CN106905435B - Method for preparing binding protein based on protein A mutant - Google Patents

Abstract

The present invention relates to a method for producing binding proteins based on protein a mutants. In addition, the present invention provides a library of nucleotides encoding the heteromultimeric protein A mutants and corresponding protein libraries, methods of screening the mutant libraries for protein A mutants having binding ability to a predetermined target molecule, methods of isolating the nucleotides encoding the protein A mutants, methods of identifying and analyzing the protein A mutants, and methods of producing the protein A mutants are disclosed. The method of the invention has the advantage of producing specific binding proteins with higher binding capacity.

Description

Method for preparing binding protein based on protein A mutant

Technical Field

The present invention relates to methods for making heteromultimeric protein a mutants having novel specific binding capabilities. The present invention provides nucleotide libraries encoding the heteromultimeric protein A mutants and corresponding protein libraries, methods of screening the mutant libraries for protein A mutants having binding ability to a predetermined target molecule, methods of isolating nucleotides encoding the protein A mutants, methods of identifying and analyzing the protein A mutants, and methods of producing the protein A mutants are disclosed. Furthermore, the present invention further provides novel binding proteins based on heteromultimeric protein A that specifically bind to a predetermined target molecule with high affinity.

Background

The antibody can specifically recognize and combine various exogenous substances, such as hapten and antigen of small molecular compounds, pollen, viruses, bacteria and the like. At the two branched ends of the Y-type antibody molecule, a complementary region (CDR) formed by six cyclic polypeptides forms a lock-like structure, and amino acids at the positions form noncovalent bond interaction with epitopes on the surface of the antigen through hydrogen bonds, van der Waals force, charge action and hydrophobic action. In the antibody gene, the DNA encoding the antigen binding site can be randomly combined and mutated, and the amino acids in this hypervariable region can be changed in a very rich manner, each specific change giving the antibody the ability to bind to a predetermined antigen.

With the development of gene recombination technology, protein engineering, recombinant antibody library in vitro affinity screening technology and structural biology, more and more antibody three-dimensional crystal structures are analyzed, and the molecular mechanism of antibody combined with antigen is better understood. The limitations of antibodies, such as large molecular weight, complex structure, etc., have prompted researchers to graft "binding domains" derived from the CDR regions of antibodies onto the surface of a number of very stable, non-antibody proteins (e.g., heteromultimeric protein A used in the present invention). Mutating the surface of these proteins to a high degree of certain adjacent amino acids, appropriate binding regions can be artificially created, thereby conferring on these proteins the ability to bind to a variety of predetermined target molecules.

WO 95/19374 describes the production of binding proteins (Affinifoods) based on monomers of one domain of protein A. Protein a is a protein derived from the cell surface of staphylococcus aureus and consists of 5 highly homologous domains that bind immunoglobulins from many mammalian species, including humans. Since the molecular weight of a single domain of protein a is very small (about 6kDa), the surface energy of the binding domain is relatively small, limiting its binding capacity. According to the prior art information, the affinity K of the binding proteins produced in WO 95/19374_DGenerally in the range of 10^-6-10^-8M range, in most cases 10^-7M, e.g., binding proteins that bind EGFR from the initial screen have an affinity of 130nM to 185nM, binding proteins that bind H-Ras have an affinity of 79nM to 283nM, binding proteins that bind rVIII have an affinity of 100nM to 200 nM.. furthermore, the binding region of a single domain of protein A is composed of amino acids on one side of two juxtaposed α helices, forming a flat binding surface, which also limits the types of epitopes of the target molecule that it can bind.

Disclosure of Invention

The invention aims to provide a method for preparing multimeric protein A mutants with higher binding capacity and capable of universally recognizing more different types of target molecule epitopes. It is a further object of the present invention to provide a method for the universal production of high affinity binding proteins by initial screening based on novel heteromultimeric protein A.

More specifically, the present invention provides a method for preparing heteromultimeric protein A mutants having novel specific binding ability, comprising the following main steps:

providing a library of monomer-modified heteromultimeric mutants of protein a, said library comprising heteromultimeric proteins comprising two or more protein a monomers linked together in a head-to-tail arrangement, wherein at least two of said monomers of said heteromultimeric proteins are encoded by a sequence that is complementary to a sequence encoded by a sequence located in SEQ ID No:1, 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32, 35, said modified monomeric protein having at least 80% amino acid sequence identity to unmodified protein a;

providing a pool of said modified protein a with potential target molecules;

contacting the library of modified proteins with the target molecule;

heteromultimeric protein A mutants were obtained by screening methods.

Definitions for terms of importance in this application

The term "protein A monomer" refers to a domain protein of protein A, including proteins that are identical to SEQ ID NO:1, or have more than 80% amino acid sequence identity.

In the present specification, the term "heteromultimeric protein" is a protein comprising two or more different modified protein a monomers. Thus, a "heteromultimer" of the invention is a fusion protein of at least two different modified protein a monomers having two independent binding regions, preferably a heterodimer or a heterotrimer.

