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WO2010104114A1 - Procédé de préparation d'une bibliothèque de polypeptides - Google Patents

Procédé de préparation d'une bibliothèque de polypeptides Download PDF

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Publication number
WO2010104114A1
WO2010104114A1 PCT/JP2010/054009 JP2010054009W WO2010104114A1 WO 2010104114 A1 WO2010104114 A1 WO 2010104114A1 JP 2010054009 W JP2010054009 W JP 2010054009W WO 2010104114 A1 WO2010104114 A1 WO 2010104114A1
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cys
polypeptide
amino acid
library
acid sequence
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Japanese (ja)
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泰 久保
忠史 木村
世吾 小野
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National Institute of Advanced Industrial Science and Technology AIST
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National Institute of Advanced Industrial Science and Technology AIST
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1044—Preparation or screening of libraries displayed on scaffold proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present invention relates to a method for preparing a polypeptide library and a method for identifying a novel polypeptide using the method.
  • Non-patent Document 1 An attempt has been made to create a combinatorial library of polypeptides using a polypeptide scaffold and to discover a novel protein that replaces an antibody (Non-patent Document 1).
  • a polypeptide library designed so that the amino acid sequence of the region involved in the interaction (for example, the loop region) is random is generated and the desired target is created.
  • a novel protein that can bind to the desired target can be identified by contacting with and selecting a protein that can bind to the target and determining its structure.
  • Poisons that snakes, scorpions, spiders, and insects use immediately for defense or attack against other organisms are generally neurotoxins and can rapidly paralyze their opponents.
  • ⁇ -type neurotoxins are often antagonists to voltage-gated ion channels.
  • These “ ⁇ -type neurotoxins” generally form a stable scaffold structure having a finger-like loop region by 4 to 5 disulfide bonds and 3 to 5 antiparallel ⁇ sheets.
  • the present inventors have previously focused on the “finger-like” “scaffold structure” in “ ⁇ -type neurotoxin”, and the three-finger (3F) scaffold possessed by polypeptide neurotoxin derived from snake venom.
  • Patent Documents 1 and 2 A method of creating a combinatorial library using a fold has been developed (Patent Documents 1 and 2).
  • a display-type polypeptide library in which disulfide bonds and ⁇ -sheet region amino acids that are involved in the formation of a stable scaffold structure are left intact, and regions that are expected to form loops are randomized. And has succeeded in obtaining a polypeptide having high affinity with the target protein by high-throughput screening combined with the emulsion PCR method.
  • the molecular weight of the polypeptide is quite large, about 7 kDa, and there are four disulfide bonds.
  • it is technical for the production of recombinant proteins such as refolding and disulfide bond formation.
  • a scaffold having a molecular weight as small as possible and having a smaller number of cysteine residues is suitable.
  • 3F scaffolds When creating a library using 3F scaffolds, not all three finger-like scaffolds are necessary, but one finger is divided so as to maintain the scaffold structure. It was confirmed that even a library using a protein is effective for screening a polypeptide having an affinity for a target protein (Patent Document 2), but its screening efficiency is considerably lower than that of the original 3F scaffold. End up.
  • a polypeptide library that has been developed in the past can be used as a high-throughput screening system for agonists and antagonists because there are many complicated steps such as preparation of cells that express the target protein when targeting a membrane protein.
  • the target protein is only soluble protein, and the development of application to membrane protein has been difficult. From the above, it is a library using a scaffold having a small molecular weight and a smaller number of cysteine residues instead of a library using a 3F scaffold derived from a snake venom, and having high screening efficiency. The rally was strongly desired.
  • provision of a polypeptide library applicable to the system has been eagerly desired.
  • the present invention relates to a polypeptide library for identifying a polypeptide having a high affinity for a desired target protein, using a scaffold having a lower molecular weight than a 3F scaffold and having a smaller number of cysteine residues.
  • An object of the present invention is to provide a polypeptide library applicable to a high-throughput screening system targeting proteins.
  • spider venom contains physiologically active polypeptides that inhibit the action of calcium channels, sodium channels, potassium channels, protein hydrolases, and the like.
  • the mature polypeptide consists of 30-40 amino acid residues and has a structural motif called Inhibitor Cystine Knots (ICK).
  • ICK Inhibitor Cystine Knots
  • This family has 6 cysteine residues and its cysteine framework is strictly conserved, but the rest of the sequence is diverse. It has been reported from mass spectrometry and three-dimensional structure analysis that this polypeptide forms three disulfide bonds and has a physicochemical and biologically stable structure.
  • Non-patent document 7 The present inventors recently determined the base sequence and the corresponding amino acid sequence of the calcium channel blocking type venom peptide (A4-1) gene derived from South American Gramostra spatulata among these spider venoms (Patent Document 3). ). In the process of examining the amino acid sequence and the three-dimensional structure of the A4-1 polypeptide 82 amino acid ORF (7 cysteines) in detail, the present inventors determined that the N-terminal side of the polypeptide is a signal peptide and a prepro sequence. The matured body was found to have 34 amino acids, a molecular weight of about 4 kDa, and 6 cysteine residues. The amino acid sequence of the mature polypeptide of A4-1 is as follows.
  • DCLGFMRKCIPDNDKCCRPNLVCSRTHKWCKYVF SEQ ID NO: 1
  • Cys-1 the first cysteine
  • Cys-2 the second cysteine
  • Cys-3 the third cysteine
  • ICK scaffold the sixth (Cys-6) at three positions ( FIG. 1). Since the ICK scaffold is relatively small in size and high in stability, it was considered to be optimal as such a polypeptide library candidate.
  • the present inventors have said that the calcium channel of the ICK scaffold target is three loop regions (loop I, N-terminal side from the N-terminal side) excluding the ⁇ sheet structure in three regions sandwiched between the respective SS bonds. Loop II and Loop III.) And the tail region from Cys-6 to the C-terminal side are predicted to be bound, and a polypeptide library is created in which the amino acid residues in each region are simultaneously randomized. (The dark gray portion in FIG.
  • the present inventors inserted a mutant polypeptide gene in which nucleotide sequences corresponding to the above four locations were randomized into an expression vector to produce a display-type library, and at the same time, targeted membrane protein genes A combinatorial library inserted into the same expression vector was constructed.
  • the library can display the target protein on the inner membrane surface of E. coli, but the library using ICK scaffold has a relatively small molecular weight and high stability, so it can be screened in the peri- mic space. It was confirmed that. It has also been demonstrated that the screening technology enables molecular evolution to a polypeptide sequence with higher affinity for the target membrane protein.
  • a polypeptide library composed of a plurality of polypeptides having an ICK scaffold, and the amino acid sequence of each polypeptide was simultaneously randomized in the following regions (i) to (iv) A polypeptide library characterized by having an amino acid sequence; (I) the amino acid sequence from the fourth C-terminal side of Cys-1 to the first N-terminal side of Cys-2 in the loop I region between Cys-1 and Cys-2; (Ii) an amino acid sequence from the third C-terminal side of Cys-2 to the first N-terminal side of Cys-3 in the loop II region between Cys-2 and Cys-3; (Iii) the amino acid sequence from the second C-terminal side of Cys-5 to the third N-terminal side of Cys-6 in the loop III region between Cys-5 and Cys-6; and (iv) from Cys-6 Amino acid sequence from the third C-terminal side to the C-terminal side of Cys-6 in the tail region on the C-termin
  • each polypeptide constituting the polypeptide library is a polypeptide comprising an amino acid sequence of the following formula [1]; Formula [1] DCLGF (X) 3 CIP (X) 4 CCRPNLVCS (X) 3 KWCKY (X) 2 (SEQ ID NO: 2) (Wherein X is any amino acid residue) [3] The polypeptide library according to [1] or [2], wherein each polypeptide constituting the polypeptide library is present in a form associated with a polynucleotide encoding the polypeptide. .
