIKG
  • Fig. 2 shows antibody binding to lOmer peptide GQVLQGAIKG and its Ala scan derivatives
  • Fig. 3 is binding and elution profile from peptide affi-prep-10 column.
  • Fig. 4 is a fluorescence spectrum recorded by excitation at 490nm showing quenching of FTTC fluorescence by anti-FTTC in the presence and absence of the 10 mer peptide;
  • Fig. 5 shows binding of HRP,GARP,Fc fragment and Fab fragment to lOmer peptide GQVLQGAIKG. Control experiments in which no peptide was present are marked by ⁇
  • Fig.6 Shows binding of goat anti rabbit peroxidase to 11 mer peptide (NDNGVDGETWY) derived from natural antibody binding protein (Proc. Natl. Acad. Sci.USA 1992, 89, 8532-8536) compared to peptide GQVLQGAIKG. Control experiments in which no peptide was present are marked by ⁇ .
  • Fig. 7 shows binding of peroxidase conjugated antibody and fragments Fab and Fc to peptide GQVLQGAIKG which has been immobilised on affinity matrix affi-preplO. Control experiments in which no peptide was present are marked by ⁇ .
  • Fig 8. Shows association and dissociation of different concentrations of Goat IgG with BSA-peptide conjugate immobilised on CM 5 chip.
  • the curves from top to bottom are for IgG concentrations 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 and 0.025 ⁇ M respectively.
  • Fig 9. Shows association and dissociation of different concentrations of Goat IgG with multimeric 10 mer peptide immobilised on CM 5 chip.
  • the curves from top to bottom are for IgG concentrations 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 and 0.00625 ⁇ M respectively.
  • Fig. 10 Shows binding of goat antirabbit peroxidase (GARP) to a 30 mer peptide (KIGQFLIQFAGAFLS ⁇ LQGLTLRAAEKQAG) and also a conjugate of 10 mer peptide (GWVLQGAIKG) with BSA measured by ELISA.
  • GARP goat antirabbit peroxidase
  • Fig. 11 Shows binding of GARP to a peptide (SRAQILQQAG) sequence taken from Flagella protein (J. Mol. Biol 1991 219: 471-480) and the same figure also shows that the protein itself does not bind.
  • SRAQILQQAG a peptide sequence taken from Flagella protein
  • Fig. 12 is a chromatographic profile indicating binding and elution of Goat IgG from multimeric peptide (GWVLQGAIKG) column.
  • Example 1 Preparation, purification and characterisation of polypeptides.
  • polypeptides by solution phase and solid phase chemistries.
  • the polypeptide can be readily prepared by solid-phase synthesis as follows using well established protocols. For instance we used Boc chemistry developed by Merrifield to synthesise a polypeptide having the sequence TRNGQVLQGAIKG. MBHA resin (0.5mMoles) was used. The side chain protecting group for Lys was 2-C1Z. Each synthetic cycle consisted of (i) a 2min and 25min deprotection with 50% TFA/DCM (ii) neutralisation with 5% DIPEA/DCM and (iii) coupling with 1.5mMoles amino acid, 1.5mMoles BOP and 4.5mmoles DIPEA in DMF for 40 mins.
  • polypeptide was cleaved with HF by known procedure. Typically the polypeptide resin was treated with 20ml HF, 0.5g thiocresol and 0.75g p-cresol and after evaporation of HF, extraction was carried out with 50% acetic acid/water. The polypeptide was purified on C-8 reverse phase Vydac semi-prep column using linear gradient of 20% acetonitrile/0.1% TFA to 80% acetonitrile/0.1% TFA over 45 mins. The product peak was lyophilised and analysed by HPLC.
  • sequences accrding to the invention could be similarly produced.
  • the side chain protected amino acids used in other sequences were Boc-Arg(Tos)-OH, Boc-Asp(OcHx)-OH, Boc-Glu(OBzl)-OH, Boc-Lys(2-CL-Z)-OH, Boc-Lys(Fmoc)-OH, Boc-Ser(BzL)-OH and Boc-Thr(Bzl)-OH.
  • Biotin could be coupled in identical manner to amino acids using BOP activation as described above.
  • the first residue to couple was Fmoc-Lysine (Fmoc)-OH.
  • the Fmoc groups were removed using 20% Piperidine in DMF. Repeating this procedure again yielded the lysine core for extending four peptide chains in the usual manner.
  • Figure 1 shows an HPLC trace of the purified polypeptide : TRNGQVLQGAIKG 25 ⁇ g polypeptide applied to a C-18 Vydac column running gradient of 0.1% TFA to 80% acetonitrile/0.1 %TFA in 30 mins. Detection wavelength was 218nm.
  • polypeptides (1) - (10) with Ala residue at positions 1-10 were screened for their binding to GARP (Goat anti-rabbit peroxidase) as described in Example 3.
  • GARP Goat anti-rabbit peroxidase
  • the absorbance reflects the binding of each analogue.
  • the polypeptides produced by this substitution technique of Fig. 2 show that the Ala scan polypeptide sequences 1 to 8 each bound the protein to a different level.
  • the protein may be a labelled protein such as antibody or avidin or any other labelled with HRP or other enzymes or reported groups.
  • concentration of protein solution will depend on the amount of label.
  • HRP Horse Radish Peroxides