According to the present invention, heteromultimeric binding proteins comprised of at least two different protein A modifying proteins are capable of binding to a predetermined antigen. The protein A modified proteins can be combined together in various ways to form "head-to-tail" fusion proteins, such as gene fusions and the like. Such fusions allow the binding domains of multiple different protein a-modified proteins to combine to form a large, unitary binding domain. This fusion, like the combination of multiple CDR regions of an antibody variable region, together forms an antibody binding site. Correspondingly, the binding regions of a plurality of different protein A modified proteins can bind to different parts of the same specific antigen, and the parts are combined to form an epitope together.

The term "head-to-tail fusion" describes the form of joining a plurality of different protein a-modifying proteins to form a fusion protein. A plurality of different protein A modified proteins are linked together in a (amino-carboxy-terminal) -n (amino-carboxy-terminal) linking orientation to form a heteromultimeric fusion protein, where n depends on the number of protein A modified proteins to be linked. In this head-to-tail linkage, two adjacent protein a-modified proteins may be linked together directly, or via a flexible linker polypeptide, such as a linker polypeptide having the amino acid sequence SGGGG, or other linker polypeptides, such as KPEVIDASELTPAVT, GGGGS, etc. Other linking polypeptides commonly used in gene fusions may also be used herein. In general, heteromultimers can be prepared by joining two or more different protein A-modifying proteins together in an end-to-end fashion to form a fusion protein of two or more protein A-modifying proteins. The binding regions on two or more protein a modifying monomers are linked by a flexible polypeptide, which provides more conformations and the possibility to recognize more types of epitopes of the target molecule.

The term "population" or "library" refers in particular to a collection resulting from a mixture of different modified heteromultimeric mutants of protein A. The mixture of DNA encoding the modified heteromultimeric mutant of protein A is also referred to as a nucleotide population. The terms "population" or "library" are synonymous and are used interchangeably.

The "specific binding region" of the present invention means: between the individual protein a monomers forming the polymer, the mutation sites due to charge, steric structure and hydrophobicity/hydrophilicity of the side chains, the mutated amino acids interact with the surrounding environment, forming a continuous exposed surface region that is capable of specifically binding to the intended target molecule, which may be a solvent, typically water, or other molecule.

In the present invention, "amino acid modification" refers to amino acid substitution, insertion, deletion, or chemical modification; amino acid substitutions are preferred. In particular embodiments, the amino acid modification is achieved by substitution of at least five amino acids at positions 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32, 35 of the protein a monomer. The term "modification" or "mutation" refers to the substitution of an amino acid at a selected position with any other of the 20 amino acids. Both of which are synonymous and can be used interchangeably.

The heteromultimeric protein library consisting of the protein A modified proteins generated by mutation can be screened for any predetermined target molecule by phage display, ribosome display, mRNA display or cell surface display, yeast display or bacteria surface display methods and then in an in vitro directional screening mode, so that heteromultimeric protein A mutants capable of specifically binding the target molecule are enriched, and the binding proteins are obtained. According to the present invention, whether the protein A mutant has quantifiable binding ability to a specific target molecule can be determined by one or more methods, such as enzyme-linked immunosorbent assay (ELISA), plasma surface resonance (SPR), immunofluorescence spectroscopy (IF), flow cytometry (FACS), Isothermal Titration Calorimetry (ITC), and analytical centrifugation.

In the phage display method employed in the present invention, the recombinant protein A mutant is displayed on the M13 filamentous phage while the DNA encoding the mutant is packaged in single stranded form in the phage envelope. Thus, in affinity screening, mutants with certain properties can be selected from the library, the genetic information of which can be amplified by infecting the bacteria and in the next round of affinity screening, respectively. The mutant protein can be displayed on phage expression by fusion to a phage capsid protein, preferably a PIII protein. In addition, the encoded fusion protein may also contain other functional elements, such as affinity tags to facilitate detection or purification by affinity chromatography, and the like.

As target molecules for said binding proteins produced according to the invention, all molecules related to biological and medical activity may be used, including but not limited to antigens and haptens, antigen refers to any substance that can be recognized by the immune system as foreign (non-self), in most immune response situations, an immune response may be elicited.haptens are generally simple, low molecular weight compounds that are not capable of eliciting an immune response, but can become intact antigens upon binding to a carrier protein preferably the target molecules may be biological receptors, preferably G protein coupled receptors (GPCRs, e.g. human EGFR, HER2, HER3, VEGF/R1-4, EpCAM) or their ligands, or domains thereof, tumor necrosis factor α (TNF- α), tumor necrosis factor β (TNF- β), interleukins (e.g. IL-2, IL-6, IL-11, IL-12), growth factors (e.g. nerve growth factor NGF) and their precursors (ProBMP, glycoprotein kinases, integrin (e.g. IIb/IIb receptor), human serum albumin, IL-6, IL-11, IL-12), growth factors (e.g. nerve growth factor NGF) and its precursors (ProBMP), glycoprotein kinases, integrin (e.g. IIb/IIb), human serum albumin, CD 3884, CD 9642, CD 9638, CD 968, CD 9636, CD 968.

In the present invention, based on the heterodimeric protein A, mutants modified at specific sites thereof are obtained, and these mutants obtain target molecule binding ability which they do not have originally. Examples of target molecules for use as heterodimeric protein A in the present invention are Procalcitonin (PCT), Human Serum Albumin (HSA), cadherin 16(CDH 16). The affinity distribution of the protein A heterodimer binding protein obtained by initial screening is 3.6nM-57.6nM, which is significantly higher than that of the binding protein prepared according to WO 95/19374 in the prior art, with an improvement of about 10-fold.