  • a polynucleotide library comprising a group of polynucleotides encoding a plurality of polypeptides having an ICK scaffold, wherein the amino acid sequences of the polypeptides encoded by the polynucleotides are the following (i) to ( a polynucleotide library characterized by having amino acid sequences randomized simultaneously in the region of iv); (I) the amino acid sequence from the fourth C-terminal side of Cys-1 to the first N-terminal side of Cys-2 in the loop I region between Cys-1 and Cys-2; (Ii) an amino acid sequence from the third C-terminal side of Cys-2 to the first N-terminal side of Cys-3 in the loop II region between Cys-2 and Cys-3; (Iii) the amino acid sequence from the second C-terminal side of Cys-5 to the third N-terminal side of Cys-6 in the loop III region between Cys-5 and Cys-6; and (iv) from Cys-6 Amino acid
  • each polynucleotide constituting the polynucleotide library is a polynucleotide encoding a polypeptide comprising the amino acid sequence of the following formula [1]; Formula [1] DCLGF (X) 3 CIP (X) 4 CCRPNLVCS (X) 3 KWCKY (X) 2 (SEQ ID NO: 2) (Wherein X is any amino acid residue).
  • Formula [1] DCLGF (X) 3 CIP (X) 4 CCRPNLVCS (X) 3 KWCKY (X) 2 (SEQ ID NO: 2) (Wherein X is any amino acid residue).
  • [6] The polynucleotide library according to [4] or [5], wherein the polynucleotide library is in a state of being incorporated into an expression vector.
  • [7] The polynucleotide library according to [6], wherein a secretion signal sequence is present upstream of the polynucleotide in the expression vector.
  • [8] The polynucleotide library according to [6] or [7], wherein a polynucleotide encoding a target protein is incorporated on the same vector in the expression vector.
  • [9] A polynucleotide library composed of a group of transformed hosts transformed with the expression vector according to any one of [6] to [8].
  • [10] The polynucleotide library according to [9], wherein the transformed host is a gram-negative bacterium.
  • a method for identifying a polypeptide having affinity for a target comprising the following steps (a) to (d): (A) A polypeptide library composed of a group of a plurality of polypeptides having an ICK scaffold, and the amino acid sequences of the respective polypeptides were simultaneously randomized in the following regions (i) to (iv) Preparing a polypeptide library characterized by having an amino acid sequence; (I) the amino acid sequence from the fourth C-terminal side of Cys-1 to the first N-terminal side of Cys-2 in the loop I region between Cys-1 and Cys-2; (Ii) an amino acid sequence from the third C-terminal side of Cys-2 to the first N-terminal side of Cys-3 in the loop II region between Cys-2 and Cys-3; (Iii) the amino acid sequence from the second C-terminal side of Cys-5 to the third N-terminal side of Cys-6 in the loop III region between Cys-5 and Cys-6; and (iv) from Cys
  • each polypeptide constituting the polypeptide library is a polypeptide comprising an amino acid sequence of the following formula [1]; Formula [1] DCLGF (X) 3 CIP (X) 4 CCRPNLVCS (X) 3 KWCKY (X) 2 (SEQ ID NO: 2) (Wherein X is any amino acid residue) [13] A step in which each polypeptide constituting the polypeptide library in the step (a) is present in a form associated with a polynucleotide encoding each polypeptide, and the amino acid sequence of the polypeptide is determined ( The method according to [11] or [12], wherein d) is performed by determining a base sequence of a polynucleotide bound to the polypeptide.
  • a method for identifying a polypeptide having affinity for a target comprising the following steps (a) to (h): (A) A polypeptide library composed of a group of a plurality of polypeptides having an ICK scaffold, and the amino acid sequences of the respective polypeptides were simultaneously randomized in the following regions (i) to (iv) A polypeptide library having an amino acid sequence and a polypeptide library in which each polypeptide constituting the polypeptide library is associated with a polynucleotide encoding each polypeptide is prepared.
  • each polypeptide constituting the polypeptide library is a polypeptide comprising an amino acid sequence of the following formula [1]; Formula [1] DCLGF (X) 3 CIP (X) 4 CCRPNLVCS (X) 3 KWCKY (X) 2 (SEQ ID NO: 2) (Wherein X is any amino acid residue) [16]
  • a method for identifying a polypeptide having affinity for a target comprising the following steps (a) to (h): (A) A polynucleotide library that exists in the state of an expression vector, comprising a group of expression vectors comprising a polynucleotide encoding a plurality of polypeptides having an ICK scaffold, wherein the polynucleotide encoded by each polynucleotide Providing a polynucleotide library, wherein the amino acid sequence of the peptide has an amino acid sequence that is simultaneously randomized in the following regions (i) to (i
  • each polynucleotide constituting the polynucleotide library is a polynucleotide encoding an amino acid sequence of the following formula [1]; Formula [1] DCLGF (X) 3 CIP (X) 4 CCRPNLVCS (X) 3 KWCKY (X) 2 (SEQ ID NO: 2) (Wherein X is any amino acid residue) [18]
  • a method for identifying a polypeptide having affinity for a target membrane protein comprising the following steps (a) to (h): (A) A polynucleotide encoding a plurality of polypeptides having an ICK scaffold is composed of a group of expression vectors inserted downstream of a secretory signal sequence, and the amino acid sequence of the polypeptide encoded by each polynucleotide is A polynucleotide library having amino acid sequences simultaneously randomized in the following regions (i) to (iv), wherein the expression vector contains a poly
  • a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 3 to 14, having a high affinity for the m2 receptor.
  • An inhibitor of a m2 receptor comprising a polypeptide having any amino acid sequence of SEQ ID NOs: 3 to 14 and having a polypeptide having a high affinity for the m2 receptor as an active ingredient.
  • a polypeptide having a high affinity for a desired target protein can be easily and efficiently screened. Became possible.
  • the polypeptide can be displayed in a correctly folded state in the periplasmic space of Gram-negative bacteria, it can also be applied to a high-throughput screening system targeting membrane proteins. Therefore, according to the present invention, a novel polypeptide that specifically binds to a desired target, particularly a membrane protein, can be created.
  • FIG. 1 is a diagram showing a spider venom-derived ICK peptide scaffold; positions of cysteine residues (C1-C6) and randomized regions (dark gray portions) according to the present invention.
  • FIG. 2 is a diagram showing an alignment of the ICK peptide family.
  • FIG. 3 is a diagram showing homology modeling based on Hainantoxin-4 of the peptide Peak A4-1.
  • FIG. 4 is a diagram showing a process of the molecular evolution technique “iPS & S method”.
  • FIG. 5A is a diagram showing an outline of the construction of plasmid pGRII-Tx
  • FIG. 5B is a photograph showing confirmation of expression of membrane proteins and peptides.