  • a substrate such as 5-amino salicylic acid dissolved in 50mM Sodium phosphate pH 6 buffer containing 0.01% (W) of fresh hydrogen peroxide.
  • W 0.01%
  • Fig 5 shows binding of immunoglobulins and two fragments where the enzyme peroxidase is used as label.
  • the label could be attached or bound secondary to the protein (step 2 above).
  • the procedure following coating of polypeptide would then comprise:
  • Biotinylated GQVLQGAIKG showed significant binding to polyclonal IgG from various sources and some proteins relative to the appropriate controls (Table 1 and 2 ). In this assay avidin exhibited some non-specific binding to the proteins. Nevertheless, signal from peptide-protein interaction was apparent.
  • biotinylated GQVLQGAIKG is an example of a polypeptide in accordance with the invention which binds more than one protein.
  • Example 3 Screening of polypeptides binding to a typical protein
  • Goat antirabbit peroxidase is used as typical protein.
  • sequence GQVLQGAIKG was compared to 11 mer sequence derived from Protein G (Proc. Natl. Acad. Sci.USA 1992, 89, 8532-8536) .
  • Figure 6 shows that the peptide derived from natural protein is unable to bind IgG in the same manner as our sequence.
  • Fig. 10 shows the binding of GARP to a 30mer peptide of sequence KIGQFLIQFAGAFLSILQGLTLRAAEKQAG. In these cases an improved response is apparent due to the longer peptide than a shorter peptide as adso ⁇ tion and or binding may be improved.
  • a 10 mer peptide of sequence GQVLQGAIKG immobilised on BSA protein as in Example 9 also shows comparable binding to the 30 mer peptide (Fig. 10).
  • Short polypeptides do not usually adsorb efficiently to microwells.
  • polypeptides can be screened for their binding ability.
  • One of the ways is to immobilise the polypeptide covalently to solid surface.
  • ELISA plates with derivatised-surfaces are commercially available for linking molecules to surfaces. Such methods can easily be applied to bind shorter sequences and then screen in the usual manner described above.
  • the sequences can be synthesised on the solid phase by well known techniques and portions of the resin removed at various stages of the synthesis . The resin can then be used instead of the microwell as a support for the protein binding polypeptide of the invention.
  • the washing steps analogous to the ELISA method reported above can be carried out by mixing the resin with desired solution and separation effected by bench top microcentrifuge.
  • the inventors were able to scan the whole of TRNGQVLQGAIKG polypeptide of the invention, and found that even short sequences, for instance AIKG, are able to bind proteins.
  • the polypeptide (GQVLQGAIKG) was assembled on acid resistant resin but using Fmoc amino acids instead of the Boc used in example 1.
  • a small amount of resin (lOmg) was removed after assembly of each amino acid and treated with 95% TFA/ 5% water mixture to cleave side chains.
  • the resin was washed with dichloromethane and methanol and dried.
  • the resin was incubated in 1ml solution of PBS-T to block non specific sites.
  • the ELISA steps as described in examples 2,3 and 4 could be performed on the resin using 1ml solution volumes followed by centrifugation to recover the resin after each wash step.
  • the response was measured by recording absorbance reading of the supernatant.
  • affinity columns with a wide range of matrices
  • c.f Immobilised affinity ligand techniques (Academic Press 1992) or Bioaffinity Chromatography (Elsevier Science Publications 1993)
  • the inventors used a commercially available preactivated Affi-prep column and immobilised the polypeptide of the invention in a high performance stainless steel column.
  • a 13mer polypeptide was synthesised with a 6 carbon spacer, aminohexanoic acid, and was immobilised in dilute buffer at pH7.8 at polypeptide concentration of lOmg/ml. The coupling was allowed to proceed overnight by recirculating the polypeptide solution through the column. The next day any remaining activated groups were treated with 0.1M ethanolari ⁇ i ⁇ e solution.
  • Proteins-HRP conjugates binding to Affiprep-10 immobilised peptide were measured by incubation of small amount of matrix (Preblocked to minimise non specific binding) in PBS-T buffer and washing off unbound material by centrifugation. The bound conjugates were then estimated by incubation with 5- aminosalicylic acid as described for ELISA measurement in example 2 except that the reaction mixture was centrifuged at low speed and supernatant used for recording the optical density. Affi-prep 10 matrix blocked with ethanolamine was used as a control matrix.
  • Figure 7 shows that the immobilised sequence is able to bind proteins.