The mutant protein of the heteromultimeric protein A obtained by the invention has binding activity in both intracellular and extracellular environments, and has wide application fields. The protein of the present invention can be used for detecting, quantitatively detecting, separating and extracting the corresponding target molecule, and for diagnosing and treating diseases involving the corresponding target molecule, etc. For example, the target molecules can be detected directly by bioanalytical tests such as ELISA, Western blot, etc., and can be used for the treatment of tumors or infectious diseases, in the food and nutrition industry, in nutritional supplements, in cosmetics, in the medical and non-medical diagnostic and analytical fields.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

The invention has the beneficial effects that: compared with the prior art, the method has the following advantages:

1. protein a heterodimer or multimer based binding proteins can provide a larger area of binding region, which in turn can provide higher binding capacity;

2. two or more protein a monomers are linked by a flexible linker polypeptide, allowing the binding domains distributed over the two protein a monomers to be flexible in spatial conformation, thereby providing the ability to recognize more target epitopes.

Drawings

FIG. 1 is a view showing the binding site region newly formed after modification on the surface of a wild-type protein A monomer;

the surface by amino acid random substitution modification, thereby obtaining new binding sites of wild type protein A monomer crystal structure diagram. Three-dimensional visualization of the secondary structure elements was performed using PyMOL software (DeLano Scientific). As an example positions 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32, 35 of the randomly substituted library are indicated by side chains.

FIG. 2 is a schematic structural diagram of heterodimeric protein A;

the end-to-end connection of two protein a monomers by flexible polypeptide chains forms the basis of the present invention for the formation of heterodimeric protein a with novel binding activity. The oval coil represents the target molecule binding region on the protein surface, formed by a modification of a specific amino acid substitution.

FIG. 3 is a phagemid plasmid pCan-dPA for screening modified proteins based on heteromultimeric protein A with novel binding properties;

pCan-dPA was used to prepare phage libraries, and under the transcriptional control of the Lac operon, sequences encoding the g3p protein signal peptide, protein A dimer, E-tag, and phage capsid protein III were expressed as fusion proteins, allowing the protein A dimer to be displayed on the phage surface.

FIG. 4 shows the results of ELISA experiments to determine the binding affinity of the modified protein PCT-P1G07 of heterodimeric protein A, which was selected by phage display, to its target molecule PCT. Determination of K under equilibrium conditions by non-linear regression_DThe value is obtained.

FIG. 5 shows the results of ELISA experiments to determine the binding affinity of the modified protein PCT-P2A08 of the heterodimeric protein A selected by phage display to its target molecule PCT. Determination of K under equilibrium conditions by non-linear regression_DThe value is obtained.

FIG. 6 shows the results of ELISA experiments to determine the binding affinity of the modified protein PCT-P2A12 of heterodimeric protein A, which was screened by phage display, to its target molecule PCT. Determination of K under equilibrium conditions by non-linear regression_DThe value is obtained.

FIG. 7 shows the results of ELISA experiments to determine the binding affinity of the modified protein HSA-P1A02 of heterodimeric protein A screened by phage display to its target molecule HSA. Determination of K under equilibrium conditions by non-linear regression_DThe value is obtained.

FIG. 8 shows the results of ELISA experiments to determine the binding affinity of the modified protein HSA-P1F11 of the heterodimeric protein A screened by phage display to its target molecule HSA. Determination of K under equilibrium conditions by non-linear regression_DThe value is obtained.

FIG. 9 shows the results of ELISA experiments to determine the binding affinity of the modified protein CDH16-2H03-2 of heterodimeric protein A screened by phage display to its target molecule CDH 16. Determination of K under equilibrium conditions by non-linear regression_DThe value is obtained.

FIG. 10 shows the results of ELISA experiments to determine the binding affinity of the modified protein CDH16-2H07-1 of heterodimeric protein A screened by phage display to its target molecule CDH 16. Determination of K under equilibrium conditions by non-linear regression_DThe value is obtained.

FIG. 11 shows the sequence information and alignment of unmodified dPA, a library of dPA mutants, and each modified heterodimeric protein A binding protein. The grey background shows the amino acid sequence of the non-mutated conserved regions, while the white background shows the amino acid sites selected for modification. X represents the randomized substitution of the amino acid at the position to prepare a modification library.

Detailed Description

Example 1: synthesis of an unmodified protein A dimer (dPA-wt) Gene

This example provides a means to obtain unmodified protein a Dimer Nucleotide (DNA) fragments by means of gene synthesis.

The following genetic engineering procedures were carried out using standard methods known to those skilled in the art. To prepare a DNA sequence (Seq ID No.3) encoding dPA-wt protein (Seq ID No.2) for use as a starting point and template for the preparation of dPA mutant libraries, the following procedure was used: to synthesize the gene fragment, PCR reactions were carried out in a volume of 50. mu.l, and bridge PCR reactions were carried out using primers (0.1. mu.M each concentration) as templates, each in 1. mu.l. The length of the primers is typically 30-59 bases, and the 3 'and 5' ends of each pair of consecutive primers overlap by approximately 15-20 bases. The PCR product was identified by agarose gel electrophoresis and used

The SV Gel and PCR Clean-Up kit (Promega) recovers the target band. The above PCRs were all reacted according to the following procedure, with pre-denaturation at 94 ℃ for 5 minutes, and then 28 cycles. Each cycle was 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 68 ℃ for 1 minute. Finally, the temperature is kept for 10 minutes at 68 ℃.