  • FIG. 5A is a diagram showing an outline of the construction of plasmid pGRII-Tx
  • FIG. 5B is a photograph showing confirmation of expression of membrane proteins and peptides.
  • FIG. 5A is a diagram showing an outline of the construction of
  • FIG. 6 shows the structure of plasmid pGRII-Tx.
  • FIG. 7 shows the structure of plasmid pGRII-USER.
  • FIG. 8 is a diagram showing a step of removing nonspecifically adsorbed peptides.
  • FIG. 9 is a diagram showing the results of a binding inhibition assay between muscarinic receptor ligand NMS and m2 receptor using peptides A-1 and A-2.
  • FIG. 10A is a diagram showing a structural outline of peptide A-1 and a non-specific binding peptide.
  • FIG. 10B is a diagram showing detection of an Escherichia coli plasmid adsorbed with a peptide.
  • FIG. 10C is a diagram showing detection of peptides expressed in the periplasm.
  • FIG. 11 is a diagram showing an outline of the peptide target specificity evaluation experiment.
  • FIG. 12 is a diagram showing the localization of plasmids, membrane proteins, and polypeptides in the “iPS & S method”.
  • ICK scaffold refers to a polypeptide consisting of 30-70 amino acid residues found in spider and scorpion venom. Tertiary structure formed by SS bonds of two cysteine residues (Cys-1-Cys-6) with Cys-1 and Cys-4, Cys-2 and Cys-5, and Cys-3 and Cys-6 (Fig. 1).
  • FIG. 2 shows an amino acid sequence alignment of a representative polypeptide having an ICK scaffold. The number at the right end of the figure is the length of the amino acid. The name of the polypeptide shown in this alignment and the accession number of the protein database are shown in the table below.
  • the polypeptide shown in the first line of FIG. 2 is a novel calcium channel blocker poison “A4-1” (Patent Document 3, Non-Patent Document 8) polypeptide isolated from Grammostola spatulata.
  • A4-1 is an ICK-type toxin containing 34 amino acids and has been found to bind to and inhibit calcium channels.
  • Many peptides containing the ICK motif are already known, and each of them is known to have various biological activities such as Na + channel, Ca 2+ channel, K + channel blocker and enzyme inhibition.
  • the gene of this peptide is characterized by the introduction of base substitutions (mutations) that actively undergo amino acid substitution in regions related to interaction with the target molecule through accelerated evolution, which creates the diversity of the target molecule It is the cause.
  • FIG. 2 shows multiple alignments of primary amino acid sequences such as A4-1 and known channel inhibitory toxins such as paultoxin (PaurTx3) and hyantoxin (HnTx4). Homologous sequences are indicated by blocks.
  • PaurTx3 paultoxin
  • HnTx4 hyantoxin
  • the tertiary structure of A4-1 was predicted using the 3D modeling software component of BioPackage (MolSoft LLC, California, USA). ) And was estimated (Fig. 3).
  • the ⁇ structure obtained from the secondary structure information is shown as a ribbon-like arrow.
  • the numbers indicate the start and stop positions of the turn forming the loop, and the first amino acid is represented as 1.
  • FIG. 1 and FIG. 2 three loops are formed by SS bonds between Cys-1 and Cys-4, Cys-2 and Cys-5, and Cys-3 and Cys-6. Regions (regions between Cys-1 and Cys-2, regions between Cys-2 and Cys-3, and regions between Cys-5 and Cys-6) are formed, loop I, loop II, and loop, respectively. It is called III (a ⁇ sheet structure exists mainly between Cys-4 and Cys-5). These three loop regions and the tail region on the C-terminal side from Cys-6 are predicted to be regions that bind to calcium channels.
  • polypeptide library In the polypeptide library of the present invention, the amino acid sequences of loops I, II, and III of the ICK scaffold and a part of the tail region are randomized and have affinity for the target protein. Used to screen for high polypeptides.
  • target proteins not only calcium channels, which are the original targets, but also various ion channels, receptors, enzymes, and other natural and non-natural compounds can be selected as targets.
  • membrane proteins such as membrane receptors involved in signal transduction on the membrane surface, drug transporters, ion channels and cell surface antigens are preferred. In particular, it is preferably used for ligand search of orphan receptors, which are G protein-coupled receptors with unknown ligands.
  • the target can be applied to saccharides, glycoproteins, nucleic acids, and low molecular weight compounds.
  • polypeptide library means a set of a plurality of polypeptides having different amino acid sequences, and a set of polynucleotides encoding each polypeptide in the polypeptide library is referred to as a polynucleotide library. Randomization refers to preparing a set of polypeptides such that two or more amino acid residues are present at any given position in the polypeptide. In a randomized polypeptide, all possible amino acid residues may be present at a given position with similar or different probabilities, and two or more specific types selected at a given position.
  • amino acid residue may be present.
  • 20 types of amino acid residues can exist at a specific position in the sequence with a probability of 5%, but the probability of the presence of each amino acid residue is not limited to 5% and may vary.
  • only a specific position of the amino acid sequence is randomized based on the sequence information obtained in the previous round, and the sequence of the remaining positions is fixed.
  • a library may be created.
  • Targets to be screened using the polypeptide library of the present invention are not limited to the original target protein (calcium channel), but rather to various different targets. Because it is intended to obtain a polypeptide having high affinity for each target, the region actually involved in target recognition and binding is a very narrow region of the tip of the loop and the tail region. As a result of trial and error by the present inventors, it has been found that not only the amino acid sequence but also the surrounding amino acid sequences need to be changed, and all of them need to be changed simultaneously. That is, in the loop I, II, III region and tail region, specific 2 to 4 amino acid residues need to be simultaneously randomized to form a polypeptide library.
  • regions (i) to (iv) As typical examples of the regions (i) to (iv), (i) from the 6th methionine to the 8th lysine in the amino acid sequence of polypeptide A4-1 shown in the first row of FIG. 2, respectively. 3 amino acid residues, (ii) 4 amino acid residues from the 12th aspartic acid to the 15th lysine, (iii) 3 amino acid residues from the 25th arginine to the 27th histidine, and (iv) the 33rd position Amino acid sequences consisting of 2 amino acid residues from valine to 34th phenylalanine.
  • the typical polypeptide library of the present invention is a library composed of a plurality of polypeptide groups derived from the amino acid sequence “A4-1” and comprising an amino acid sequence represented by the following formula [1].
  • a polypeptide having an ICK scaffold for example, the polypeptide shown in FIG. 2, (i) to (iv) in the same manner as the polypeptide library based on A4-1 with reference to the alignment of FIG. Polypeptide libraries with randomized regions can be produced.
  • the polypeptide library of the present invention can be produced manually or automatically using a normal polypeptide synthesis technique. Or 3. It is preferable that a corresponding polynucleotide library described below is prepared and the polynucleotide library is prepared by being expressed in a transformed host.
  • each polypeptide which comprises the polypeptide library of this invention exists in the form matched with the polynucleotide which codes each polypeptide. That is, a polypeptide and a polynucleotide encoding the polypeptide are associated one-to-one by “display technology”.
  • display technology various known methods can be applied. For example, a method of binding each member polypeptide of a library to a polynucleotide encoding each polypeptide via a chemical substance such as puromycin. Can be used (in vitro virus method).