  • Immobilisation of multimeric GQVLQGAIKG peptide on amino sepharose matrix was done as follows, lg of AH-Sepharose in 4ml of PBS was treated with 0.5ml of 8% Glutaraldehyde solution, for 30 mins. Excess reagent was removed by washing the resin with distilled water on a sintered funnel. A 2 fold molar excess of multimeric peptide in 50mM bicarobnate buffer pH 9.6 containing 10% DMSO was coupled to this activated matrix for 3hrs. Unbound peptide was washed by filtration with buffer followed by 10% acetic acid followed by water and ethanol.
  • the matrix was resuspended in 25ml buffer and few crystals of sodium borohydride added. The washings used to remove unbound peptides were repeated. The coupling of peptide was qualitatively measured using a ninhydrin test. The matrix was packed into a short column (0.8cm X 10cm) and equilibrated with binding buffer.
  • Example 6 Separation and/or screening of proteins using polypeptide affinity column
  • Example 6 The column produced in Example 6 was attached to the HPLC system and proteins were detected by using UV detector fixed at 280nm wavelength.
  • protein 0.2 to 0.5 to mg
  • suitable buffer such as lOmM Tris-HCL pH 8, by rheodyne injector at a flow rate of 0.2ml/min.
  • the effluent was continuously monitored at wavelength of 280nm and an elution profile obtained.
  • the bound protein was then eluted by applying elution buffer such as 3M Guanidine hydrochloride or 0.1% TFA.
  • elution buffer such as 3M Guanidine hydrochloride or 0.1% TFA.
  • the multimeric peptide column prepared in example 5 was equilibrated with 6ml of Phosphate buffered saline (PBS) at pH 7.4. A 0.2mg amount of Goat IgG(Sigma) in 0.5ml PBS was applied to the column at a flow rate of 0.4ml/min. After loading the sample the binding buffer was applied to wash off unbound protein and absorbance measured continually at 280nm. We then applied 3ml solution of 3M Guanidine hydrochloride to elute off the bound protein.
  • Figure 12 shows the chromatographic profile indicating binding and elution of typical protein from this column.
  • Example 7 Screening different compounds for binding to polypeptides.
  • anti-FTTC antibody This antibody is known to quench >95% fluorescence of fluorescein upon specific binding at the antigen binding site.
  • This quenching assay was performed in 2ml of lOmM Tris-HCL pH 7.4 buffer containing_3:l of lO ⁇ g/ml fluorescein solution in the presence and absence of antibody.
  • 5:1 of antibody was able to quench most of the fluorescence upon binding. The same level of binding was observed in the presence of 10:g of polypeptide (GQVLQGAIKG).
  • the peptide sequence GQVLQGAIKG was immobilised on BSA using the glutaraldehyde method as follows. Bovine Serum albumin (4mg) was dissolved in 0.75ml of lOmM Sodium Phosphate pH 7.4 buffer. Glutaraldehyde(0.25ml of 8% solution) was added and mixture stirred for 30mins at room temperature. The Excess Glutaradehyde was removed by Gel filtration on PD-10 column. The peptide (lOmg dissolved in minimum volume of DMSO) was added to the activated BSA and conjugation allowed to proceed for 3 hrs. Unconjugated Peptide was removed by dialysis, centrifugation and further Gel filtration.
  • the conjugate was immobilised on CM5 chip .using EDC coupling, by flowing across the sensor chip according to the manufacturers description (BIAcore).
  • BSA was used in the control flow cell.
  • the peptide protein interaction was studies using different concentrations of proteins in order to obtain optimum conditions for measuring binding constants.
  • the antibody was bound in Phosphate buffered saline and regeneration effected with 3M Guanidine hydrochloride solution.
  • the binding affinities (kD) were determined.
  • Figure 8 and 9 shows the association and dissociation progress curves.
  • the multimeric peptide could be immobilised in identical manner and binding evaluated.
  • the kD values obtained were estimated to be 4 x 10" 7 M and 1 x 10 "7 M respectively for the multimeric and BSA conjugated 10 mer peptide.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Neurosurgery (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Hospice & Palliative Care (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Psychiatry (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne un polypeptide de liaison de protéines non directement dérivé d'une protéine de liaison de ligand naturelle connue pour lier une protéine, le polypeptide liant la protéine comprenant 2 à 30 acides aminés, ainsi que les utilisations du polypeptide.
EP98962582A 1997-12-19 1998-12-21 Polypeptides de liaison de proteines Withdrawn EP1040122A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9726956 1997-12-19
GBGB9726956.7A GB9726956D0 (en) 1997-12-19 1997-12-19 Protein binding polypeptides
PCT/GB1998/003855 WO1999032513A1 (fr) 1997-12-19 1998-12-21 Polypeptides de liaison de proteines