The expected PCR product was separated into target bands by agarose gel electrophoresis, and the bands were used

The SV Gel and PCRClean-Up kit (Promega) recovered the target band. The recovered DNA fragments were digested separately with sfiI/NotI (New England Biolabs), purified and recovered DNA fragments were inserted into the phagemid vector pCANTAB which was also digested with sfi/NotI to give plasmid pCan-dPA, the structure of which is shown in FIG. 3, which was used for constructiondPA mutant library.

Example 2: preparation of dPA library of modified mutants

The dPA modified mutant library can be obtained by modification by methods established and known in the art. In the present invention, the "library of modified mutants" refers to any modification of the nucleotide or amino acid sequence at a selected site, which results in completely random substitution of nucleotides or amino acids, and the final expression product is a completely random sequence with an even distribution of different amino acids. In the invention, the dPA modified mutant library (Seq ID No.4) used for construction comprises 11 modified sites on the front and back protein A monomers respectively, and the modified sites are positioned at the 9 th, 10 th, 13 th, 14 th, 17 th, 18 th, 24 th, 27 th, 28 th, 32 th and 35 th amino acid positions of the monomers respectively.

According to the present invention, amino acid modifications are preferably obtained by modification at the gene level, preferably by randomized substitution of amino acids, resulting in mutations. Preferably, the modification of protein a is achieved by means of genetic engineering techniques. Preferably, in order to properly introduce the randomized sequence into the dPA gene fragment, a library of mutants can be generated by multiple rounds of bypass PCR by synthesizing and using primers with randomized mutations. For example, a first set of forward and reverse primers can be constructed around the desired restriction enzyme recognition site, and a second set of primers containing randomized mutations, i.e., forward and reverse mutation primers, can be constructed upstream and downstream of the codon to be mutated. With these primers in place, a first intermediate fragment and a second intermediate fragment can be constructed. Both PCR reactions produced linear intermediate fragments. Each fragment contains at least one selected codon mutation, a consensus sequence fused to the upstream and downstream intermediate fragments. The fused complete fragment also contains restriction enzyme cutting sites and protecting bases at the most upstream and most downstream, so that after the fusion, a sticky end is formed, and the sticky end can be connected with a vector fragment through DNA ligase to generate a circular nucleotide product, such as a circular plasmid. Other methods may be utilized in the art and may be used instead.

In particular, the inclusion of mutations is obtainedAfter the intermediate fragments of the sites were mixed in equimolar amounts, the primer pairs (Seq ID No.5 and Seq ID No.6) containing the cleavage sites at the most upstream and most downstream were added to carry out the final PCR amplification reaction, followed by use of

After the DNA fragment was purified and recovered by SV Gel and PCR Clean-Up kit, the fragment was digested with SfiI and NotI in a double-restriction manner, and the DNA fragment of about 400bp was separated and recovered by agarose Gel electrophoresis.

To prepare the phagemid vector, the plasmid pCANTAB was digested with SfiI and NotI, respectively, according to the manufacturer's instructions, and the larger vector fragments were separated by agarose gel electrophoresis. By using

The vector DNA was purified using the SVGEl and PCR Clean-Up kit and finally dissolved in water at 50 fmol/ul. For the ligation reaction, 100. mu.l of 10 XT 4 DNA ligase buffer, 3pmol of the SfiI and NotI double digested dPA library DNA fragment, 9pmol of pCANTAB vector fragment and 8000U T4 DNA ligase (New England Biolabs) were incubated in 1ml reaction volume for 24 hours at 16 ℃. Heating at 65 deg.C for 10 min to inactivate T4 DNA ligase

The SV Gel and PCR Clean-Up kit purified the ligation product, and the DNA was finally eluted with 50ul of sterile water for storage for electrotransformation experiments.

For transformation of the ligation products into E.coli XL1 blue (Stratagene), a Gene Pulser II (Bio-rad) electrotransformer and 2 mm-spaced electrotransformation cups (Bio-rad) were used. The solution obtained from the ligation product was mixed with electrotransformation competent E.coli XL1 Blue and the transformation experiments were carried out according to the manufacturer's instructions. The resulting cell mixture was applied to 10 plates containing 2 XYT/ampicillin solid medium, the plates were incubated overnight at 37 ℃ and the grown colonies after gradient dilution were counted to find that the constructed library contained 3.1X 10¹⁰Individual clones were cloned independently. Scraping the colonies from the plate with 2 XYT medium, addingGlycerol was added to give a final concentration of 20% (v/v), and the mixture was divided into 1ml portions and stored at-80 ℃. 50 single clones are randomly picked, and sequencing analysis shows that 26 clones have functional target DNA sequences, completely different amino acid substitutions with different properties are arranged at the preset mutation sites, and the DNA sequences are different, so that the accuracy of the library is proved to be about 52%.