  • Corresponding techniques using such a cell-free translation system include cDNA display, ribosome display, STABLE (non-covalent DNA display), microbead droplet method, and covalent DNA display.
  • display technologies such as phage display, yeast display, and bacterial display
  • each phage or cell contains a pair of each member polypeptide of the library and a polynucleotide encoding each polypeptide. Also good.
  • a polynucleotide library consisting of each group of these polynucleotides is Corresponds to a polypeptide library expressed in the transformed cells.
  • polynucleotide library generally refers to a polynucleotide library consisting of a group of each polynucleotide.
  • a group of cells can also be referred to as a polynucleotide library.
  • the polynucleotide library of the present invention expresses a polypeptide library in which amino acids at specific positions of a polypeptide having an ICK scaffold are simultaneously randomized.
  • the base sequence corresponding to the amino acid sequence to be randomized needs to be randomized.
  • a specific method for producing a polynucleotide a well-known method for introducing a mutation into a gene is used.
  • PCR is performed by combining oligonucleotide primers into which a triplet codon NNS (N is G, C, T, or A. S is G or C) is introduced at the position of the desired amino acid residue into which a random sequence is inserted.
  • the polynucleotide library of the present invention is typically provided as a gene library that encodes a polypeptide library having an ICK scaffold, and is incorporated into an expression vector so that the polypeptide is expressed and associated. ing. For example, if a polypeptide can be displayed on the surface of a phage or a transformed host, the affinity between the polypeptide and the target protein can be observed, and a polynucleotide sequence encoding a high affinity polypeptide can be determined. It can be used for a molecular evolution system.
  • each polypeptide constituting the polypeptide library of the present invention is an expression vector containing a polynucleotide encoding each polypeptide in one Gram-negative bacterial host (for example, E. coli). Since (plasmid) is introduced by transformation, only one type of polypeptide is expressed per host bacterium.
  • the expression vector to be used is incorporated with a DNA encoding the target membrane protein in advance (or simultaneously), and a secretory signal sequence is present upstream of the position where the DNA encoding the ICK polypeptide is introduced (
  • the target membrane protein is expressed on the surface of the bacterial inner membrane in the host bacterium, and the ICK polypeptide is secreted into the periplasmic space (FIG. 5, See FIG.
  • any gram-negative bacterium having a periplasmic space can be applied, but Escherichia coli is preferable.
  • an expression vector used for introducing the polynucleotide library an appropriate known expression vector suitable for a host bacterium, preferably an expression plasmid can be used.
  • a method for preparing a typical plasmid for E. coli expression is described below.
  • a plasmid is designed so that a control sequence including a promoter sequence is added and a stop codon is added to the 3 ′ side so that it is expressed as a fusion protein with an interstitial enzyme (such as DsbC).
  • the DNA encoding the fusion protein of the target membrane protein molecule (muscarinic acetylcholine m2 receptor) and maltose binding protein is designed to be inserted into the same expression plasmid together with the promoter sequence.
  • the cDNA base sequence of the human m2 receptor is obtained from the DNA database Accession No. BC106742.
  • the host microorganism in this case is a gram-negative bacterium having a periplasmic space, and any bacterium can be used as long as its expression vector is available, but typically E. coli is used.
  • a normal gene recombination technique can be applied to the transformation technique and culture conditions.
  • the present invention provides a method for identifying a polypeptide that can bind to a target protein using the polypeptide library of the present invention.
  • the polypeptide library of the present invention is first contacted with a target.
  • Contact between the polypeptide library and the target can be performed by co-expressing the polypeptide library and the target in the microbial cells.
  • the contact condition can be set so that a polypeptide having a high affinity for the target remains bound to the target and a polypeptide having a low affinity does not bind.
  • polypeptides that do not bind to the target are removed from the polypeptide library, and the polypeptides that bind to the target are selected.
  • One method is to immobilize either the polypeptide library or the target on a solid phase.
  • the solid phase any known in the art such as glass or plastic plate, beads, porous particles, membranes, magnetic particles, etc. can be used.
  • a polypeptide library may be produced in a microarray format so that it can be seen what sequence of polypeptide is present in each compartment and contacted with the target. After adding the labeled target to the microarray and incubating, unbound polypeptide is washed away and the label remaining on the microarray is detected. From the position of the compartment to which the target is bound, the sequence of the polypeptide that can bind to the target can be known.
  • each polypeptide constituting the polypeptide library of the present invention corresponds to a polynucleotide encoding each polypeptide.
  • Polypeptide libraries are produced so that they exist in a labeled form. By using such a polypeptide / polynucleotide library, the amino acid sequence of the polypeptide bound to the target can be easily determined by determining the base sequence of the polynucleotide.
  • a polypeptide library in a form associated with a polynucleotide when used, after selecting a group of polypeptides bound to a target, a group of polynucleotides encoding the polypeptide is recovered and amplified.
  • the second polypeptide library can be produced by transcribing, translating, and translating.
  • individual polypeptides have the same randomized and framework regions as the original polypeptide library, but the overall library has increased affinity for the target and the randomized regions
  • the sequence diversity should be reduced. This reduction in diversity is referred to herein as “concentration”.
  • a polypeptide that binds to the target is selected in the same manner as in the first step, and a third polypeptide library is produced from the polynucleotide encoding this polypeptide.
  • a collection of polypeptides with higher affinity for the target can be obtained by multiple rounds of selection and library generation.
  • a technique for preparing a so-called progeny library in which a part of the sequence is further randomized Randomization of a part of the sequence is designed, for example, by introducing one or a plurality of mutations into the amino acid sequence of the randomized region of the selected polypeptide, and designing the amino acid sequence in which the remaining amino acid sequences are fixed.
  • the amino acid sequence of a certain region (for example, loop I) is fixed, and an amino acid sequence in which one or more mutations are introduced into the amino acid sequence of another region (for example, loop II) is selected. It is preferable to carry out by a method such as designing a new amino acid sequence in which amino acid residues appearing in the randomized region of the polypeptide are shuffled to hybridize them. In this way, it is possible to produce a second polypeptide library consisting of a group of a plurality of polypeptides in which some amino acid sequences are the same as those of the polypeptides selected in the previous round and the remaining amino acids are different. it can.
  • This second polypeptide library can then be used to select polypeptides that bind to the target, and so on, for multiple rounds of selection and further randomization of the library. In this way, it is possible to further introduce a mutation into a polypeptide that can bind to a target to promote artificial molecular evolution.
  • a polypeptide having a higher target affinity can be obtained by preferably repeating 2 to 10 rounds, more preferably 3 to 8 rounds.
  • the library of the present invention is a library having a large size such as 10 15 types, it is preferable to apply the molecular evolution method in a round after sufficiently repeating selection and enrichment.
  • a polypeptide library composed of a polynucleotide and a polypeptide corresponding thereto is a vector introduced into E. coli and a polypeptide expressed thereby bound to a target.
  • the target membrane protein is expressed in the inner membrane of E. coli, and the polypeptide is expressed in the periplasmic space (FIG. 12).
  • the polypeptide binds to the target, the spheroplast and the polypeptide form a complex, and the polynucleotide and the polypeptide are indirectly associated with each other.