Publications (1)

Publication Number Publication Date
EP1040122A1 true EP1040122A1 (fr) 2000-10-04

Family

ID=10823972

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98962582A Withdrawn EP1040122A1 (fr) 1997-12-19 1998-12-21 Polypeptides de liaison de proteines

Country Status (6)

Country Link
EP (1) EP1040122A1 (fr)
JP (1) JP2002508301A (fr)
AU (1) AU1771599A (fr)
CA (1) CA2315301A1 (fr)
GB (1) GB9726956D0 (fr)
WO (1) WO1999032513A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201917630D0 (en) * 2019-12-03 2020-01-15 Ge Healthcare Bio Sciences Ab Method for determination of aggregates
CN119930760B (zh) * 2025-04-09 2025-06-13 深圳大学 一种结合并染色头发角蛋白的多肽、制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9932513A1 *

Also Published As

Publication number Publication date
AU1771599A (en) 1999-07-12
JP2002508301A (ja) 2002-03-19
WO1999032513A1 (fr) 1999-07-01
GB9726956D0 (en) 1998-02-18
CA2315301A1 (fr) 1999-07-01

Similar Documents

Publication Publication Date Title
US6875594B2 (en) Methods of ligating expressed proteins
US8735540B2 (en) Peptides with sequentially arranged streptavidin binding modules
EP0871658B1 (fr) Molecules modifiees d'avidine et de streptavidine, et leur application
EP2949750B1 (fr) Peptide liant les anticorps
AU710533B2 (en) Affinity labeling libraries and applications thereof
AU2002254683A1 (en) Binding molecules for Fc-region polypeptides
EP1497318A2 (fr) Molecules de liaison pour les polypeptides de zone fc
JP3811524B2 (ja) リガンドとして有用なペプチド
EP2614371B1 (fr) Procédé pour marquer des composés
WO1999032513A1 (fr) Polypeptides de liaison de proteines
Katayama et al. Application of 2, 2′‐dipyridyl disulfide‐mediated thiazolidine ring‐opening reaction to glycoprotein synthesis: Total chemical synthesis of evasin‐3
JP2005112827A (ja) 抗体アフィニティ担体
US11008365B2 (en) Polypeptide exhibiting affinity to antibodies forming non-native three-dimensional structure
US5932692A (en) Antigenic peptides
JP4258271B2 (ja) ポリアミノ酸担体
US5254672A (en) Synthetic peptides which contain sequences from factor VIIa, and the use thereof
JP4618751B2 (ja) ルシフェラーゼに対して結合性を有するペプチド
EP1621546B1 (fr) Ligands peptidiques avec affinité spécifique pour les immunoglobulines
KR101664338B1 (ko) 면역글로불린 Ｇ의 Ｆｃ 부위에 선택적으로 결합하는 신규 펩타이드
CA2245558C (fr) Bibliotheques de marquage par affinite et leurs applications
WO1998015572A1 (fr) Composition comprenant un peptide acyle et immobilise
JPWO2004022599A1 (ja) 融合抗体及びその作成方法
EP1432810A2 (fr) Production de proteines recombinantes in vivo et utilisation associee permettant de produire des anticorps
CS258289B1 (cs) Peptid s vlastnostmi antigennfch determinant a způsob jeho výroby

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20000616

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Withdrawal date: 20021104