Example 3: preparation of dPA modified protein phage display library

To produce phages displaying different dPA mutants on their surface, the glycerol bacteria obtained in example 2 were inoculated in 400ml of 2 XYT/ampicillin medium to bring the cell density to OD₆₀₀0.05, the cells were cultured with shaking at 37 ℃ and 200rmp until the cell density reached OD₆₀₀0.6. By 10¹²cfu of helper phage M13KO7 was infected and incubated at 30 ℃ and 50rpm for 30 minutes. After adding 50mg/l kanamycin and shaking culture at 30 ℃ and 200rmp for 30 minutes, the pellet was separated by centrifugation (15 minutes, 1,600 Xg, 4 ℃), resuspended in 400ml of 2 XYT/ampicillin/kanamycin medium and shaking culture at 30 ℃ and 200rmp for 16 hours. Finally the cells were pelleted by centrifugation (20 min, 8,000 Xg, 4 ℃) and discarded, and the supernatant was filtered through a 0.45 μ M format filter, 1/4 vol of 20% (w/v) PEG8000, 2.5M NaCl solution was added and the phage pelleted in ice for 1 h. The pellet was then collected by centrifugation (20 min, 12,000 Xg, 4 ℃ C.), and the phage resuspended in 25ml of precooled PBS (137mM NaCl, 2.7mM KCl, 8mM Na)₂HPO₄，2mM KH₂PO₄) After 30 minutes on ice, centrifugation (5 minutes, 20,000 Xg, 4 ℃ C.). 1/4 vol 20% (w/v) PEG8000, 2.5M NaCl solution were added to the supernatant and the phage were again precipitated in ice bath for 30 minutes. The pellet was centrifuged (30 min, 20,000 Xg, 4 ℃), and the phage pellet resuspended in 4ml of pre-cooled PBS, kept on ice for 30 min and centrifuged (30 min, 17,000 Xg, 4 ℃). The supernatant was mixed with the blocking solution at 1:1, placed on a rotary mixer, incubated at room temperature for 10 minutes, and then used directly for screening.

Example 4: enrichment of phagemids capable of specifically binding to target molecules by in vitro screening

To screen dPA mutants that specifically bind to a predetermined target molecule from phage display libraries containing various dPA mutants, screening and enrichment experiments were performed against predetermined different target molecules (antigen or hapten), respectively, using the phage display library prepared in example 3 above.

The target molecules were diluted with PBS and coated overnight in an immune tube at 4 ℃. After discarding the supernatant the next day, 5ml PBS-B (B stands for blocking solution) was added and incubated at room temperature for 90 minutes, thereby blocking the binding sites on the inner surface of the immune tube that were not occupied by the antigen. Then, 4ml of the prepared phage solution was aspirated, added to an immunization tube, and incubated at room temperature for 2 hours. Unbound phage were removed by five washes with PBST (T for 0.1% Tween-20) and PBS, respectively. Finally, the incubation was carried out at room temperature for 5 minutes with 2ml of glycine-hydrochloric acid, the phages bound to the antigen immobilized on the immunotubes were eluted, and 300. mu.l of a 1M Tris solution were added. Phage solution eluted in each case with a cell density OD of 10ml₆₀₀In a culture of 0.6, E.coli XL1 blue was infected and incubated at 37 ℃ for 30 minutes at 220 rpm. The cell suspensions were plated on plates containing 2 XYT/ampicillin solid medium, the plates were incubated overnight at 37 ℃ and the grown colonies after the gradient dilution were counted. Colonies were scraped from the plate using 2 XYT medium, glycerol was added to a final concentration of 20% (v/v), and 1ml was dispensed into each tube and stored at-20 ℃.

To increase the enrichment of dPA-binding proteins in the screening system, the targeted screening for the target molecule is performed for a total of 3-5 cycles, typically 4 cycles. Starting from the second round of screening, culture and phage rescue were performed in 100ml of medium each time, and more severe washing conditions were used, i.e. 15 and 5 washes with PBST and PBS in the second round of screening, 25 and 5 washes in the third round, and 35 and 5 washes in the fourth round, respectively.

Example 5: identification and isolation of phagemids that specifically bind to different target molecules

After 3-5 rounds of in vitro affinity screening for different predetermined target molecules, 96 clones are randomly picked from the obtained clones, and single phase ELISA method is used for identifying the target moleculesThe situation of binding. The specific operation steps are as follows: each single colony was inoculated into 600. mu.l of 2 XYT/ampicillin medium and cultured at 37 ℃ and 220rpm for 16 hours. The following day, 30. mu.l of each culture was inoculated into 800. mu.l of 2 XYT/ampicillin medium, incubated at 37 ℃ and 220rpm for 2 hours, followed by addition of 10 per well⁹cfu M13KO7 helper phage. After incubation at 37 ℃ for 30 minutes at 200rpm, 50ml/l kanamycin was added, followed by further incubation at 37 ℃ for 16 hours at 220 rpm. The bacteria were finally pelleted by centrifugation for 30 minutes (3,000 Xg, 4 ℃), the supernatant was transferred to a new 96-well plate previously supplemented with an equal volume of 20% (w/v) PEG8000, 2.5M NaCl solution, the phage particles in the supernatant were pelleted by incubation on ice for 1 hour, the pellet was centrifuged again (5,000 Xg, 4 ℃, 30 minutes), and the pellet was resuspended in 200. mu.l PBS for subsequent ELISA experiments.