  • the polypeptide When the polypeptide does not bind to the target, it cannot form a complex with spheroplasts, and thus a library of polynucleotides cannot be constructed even by PCR. Therefore, only the polynucleotide library constituting the polypeptide library becomes the second polynucleotide library, and molecular evolution in vitro can be applied very simply, and a polypeptide with higher target affinity can be efficiently obtained.
  • bonded with the membrane protein currently expressed on the inner membrane surface is selected.
  • a method of selecting with Streptactin beads using a tag (Strep-tag, etc.) added to the N-terminal side is typical.
  • Other tags include T7, histidine, FLAG There are tags, etc., and magnetic beads, sepharose beads, agarose beads, porous beads, plates, or membranes may be used.
  • the plasmid DNA inside the spheroplast cell of the selected “polypeptide-membrane protein-spheroplast complex” fixed to the beads is isolated, and the polynucleotide constituting the polynucleotide library in the plasmid DNA is isolated.
  • the nucleotide sequence is amplified by PCR. Based on the amplified polynucleotide sequence, a similar expression plasmid is prepared, and the cycles of transformation, expression, and selection are repeated multiple times, and the polynucleotide with higher affinity for the target membrane protein is concentrated. To do.
  • polypeptide with higher binding affinity can be concentrated by increasing the selection pressure sequentially.
  • various signal transductions that occur through membrane proteins such as changes in intracellular calcium, cAMP, and arachidonic acid concentrations and potential changes, are measured with each sensitive fluorescent dye, and intracellular and extracellular fluxes due to radioisotopes, etc.
  • SPR surface plasmon resonance
  • QCM quartz oscillation microbalance
  • the target of the present invention various targets such as proteins, saccharides, lipids, low-molecular compounds are targeted. Since a polypeptide to be recognized can be provided, the obtained polypeptide can be used as a detection reagent for detecting a target in a sample, an affinity carrier for protein purification, and the like. For example, it can be used for detection of viral antigen proteins and cancer antigens, identification of glycoprotein sugar chains, and the like. Moreover, the preferable target in this invention is the membrane protein which is concerned with various information transmission on the biological membrane surface.
  • a typical membrane protein, muscarinic acetylcholine receptor m2 subtype (m2) receptor is used as a target and has high affinity for m2 receptor after 6 rounds of screening steps.
  • Ten polypeptides of group AF (PepA-PepF) could be identified.
  • the m2 receptor is a receptor that is localized in the brain, heart, and smooth muscle among muscarinic receptors, and in particular, the m2 subtype is expressed exclusively in the heart, so that the action of the receptor is blocked, or By using it as a lead compound of a drug to be activated, it becomes possible to provide more effective therapeutic agents for neurological diseases, heart diseases (such as antiarrhythmia), and digestive system diseases (such as peptic ulcer).
  • Polypeptide library A polypeptide library prepared based on the ICK scaffold of A4-1 is composed of a group of polypeptides of the formula [1] consisting of the following amino acid sequences.
  • Formula [1] DCLGF (X) 3 CIP (X) 4 CCRPNLVCS (X) 3 KWCKY (X) 2 (SEQ ID NO: 2) (Wherein X is any amino acid residue)
  • the formula [1] has a structure in which three loop regions including amino acids presumed to be involved in calcium channel binding and a tail region are randomized. As shown in FIG. 1, the region predicted to form a loop and the surrounding amino acid residues were randomized. In addition, tail region amino acid residues were also randomized. The amino acids forming the beta sheet are not changed.
  • the muscarinic acetylcholine receptor m2 subtype is expressed in E. coli as a fusion protein with maltose binding protein (MBP) according to the method of Furukawa et al. (H. Furukawa and T. Haga: J. Biochem. (2000) 127, 151-161). Expressed in the intima.
  • MBP maltose binding protein
  • the polypeptide having the ICK motif is a fusion protein with the disulfide isomerase DsbC by placing the signal peptide sequence of the outer membrane protein OmpA of E. coli followed by the purification tag sequence Strep tag II on the amino terminal side.
  • a plasmid was constructed so as to be expressed in the periplasm (FIG.
  • Both the ICK library of the present invention and the m2 receptor are expressed in the periplasmic space in E. coli. Thereafter, the outer membrane of Escherichia coli is removed to form a spheroplast and incubated with Strep ⁇ tactin beads. After several washes, ICK containing the polypeptide bound to the m2 receptor is eluted. The eluted product is amplified by PCR, introduced into a vector after subtraction with a non-specific binding component (Subtraction). This vector is transformed into E. coli and ICK and m2 receptor are co-expressed as described above. In this way, multiple rounds of selection and polypeptide library generation can be performed. In the second and subsequent rounds of selection, decreasing the incubation time between the ICK and the target and increasing the number of washes increase the selection pressure in order and concentrate the polypeptide with higher binding affinity. be able to.
  • m2 receptor binding peptides were screened by the following procedure.
  • the ICK peptide expressed in each periplasm interacts with the m2 receptor.
  • those having strong binding activity were selected.
  • the outer membrane of Escherichia coli is removed to form a spheroplast and the non-binding peptide is removed.
  • Peptides bound to the receptor are selected with Streptactin beads using the Strep-tag added to the N-terminal side. That is, a complex of Streptactin- [Strep-tag-peptide]-[m2 receptor-Escherichia coli (spheroplast)] is selected.
  • sequence information of the peptide that binds to the m2 receptor is contained in the plasmid DNA of Escherichia coli that is caught at the same time, this sequence is amplified by PCR, and the expression and selection cycles after the second round are continued, The sequence gene of interest is concentrated (FIG. 4). As a result of 6 rounds of selection cycles, the following 6 groups and 10 types of peptides were identified.
  • DCLGF RRG CIP VGEL CCRPNLVCS VX 1 X 2 KWCKY X 3 X 4 (SEQ ID NO: 3) (Wherein X 1 is V, L or G, X 2 is A or G, X 3 may be A, P, Y or absent, and X 4 may be N, I, L or absent. Good.) Specifically, it is the following four polypeptides.
  • PepA-1 DCLGF RRG CIP VGEL CCRPNLVCS VVA KWCKY (SEQ ID NO: 4)
  • PepA-2 DCLGF RRG CIP VGEL CCRPNLVCS VVA KWCKY AN (SEQ ID NO: 5)
  • PepA-3 DCLGF RRG CIP VGEL CCRPNLVCS VLG KWCKY PI (SEQ ID NO: 6)
  • PepA-4 DCLGF RRG CIP VGEL CCRPNLVCS VGA KWCKY YL (SEQ ID NO: 7)
  • B Group B
  • PepB DCLGF RWR CIP GINL CCRPNLVCS NSK KWCKY VM (SEQ ID NO: 8)
  • C Group C PepC: DCLGF SMG CIP NQVR CCRPNLVCS VDL KWCKY SH (SEQ ID NO: 9)
  • D Group D PepD: DCLGF RWS CIP WEAS CCRPNLVCS DWK KWCK
  • PepF-1 DCLGF EVV CIP GMLD CCRPNLVCS TVS KWCKY AL (SEQ ID NO: 13)
  • m2 receptor binding peptides those with sufficient expression levels were characterized for m2 receptor binding peptides by the following procedure.