Each phage of the clone to be analyzed is subjected to ELISA detection simultaneously with respect to its corresponding screening target molecule and negative control protein (usually bovine serum albumin or lysozyme). The corresponding antigen and lysozyme were coated on 96-well plates and blocked using blocking solution to saturate the remaining binding sites on the plastic surface. After three PBST washes, the prepared phage samples were applied to each well of the ELISA plate. After 2 hours incubation at room temperature, washed three times with PBST. To detect bound phage, 100. mu.l of anti-M13 phage antibody-peroxidase conjugate (GEHealthcare) was added to each well at a 1:5000 dilution, and after 1 hour of incubation at room temperature, washed three times with PBST and PBS, respectively. Finally 50. mu.l of TMB substrate was pipetted into the wells and developed for 10 minutes at room temperature, followed by 50. mu.l of 2M H per well₂SO₄The color reaction was terminated. The absorbance was measured at 450nm using a Microplate reader (Bio-Rad). In ELISA experiments, the phage that showed a strong binding signal to the target molecule and no binding signal to the control protein, whose corresponding monoclonal was sequenced with primer Seq ID No. 7. The amino acid sequence of the modified protein thus obtained and further analyzed dPA, the amino acid positions and sequences at which substitutions occurred are exemplarily listed in table 1, wherein 9-35 indicate the amino acid positions at which substitutions were modified in the first protein a monomer of dPA protein; 9 '-35' marks the second eggThe amino acid position in the white a monomer at which the substitution modification occurs.

TABLE 1 examples of amino acid positions and sequences at which substitutions occur

In the example targeting PCT, the amino acid substitutions of the resulting protein A heterodimer binding proteins PCT-P1G07(Seq ID No.8), PCT-P2A08(Seq ID No.9), and PCT-P2A12(Seq ID No.10) are exemplarily set forth in Table 1. In the example targeting HSA, the resulting amino acid substitutions for the protein A heterodimer binding proteins HSA-P1A02(Seq ID No.11) and HSA-P1F11(Seq ID No.12) are exemplarily set forth in Table 1. In the example targeting CDH16, the resulting amino acid substitutions for protein A heterodimer binding proteins CDH16-2H03-2(Seq ID No.13) and CDH16-2H07-1(Seq ID No.14) are exemplarily set forth in Table 1.

Example 6: preparation and purification of dPA-based modified proteins

The dPA modified protein gene with the target molecule specificity binding activity obtained by screening is respectively cloned into an expression vector pET22b (+) after being subjected to PCR amplification through a primer pair (SeqID No.15 and Seq ID No.16), and the plasmid is transferred into escherichia coli BL21(DE 3). in the laboratory level, shake flasks and conventional isopropyl- β -D-thiogalactopyranoside (IPTG) are used for induction expression, so that dPA modified protein can be expressed in large quantity.

To produce dPA modified proteins with novel binding activity, single colonies were picked and inoculated into 5ml of 2 XYT/ampicillin medium and shake-cultured at 37 ℃ and 220rpm for 16 hours. The culture was transferred to 200ml of 2 XYT/ampicillin medium at 1:100 and cultured with shaking at 37 ℃ and 220rpm until the cell density reached about OD₆₀₀0.6 addition of 1mM isopropyl- β -D-thiogalactopyranoside (IPTG) to induce expression of the exogenous gene, shaking culture at 30 ℃ and 220rpm for 6 hours, centrifugation (4,000 Xg, 4 ℃,1 ℃)5 min) cell pellets were collected and resuspended in 15ml NPI-20 solution (50mM MaH)₂PO₄150mM NaCl,20mM imidazole, pH 8.0). Add 200U g/ml lysozyme and 80U Benzonase at room temperature mixing for 30 minutes, then use the ultrasonic cell disruption, in ice water bath every 15 seconds interval 30 seconds, repeat 5 times. The cell debris pellet was removed by centrifugation (15,000 Xg, 4 ℃, 30 minutes) and the soluble target protein was present in the supernatant and was used directly for the next immobilized metal affinity chromatography.

5ml HisTrap HP purification column was pre-equilibrated using 5 column volumes of the NPI-20 buffer, and the cell lysis supernatant was then passed through the purification column. Using 8 column volumes of NPI-50 buffer (50mM MaH)₂PO_4,500mM NaCl,50mM imidazole, pH 8.0) to wash away unbound hetero-proteins, followed by NPI-250 buffer (50mM MaH)₂PO₄150mM NaCl,250mM imidazole, pH 8.0) to elute the bound protein of interest. Protein samples were dialyzed into PBS buffer, and the purified proteins were analyzed for composition and purity by SDS polyacrylamide gel electrophoresis, and the protein concentration was determined by BCA method. The yield of dPA modified protein is typically 10-150mg/l cell culture under shake flask culture conditions.