  • mAChR muscarinic acetylcholine receptor
  • PepA-1 DCLGF RRG CIP VGEL CCRPNLVCS VVA KWCKY (SEQ ID NO: 4)
  • PepA-2 DCLGF RRG CIP VGEL CCRPNLVCS VVA KWCKY AN (SEQ ID NO: 5)
  • PepC DCLGF SMG CIP NQVR CCRPNLVCS VDL KWCKY SH (SEQ ID NO: 9)
  • the ICK library of the present invention a plurality of types of polypeptides having high affinity for the target protein can be easily obtained.
  • the obtained polypeptides for example, the m2 receptor high affinity polypeptides obtained in this example, are considered to be useful for the treatment of various diseases as inhibitors of binding to the m2 receptor. .
  • Example 1 Preparation of polypeptide library having molecular backbone of spider venom polypeptide
  • polypeptide of ICK motif A polypeptide library in which the three loop regions and the tail region of Peak A4-1 were randomized was prepared.
  • the cysteine framework of spider venom-like polypeptides is known to be conserved among several short neurotoxins.
  • the amino acid residues in and around the part containing amino acids presumed to be involved in binding to calcium ion channels were randomized.
  • the end of the polypeptide coding region was designed to introduce two stop codons (TGA and TAG) and to add an additional 19 base pair 3'-noncoding region.
  • the above nucleotide sequence using PeakA4-1toxin as a template was synthesized with two oligonucleotides containing 15 bp overlap, and the full length was synthesized by PCR. Oligonucleotides were synthesized by conventional operon biotechnology. Both fragments contain randomized nucleotides corresponding to the loop sequence. The first fragment contains three randomized regions. The second fragment contains one randomized region at the C-terminus, a stop codon and a 3 ′ non-coding region. These 2 fragments contain an overlapping sequence 3 '.
  • N represents A, G, C or T
  • S represents C or G.
  • F1 5'-GACTGTTTAGGATTT (NNS) 3 TGCATCCCC (NNS) 4 TGCTGTCGTCCAAACCTTGTATGCAGT (NNS) 3 AAATGGTGTAAATAT -3 '(SEQ ID NO: 15)
  • F2 5'-ATCTGCAGAATTCGCCCTTCTATCA (SNN) 2 ATATTTACACCATTT -3 '(SEQ ID NO: 16)
  • Underlined sequences indicate overlapping sequences for PCR.
  • PCR was performed in buffer supplied by the manufacturer with 2.5 U / ⁇ l PfuTurboCx Hotstart DNA polymerase (STRATAGENE) and 100 pmol fragment. Three cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 30 seconds were performed.
  • STRATAGENE PfuTurboCx Hotstart DNA polymerase
  • Example 2 Construction of Plasmid Vector Expressing Membrane Protein and Polypeptide Library It was designed so that m2 expressed in E. coli was expressed in the inner membrane and the polypeptide was expressed in the periplasmic space. In addition, in order to prevent a large difference between their expression levels and individual E. coli, the expression was arranged in tandem on one vector.
  • PCR was performed on the T7terminator sequence from rbs of this vector.
  • the PCR product was cloned into the above vector using In-Fusion PCR Cloning System (Clontech) (pGRII-IF).
  • PCR forward 5'-GTCTGCAGGCAAGCTTCTAGAAATAATTTTGTTTAA-3 '(SEQ ID NO: 17)
  • PCR reverse 5'-GGCCAGTGCCAAGCTTTATGCTAGTTATTGCTCAG-3 '(SEQ ID NO: 18)
  • pET40b (+) is a vector containing 468 nt (base sequence number) to 1115 nt of a DsbC sequence involved in transferring a polypeptide expressed in E.
  • This DsbC sequence was prepared by PCR using pET40b (+) as a template (PCR forward 1 and reverse 1) and cleaved with SacII (fragment 1). PCR was performed by PCR forward 2 and reverse 2 using A4-1 as a template, and cleaved with SacII (fragment 2). Thereafter, the PCR1 fragment and the PCR2 fragment were ligated, and PCR was performed using PCR forward 3 and PCR reverse 2 as a template (fragment 3). This PCR produced a Strep • tagII-DsbC-A4-1 fragment, which was cleaved with NheI.
  • the XbaI site (380 nt) on pET27b (+) was changed to TGTAGA using the QuikChange Site-Directed Mutagenesis Kit (STRATAGENE), and the signal sequence of pelB and the signal sequence of OmpA were exchanged.
  • PCR was performed by PCR forward 4 and PCR reverse 3.
  • a fragment containing OmpA was prepared from the T7 promoter (fragment 4) and cleaved with NheI. Fragments 3 and 4 were ligated, PCR was performed with PCR forward 4 and PCR reverse 2 using this as a template, and then cloned into pCR2.1.
  • PCR forward 1 5'-CTCAGTTCGAAAAAGGCGCCGATGACGCGGCAATT-3 '(SEQ ID NO: 19)
  • PCR reverse 1 5'-TTTTGCCGCGGCTTCTTTACCGCTGGT-3 '(SEQ ID NO: 20)
  • PCR forward 2 5'-GAAGCCGCGGCAAAAGACTGTTTAGGA-3 '(SEQ ID NO: 21)
  • PCR reverse 2 5'-GAATTCTTAAAATACATA-3 '(SEQ ID NO: 22)
  • PCR forward 3 5'-CTAGCTAGCTGGAGCCACCCTCAGTTCGAAAAA-3 '(SEQ ID NO: 23)
  • PCR reverse 3 5'-CTGAGGGTGGCTCCAGCTAGCGGCCTGCGCAA-3 '(SEQ ID NO: 23)
  • the USER cassette sequence of the USER friendly DNA engineering method was synthesized into two fragments by Operon Biotechnology Co., Ltd. PCR was performed with PCR forward 1 and PCR reverse to prepare a USER cassette. Using this as a template, PCR was performed by PCR forward 2 and reverse. The PCR product was cleaved with SacII (SacII-USER). This fragment was ligated with pGRII-Tx cleaved with SacII, PCR was performed with the above PCR forward 4 (SEQ ID NO: 25) and the following PCR reverse (SEQ ID NO: 27), and cloned into pCR2.1.
  • the plasmid obtained was excised with PstI and EcoRI and cloned into the same site of pGRII-Tx (see pGRII-USER, see FIG. 7).
  • the sequence of the obtained vector was confirmed with a DNA sequencer.
  • PCR forward 1 5′-CTGCAGGCTGAGGAGACATCTAGAGGATCCT-3 ′ (SEQ ID NO: 26)
  • PCR reverse 5'-GCTGAGGGAAAGTCTAGAGGATCCTCT-3 '(SEQ ID NO: 27)
  • PCR forward 2 5'-TCCCCGCGGCTTTTGCTGAGGAGACATCT-3 '(SEQ ID NO: 28)
  • This vector was cut with XbaI and Nt.BbvCI (New England Biolabs) (m2-USER). This vector was used in the following experiments as a cloning vector using the USER friendly DNA engineering method.
  • PCR forward 5'-GGAGACAUCAGACTGTTTAGGA-3 '(SEQ ID NO: 29)
  • PCR reverse 5'-GGGAAAGUATCTGCAGAATT-3 '(SEQ ID NO: 30)
  • E. coli was collected at 6000 ⁇ g for 10 minutes and washed with 20 mM Tris-HCl (pH 7.5). The periplasmic fraction and spheroplast fraction were prepared using the cells. The spheroplast fraction was suspended in a buffer solution (20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 25% Sucrose, 0.5 mM phenylmethylsulphonyl fluoride) and sonicated.
  • a buffer solution (20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 25% Sucrose, 0.5 mM phenylmethylsulphonyl fluoride
  • the cells were collected by centrifugation at 150000 ⁇ g for 1 hour and suspended in a buffer solution (20 mM Tris-HCl (pH 7.4), 500 mM NaCl, and 1 mM EDTA). Cells were collected by centrifugation at 150,000 xg for 1 hour, and suspended in 20 mM Tris-HCl (pH 7.4) to obtain a membrane fraction.
  • the periplasmic fraction and membrane fraction were subjected to electrophoresis (SDS-PAGE), and expression was confirmed by Western blot. The periplasm fraction was detected for polypeptide expression using Strep • tagII antibody, and the membrane fraction was detected for m2 using MBP antibody.
  • Example 3 Selection of m2 receptor binding polypeptide from spider venom-like polypeptide library (3-1) Expression of spider venom-like polypeptide and binding to m2 receptor m2 and spider-like expressed in E. coli
  • the polypeptide reacts in the periplasmic space within the cell during culture. By using the periplasmic space as a reaction field, each E. coli can react with the polypeptide and m2.
  • the vector containing the randomized polypeptide was transformed into E. coli, and the cells were collected from the overnight culture plate in a liquid medium of LB-ampicillin and grown until OD 600 reached 0.6. Cultures were induced with 0.5 mM IPTG for 24 hours at 20 ° C.
  • the collected cells were suspended in a hypertonic solution (20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 20% Sucrose, 1 mM phenylmethylsulphonyl fluoride) and incubated on ice for 10 minutes. Cells were harvested by centrifugation at 12000 xg for 10 minutes. This was suspended in a hypotonic solution (50 mM Tris-HCl (pH 7.5)) and incubated on ice for 10 minutes. The fraction collected by centrifugation at 12000 ⁇ g for 10 minutes was used as spheroplast.
  • Strep • tagII added to the spider-like polypeptide was bound with Strep tactin (Magnetic Beads, QIAGEN). This was sufficiently washed with a buffer solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 M NaCl, 0.1% TritonX-100) to remove non-specifically bound substances (FIG. 4). .
  • the polypeptide spheroplast complex that was specifically bound by elution with a biotin solution was recovered from Strep tactin. Using this collected solution, PCR (95 ° C.
  • PCR forward 5 '-[Bio] -GAGGGACATCAGACTGTTTAGGA-3' (SEQ ID NO: 31)
  • PCR reverse 5'-TTCGCCCTTCGACTGAGGGAAAGT-3 '(SEQ ID NO: 32)
  • the sequence was commissioned to Bex Corporation to confirm the DNA sequence of the polypeptide contained in the plasmid. Based on randomized sequence similarity, it could be divided into 6 groups. There were no mutations in the non-randomized sequences on the polypeptide sequences of these groups. Ten peptides were identified after 6 rounds of selection cycle. If 2 to 4 amino acid mutations are considered as 1 group, they can be roughly divided into 6 groups.
  • each is referred to as Group A to F, and the polypeptides within the group are referred to as PepA to F. (Note that the region randomized in the original library is underlined)
  • A The group A polypeptide is represented by the following formula [2].
  • DCLGF RRG CIP VGEL CCRPNLVCS VX 1 X 2 KWCKY X 3 X 4 (SEQ ID NO: 3) (Wherein X 1 is V, L or G, X 2 is A or G, X 3 may be A, P, Y or absent, and X 4 may be N, I, L or absent. Good.) Specifically, it is the following four polypeptides.
  • PepA-1 DCLGF RRG CIP VGEL CCRPNLVCS VVA KWCKY (SEQ ID NO: 4)
  • PepA-2 DCLGF RRG CIP VGEL CCRPNLVCS VVA KWCKY AN (SEQ ID NO: 5)
  • PepA-3 DCLGF RRG CIP VGEL CCRPNLVCS VLG KWCKY PI (SEQ ID NO: 6)
  • PepA-4 DCLGF RRG CIP VGEL CCRPNLVCS VGA KWCKY YL (SEQ ID NO: 7)
  • B Group B
  • PepB DCLGF RWR CIP GINL CCRPNLVCS NSK KWCKY VM (SEQ ID NO: 8)
  • C Group C PepC: DCLGF SMG CIP NQVR CCRPNLVCS VDL KWCKY SH (SEQ ID NO: 9)
  • D Group D PepD: DCLGF RWS CIP WEAS CCRPNLVCS DWK KWCK
  • PepF-1 DCLGF EVV CIP GMLD CCRPNLVCS TVS KWCKY AL (SEQ ID NO: 13)
  • polypeptides were prepared from all clones.
  • the polypeptide was prepared as a thioredoxin fusion protein using a partially modified pBAD / TOPOThio fusion expression kit (Invitrogen).
  • the modified part is the point where Strep • tagII was inserted into the N-terminal side of thioredoxin of the pBAD / TOPOThio vector and the recognition sequence of PreScission enzyme (GE Healthcare) was inserted into the C-terminal side of the enterokinase recognition sequence.
  • DNA was amplified from a plasmid encoding an m2-binding polypeptide, cloned into a pBAD / TOPOThio modified vector, and transformed into E. coli.
  • the frame was confirmed by sequencing. Positive clones were cultured in LB-ampicillin medium until OD 600 reached 0.5. Cultures were induced with 0.02% arabinose at 25 ° C. for 3 hours. Cells were collected for 20 minutes at 3000 rpm and sonicated. The lysate was centrifuged, separated into a supernatant (soluble fraction) and a pellet (insoluble fraction) and analyzed by SDS-PAGE.
  • the soluble fraction is expressed as Strep tactin (IBA GmbH) was coupled with an affinity column under non-denaturing conditions, and the fusion protein was cleaved with PreScission enzyme. The polypeptide from which the fusion protein had been removed was recovered, and the protein was quantified by the Lowry method.
  • expression levels of PepA-1, PepA-2 and PepC were sufficiently ensured. Therefore, these 3 polypeptides were subjected to the following binding experiments, and were used with the m2 receptor. Objectively evaluate affinity and specificity.
  • the muscarinic acetylcholine receptor includes m1, m3 and m4 subtypes in addition to m2.
  • binding experiments for subtypes were performed. CRNA of each of these receptors was prepared, and binding inhibition to the subtype was measured by the same method as described above.
  • PepC has a concentration of 438 nM, 6.0% inhibition for m1, 10.4% for m3 and 6.2% inhibition for m4, and 37.5% inhibition of binding for m2 at 365 nM. It was. This indicates that PepC selectively inhibits m2 among muscarinic acetylcholine receptor subtypes.
  • spheroplasts Bacterial cells were collected by centrifugation to prepare spheroplasts. This was recovered with Strep tactin, and PCR was performed with F1 and F2 for 30 cycles (95 ° C. for 30 seconds, 50 ° C. for 30 seconds, 72 ° C. for 30 seconds) as non-specifically adsorbed DNA. In this PCR, biotin was added to the 5 ′ side of the reverse primer. Nonspecifically adsorbed DNA binds to avidin-coated magnetic beads (MAGNOTEX-SA, TAKARA BIO INC.) Strand DNA (Non-speDI) was prepared (FIG. 8). The sequences of F1 and F2 used for PCR are shown below. [Bio] indicates that biotin is attached.