Example 7 identification of binding Activity of dPA modified proteins with novel binding Properties

The binding activity of the dPA modified proteins to different target molecules was determined by concentration gradient ELISA experiments. For this purpose, 0.1M NaHCO was used₃(pH 9.6) target molecules were diluted in coating solution, coated 200ng per well, 50. mu.l per well, coated overnight at 4 ℃ and blocked with PBST containing blocking solution for 2 hours at room temperature. The plates were then rinsed three times with PBST and removed. Subsequently, 100 μ l of PBST solution containing a range of concentrations of each dPA modified protein was added to each well plate and each sample assayed using parallel three-well assays. After incubation at 37 ℃ for 2 hours, the cells were rinsed three times with PBST, followed by addition of 100. mu.l/well of HRP-labeled murine anti-E-tag antibody (purchased from Kinseri) diluted 1:5000, and reacted at 37 ℃ for 1 hour. For detection, wells were rinsed three times with PBST, then three times with PBS, and finally 15 minutes with TMB addition, with 50. mu.l of 2M H per well₂SO₄Terminating the chromogenic reaction and performing enzyme-linked immunizationThe absorbance was measured at 450nm by a plague detector (Bio-Rad). The resulting absorbance values were evaluated by Sigma Plot software and the binding strength of the antibody was calculated. For this purpose, the measured extinction values in each case are plotted against the corresponding binding protein concentration, and the resulting curves are fitted using the following non-linear regression.

Wherein the association/dissociation balance between the identification of the immobilized target molecule and the dPA modified protein is:

concentration of the modified protein-dPA

Concentration of target molecule/dPA modified protein complex (measured indirectly by absorbance after color development reaction)

a is the total concentration of immobilized target molecules

b is dissociation constant (K)_DValue)

SEQUENCE LISTING

<110> Wuhan Haisha Baide Biotech Co., Ltd

<120> a method for preparing a protein a mutant-based binding protein

<130>2017-03-10

<160>16

<170>PatentIn version 3.3

<210>1

<211>58

<212>PRT

<213> Artificial sequence

<400>1

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210>2

<211>126

<212>PRT

<213> Artificial sequence

<400>2

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Val Asp Asn Lys Phe AsnLys Glu Gln Gln Asn Ala

65 70 75 80

Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn

85 90 95

Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu

100 105 110

Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210>3

<211>378

<212>DNA

<213> Artificial sequence

<400>3

gtagacaaca aattcaacaa agaacaacaa aacgcgttct atgagatctt acatttacct 60

aacttaaacg aagaacaacg aaacgccttc atccaaagtt taaaagatga cccaagccaa 120

agcgctaacc ttttagcaga agctaaaaag ctaaatgatg ctcaggcgcc gaaatctggt 180

ggtggcggta gtggaggtgg tggagtggac aataaattta acaaggagca gcagaacgct 240

ttctacgaaa tcctgcacct gccgaacctg aacgaagaac agcgtaacgc gttcattcag 300

tctctgaagg acgacccgtc gcagtctgcc aacctgctgg ctgaagccaa gaaactgaac 360

gatgcgcagg ccccgaaa 378

<210>4

<211>128

<212>PRT

<213> Artificial sequence

<220>

<221>misc_feature

<222>(9)..(10)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(13)..(14)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(17)..(18)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(24)..(24)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(27)..(28)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(32)..(32)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(35)..(35)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(79)..(80)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(83)..(84)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(87)..(88)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(94)..(94)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(97)..(98)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(102)..(102)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(105)..(105)

<223>Xaa can be any naturally occurring amino acid

<400>4

Val Asp Asn Lys Phe Asn Lys Glu Xaa Xaa Asn Ala Xaa Xaa Glu Ile

1 5 10 15

Xaa Xaa Leu Pro Asn Leu Asn Xaa Glu Gln Xaa Xaa Ala Phe Ile Xaa

20 25 30

Ser Leu Xaa Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Xaa Xaa

65 70 75 80

Asn Ala Xaa Xaa Glu Ile Xaa Xaa Leu Pro Asn Leu Asn Xaa Glu Gln

85 90 95

Xaa Xaa Ala Phe Ile Xaa Ser Leu Xaa Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210>5

<211>35

<212>DNA

<213> Artificial sequence

<400>5

tcatggccca gccggccatg gtagacaaca aattc 35

<210>6

<211>26

<212>DNA

<213> Artificial sequence

<400>6

agatgcggcc gctttcgggg cctgcg 26

<210>7

<211>24

<212>DNA

<213> Artificial sequence

<400>7

caacgtgaaa aaattattat tcgc 24

<210>8

<211>128

<212>PRT

<213> Artificial sequence

<400>8

Val Asp Asn Lys Phe Asn Lys Glu Tyr Leu Asn Ala Phe Phe Glu Ile

1 5 10 15

Ser Ile Leu Pro Asn Leu Asn Arg Glu Gln Lys His Ala Phe Ile Arg

20 25 30

Ser Leu Gly Asp Asp Leu Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Thr Gly

65 70 75 80

Asn Ala Leu Thr Glu Ile Val Leu Leu Pro Asn Leu Asn Ile Glu Gln

85 90 95

Ile Leu Ala Phe Ile Val Ser Leu Gly Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210>9

<211>128

<212>PRT

<213> Artificial sequence

<400>9

Val Asp Asn Lys Phe Asn Lys Glu Leu Tyr Asn Ala Gly Leu Glu Ile

1 5 10 15

Asn Arg Leu Pro Asn Leu Asn Arg Glu Gln Trp Val Ala Phe Ile Val

20 25 30

Ser Leu Leu Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Arg Asp

65 70 75 80

Asn Ala Leu Val Glu Ile Thr Gln Leu Pro Asn Leu Asn Ile Glu Gln

85 90 95

Arg Lys Ala Phe Ile Arg Ser Leu Ile Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210>10