  • PCR forward 5'-GAGGAGACATCAGACTGTTTAGGA-3 '(SEQ ID NO: 31)
  • PCR reverse 5 '-[Bio] -TTCGCCCTTCGACTGAGGGAAAGT-3' (SEQ ID NO: 32)
  • DNA encoding the m2-binding polypeptide obtained by preparing with a vector containing m2 was prepared by PCR.
  • This PCR product was made into a single strand by heat treatment and annealed with Non-speDI at 50 ° C.
  • Non-speDI DNA forms a double strand with Non-speDI DNA.
  • the DNA that did not form a double strand after annealing was recovered with magnetic beads, and the PCR was performed using the recovered DNA as a template (FIG. 8).
  • Colonies were collected from the cultured plates and induced with 0.5 mM IPTG for 24 hours.
  • Spheroplasts were prepared from the collected cells by centrifugation and collected with Strep • tactin. PCR was performed using the collected spheroplasts to see if bands were detected by electrophoresis. On the other hand, a band appeared in the Truncated peptide, while no band was seen in the sample that expressed PepA-1. It was confirmed that they were specifically bound (see FIGS. 10B and 11). At this time, it was confirmed that both peptides were expressed in the periplasm by analyzing the collected periplasmic fraction by electrophoresis and Western blotting (FIG. 10C).
  • PepA-2 DCLGFRRGCIPVGELCCRPNLVCSVVAKWCKYAN 6). PepA-3 DCLGFRRGCIPVGELCCRPNLVCSVLGKWCKYPI 7). PepA-4 DCLGFRRGCIPVGELCCRPNLVCSVGAKWCKYYL 8).
  • PepB (Group B) DCLGFRWRCIPGINLCCRPNLVCSNSKKWCKYVM 9.
  • PepC Group C) DCLGFSMGCIPNQVRCCRPNLVCSVDLKWCKYSH 10.
  • PepD (Group D) DCLGFRWSCIPWEASCCRPNLVCSDWKKWCKYIL 11.
  • PepE (Group E) DCLGFLGWCIPREELCCRPNLVCSNNWKWCKYTI 12
  • PepF-1 DCLGFEVVCIPGMLDCCRPNLVCSTVSKWCKYAL 14
  • PepF-2 DCLGFEVVCIPGMLDCCRPNLVCSTVSKWCKYDY 15.

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Abstract

L'invention porte sur une bibliothèque de polypeptides pour identifier un polypeptide qui a une haute affinité pour une cible désirée. La bibliothèque de polypeptides a un échafaudage, qui a une masse moléculaire relativement faible et porte un petit nombre de résidus cystéine, est applicable à un système de criblage à haut débit ciblant une protéine de membrane, et est caractérisée en ce qu'elle consiste en une pluralité de polypeptides ayant un échafaudage ICK, et que la séquence d'acides aminés de chacun des polypeptides précités comprend une séquence d'acides aminés qui a été simultanément randomisée dans les régions suivantes (i) à (iv) : (i) la séquence d'acides aminés de la quatrième position à partir de l'extrémité C terminale de Cys-1 à la première position à partir de l'extrémité N terminale de Cys-2 dans la région de boucle I entre Cys-1 et Cys-2; (ii) la séquence d'acides aminés de la troisième position à partir de l'extrémité C terminale de Cys-2 à la première position à partir de l'extrémité N terminale de Cys-3 dans la région de boucle II entre Cys-2 et Cys-3; (iii) la séquence d'acides aminés de la deuxième position à partir de l'extrémité C terminale de Cys-5 à la troisième position à partir de l'extrémité N terminale de Cys-6 dans la région de boucle III entre Cys-5 et Cys-6; et (iv) la séquence d'acides aminés de la troisième position à partir de l'extrémité C terminale de Cys-6 à l'extrémité C terminale dans la région de queue de Cys-6 à l'extrémité C terminale.
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Cited By (8)

* Cited by examiner, † Cited by third party
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WO2012125973A3 (fr) * 2011-03-16 2013-01-03 Amgen Inc. Inhibiteurs puissants et sélectifs de nav1.3 et nav1.7
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WO2020039984A1 (fr) * 2018-08-20 2020-02-27 国立大学法人名古屋大学 Banque de composés
WO2022030539A1 (fr) 2020-08-05 2022-02-10 国立研究開発法人産業技術総合研究所 Procédé de criblage d'un polypeptide agissant sur une protéine cible

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JP2014509859A (ja) * 2011-03-16 2014-04-24 アムジエン・インコーポレーテツド １７ｎａｖ１．３およびｎａｖ１．７の強力かつ選択的阻害剤
CN103930437A (zh) * 2011-03-16 2014-07-16 安姆根有限公司 Nav1.3和Nav1.7的强效及选择性抑制剂
US9340590B2 (en) 2011-03-16 2016-05-17 Amgen Inc. Potent and selective inhibitors of NaV1.3 and NaV1.7
JP2017031157A (ja) * 2011-03-16 2017-02-09 アムジエン・インコーポレーテツド １７ｎａｖ１．３およびｎａｖ１．７の強力かつ選択的阻害剤
WO2012125973A3 (fr) * 2011-03-16 2013-01-03 Amgen Inc. Inhibiteurs puissants et sélectifs de nav1.3 et nav1.7
CN103304630A (zh) * 2012-03-07 2013-09-18 中国科学院大连化学物理研究所 东亚钳蝎蝎毒中的gpcr活性多肽及其提取分离和应用
CN103304630B (zh) * 2012-03-07 2014-09-17 中国科学院大连化学物理研究所 东亚钳蝎蝎毒中的gpcr活性多肽及其提取分离和应用
EP2831316A4 (fr) * 2012-03-31 2016-03-30 Abmart Shanghai Co Ltd Bibliothèques de peptides et d'anticorps et leurs utilisations
US9636418B2 (en) 2013-03-12 2017-05-02 Amgen Inc. Potent and selective inhibitors of NAV1.7
US10344060B2 (en) 2013-03-12 2019-07-09 Amgen Inc. Potent and selective inhibitors of Nav1.7
EP3387007A4 (fr) * 2015-12-09 2019-09-11 The University of Queensland Molécules protéiques et procédés d'utilisation
US11124550B2 (en) 2015-12-09 2021-09-21 The University Of Queensland Proteinaceous molecules and methods of use
WO2020039984A1 (fr) * 2018-08-20 2020-02-27 国立大学法人名古屋大学 Banque de composés
JPWO2020039984A1 (ja) * 2018-08-20 2021-08-26 国立大学法人東海国立大学機構 化合物ライブラリ
JP7389488B2 (ja) 2018-08-20 2023-11-30 国立大学法人東海国立大学機構 化合物ライブラリ
WO2022030539A1 (fr) 2020-08-05 2022-02-10 国立研究開発法人産業技術総合研究所 Procédé de criblage d'un polypeptide agissant sur une protéine cible
EP4194562A4 (fr) * 2020-08-05 2024-08-14 National Institute Of Advanced Industrial Science and Technology Procédé de criblage d'un polypeptide agissant sur une protéine cible

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