<211>128

<212>PRT

<213> Artificial sequence

<400>10

Val Asp Asn Lys Phe Asn Lys Glu Val Trp Asn Ala Tyr Lys Glu Ile

1 5 10 15

Ser Val Leu Pro Asn Leu Asn Met Glu Gln Thr Ile Ala Phe Ile Val

20 25 30

Ser Leu Val Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Val Leu

65 70 75 80

Asn Ala Leu Leu Glu Ile Thr Arg Leu Pro Asn Leu Asn Ser Glu Gln

85 90 95

Ile Lys Ala Phe Ile Leu Ser Leu Ile Asp Asp Pro Ser Gln Ser Ala

100105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210>11

<211>128

<212>PRT

<213> Artificial sequence

<400>11

Val Asp Asn Lys Phe Asn Lys Glu Pro Trp Asn Ala Trp His Glu Ile

1 5 10 15

Ser Lys Leu Pro Asn Leu Asn Leu Glu Gln Glu Phe Ala Phe Ile Phe

20 25 30

Ser Leu Arg Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Phe Ala

65 70 75 80

Asn Ala Val Ser Glu Ile Lys Gln Leu Pro Asn Leu Asn Gly Glu Gln

85 90 95

Arg Val Ala Phe Ile Val Ser Leu His Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp AlaGln Ala Pro Lys

115 120 125

<210>12

<211>128

<212>PRT

<213> Artificial sequence

<400>12

Val Asp Asn Lys Phe Asn Lys Glu Phe Thr Asn Ala Thr Tyr Glu Ile

1 5 10 15

Gln Asp Leu Pro Asn Leu Asn Met Glu Gln Glu Asp Ala Phe Ile Trp

20 25 30

Ser Leu Arg Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Met Glu

65 70 75 80

Asn Ala Val Tyr Glu Ile Gly Val Leu Pro Asn Leu Asn Val Glu Gln

85 90 95

His Met Ala Phe Ile Thr Ser Leu Arg Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210>13

<211>128

<212>PRT

<213> Artificial sequence

<400>13

Val Asp Asn Lys Phe Asn Lys Glu Tyr Gln Asn Ala Asn Ile Glu Ile

1 5 10 15

Ile Ala Leu Pro Asn Leu Asn Phe Glu Gln Ala Gly Ala Phe Ile Tyr

20 25 30

Ser Leu Thr Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Ala His

65 70 75 80

Asn Ala Tyr Ser Glu Ile Ile Ile Leu Pro Asn Leu Asn His Glu Gln

85 90 95

Leu Ile Ala Phe Ile Thr Ser Leu Asn Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210>14

<211>128

<212>PRT

<213> Artificial sequence

<400>14

Val Asp Asn Lys Phe Asn Lys Glu Gly Trp Asn Ala Phe Ile Glu Ile

1 5 10 15

Lys Ile Leu Pro Asn Leu Asn Gln Glu Gln Pro Leu Ala Phe Ile Met

20 25 30

Ser Leu Tyr Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Gly Gly Gly Gly Ser

50 55 60

Gly Gly Gly Gly Ile Gly Val Asp Asn Lys Phe Asn Lys Glu Gln Tyr

65 70 75 80

Asn Ala Phe Met Glu Ile Val Thr Leu Pro Asn Leu Asn Tyr Glu Gln

85 90 95

Asn Val Ala Phe Ile Arg Ser Leu Met Asp Asp Pro Ser Gln Ser Ala

100 105 110

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

115 120 125

<210>15

<211>43

<212>DNA

<213> Artificial sequence

<400>15

taagaaggag atatacatat ggtagacaac aaattcaaca aag 43

<210>16

<211>31

<212>DNA

<213> Artificial sequence

<400>16

tggtggtggt gctcgagttt cggggcctgc g 31

Claims (8)

1. A method of making a protein a mutant-based binding protein comprising the steps of:

a) providing a library of monomer-modified heteromultimeric mutants of protein a, said library comprising heteromultimeric proteins comprising two or more protein a monomers linked together in a head-to-tail arrangement, wherein at least two of said monomers of said heteromultimeric proteins are encoded by a sequence that is complementary to a sequence encoded by a sequence located in SEQ ID No:1, 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32, 35, said modified monomeric protein having at least 80% amino acid sequence identity to unmodified protein a;

b) providing a pool of said modified protein a with potential target molecules;

c) contacting the library of modified proteins with the target molecule;

d) obtaining heteromultimeric protein A mutants by screening methods, said mutants being represented by K_DRange is 10^-7-10^-12The affinity of M binds to the target molecule.

2. The method of claim 1, wherein the number of amino acid substitutions is between 8 and 11 amino acid substitutions.

3. The method of claim 1, further comprising a nucleotide pool of said heteromultimeric protein a mutant pool.

4. A combination of a library of mutant proteins according to claim 1 and a library of nucleotides according to claim 3, each member of said library of mutant proteins being physically bound to the nucleotide encoding that member by means for genotype-phenotype coupling.

5. The combination of claim 4, wherein the means for genotype-phenotype coupling comprises a phage display system, yeast display, bacterial display, cell surface display, or ribosome display system.

6. The method of any one of claims 1-3, wherein the target molecule is an antigen or a hapten.

7. A protein library obtained by expression of the nucleotide library of claim 3.

8. A prokaryotic or eukaryotic cell or phage library comprising the nucleotide library of claim 3.

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