CN117417456A - Multivalent Fab binding peptide and preparation and application thereof - Google Patents
Multivalent Fab binding peptide and preparation and application thereof Download PDFInfo
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- CN117417456A CN117417456A CN202310871132.0A CN202310871132A CN117417456A CN 117417456 A CN117417456 A CN 117417456A CN 202310871132 A CN202310871132 A CN 202310871132A CN 117417456 A CN117417456 A CN 117417456A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention discloses a multivalent Fab binding peptide, which is a fusion peptide formed by fusing a Fab binding peptide with a trimerization domain Tri through a linker; the amino acid sequence of the trimerization domain Tri is shown in SEQ ID NO. 4; the amino acid sequence of the linker is shown as SEQ ID NO. 6. The invention fuses the Fab binding peptide with the linker and the trimerization domain tri to form the multivalent Fab binding peptide, which is used for separating and purifying Fab, has high specificity and broad spectrum, high purification efficiency, and the obtained Fab has high purity, which is proved by experiments to be obviously superior to the Protein G-HP and Protein L-HP of similar products in the current market, and has practical popularization and application values.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a multivalent Fab binding peptide, and preparation and application thereof.
Background
Monoclonal antibodies based on immunoglobulin G (IgG) have been widely used for diagnosis and treatment of diseases. To date, more than 100 human or humanized monoclonal antibodies have been approved by the U.S. food and drug administration (Food and Drug Administration, FDA), and more than 800 monoclonal antibodies are undergoing clinical trials. In addition, monoclonal antibodies of the species rat, mouse and rabbit are also widely used in experimental treatment and diagnosis of diseases (Kaplon et al, mAbs,2022, 14:2014296). However, igG Antibodies are often poorly able to cross the blood brain barrier and penetrate solid tumors due to their relatively large molecular weight (150 kDa), and have certain limitations in the treatment of related diseases (Bates et al, antibodies,2019,8,28). Meanwhile, in patients requiring neutralization of pro-inflammatory cytokines or neutralizing toxins with IgG, igG may exacerbate tissue and organ damage by causing immune side reactions through Fc (Fragment crystallizable) (Ji et al, int.j. Mol. Sci.,2021, 22:5770; kurtovic et al, PLoS negl. Trop. Dis.,2019,13:e 0007431). In addition, in solid tumor molecular imaging diagnosis, igG may also bind to immune cells in normal organ tissues through the Fc segment, thereby increasing nonspecific signals, which may easily lead to misdiagnosis (Dijkers et al, clin.Pharm. Ther.,2010,87:586-592; mortimer et al, J.Nucl. Med.,2014, 55:23-29). Obviously, the presence of the Fc fragment will limit the use of intact IgG in the treatment and diagnosis of these diseases.
To overcome the drawbacks of intact IgG antibodies, the preparation of antigen binding fragments (Fragment antigen binding, fab) free of Fc fragments for the diagnosis and treatment of specific diseases is of increasing interest. For example, as potent antagonists of tumor necrosis factor alpha (Tumor necrosis factor alpha, tnfalpha), full-length adalimumab (adalimumab) may be replaced by its long-acting Fab (Zhang et al, eur.j. Pharm.,2021, 904:174152). Treatment of snake venom or drug poisoning with Fab in place of intact antibodies has been used clinically for more than ten years (Kurtovic et al, PLoS Negl. Trop. Dis.,2019,13: e0007431; yusakul et al, J. Biotechnol.,2018,288: 41-47). The diagnosis is more accurate with Fab's shorter time than the acquisition of high definition tumor images with IgG carrying radiocontrast agent (Wong et al, EJNMI Res,2011,1:1; kang et al, eur.J. Nucl. Med. Mol. Imaging,2021, 49:1470-1481). These facts indicate that Fab has wide application prospect in the treatment and diagnosis of diseases.
Fab can be produced by protease digestion of full length IgG or by recombinant gene expression. In either method, pure Fab product cannot be obtained directly, and the Fab needs to be purified by corresponding means. Affinity chromatography is the most concise and most efficient purification means. At present, the separation and purification of Fab mainly depend on the affinity chromatography of Protein G and Protein L. Since Protein G has affinity for both Fab and Fc, purification of Fab from IgG cleavage products is not favored. Furthermore, protein G has low affinity for rat Fab and its utility will be limited. Protein L exhibits high affinity only for IgG kappa light chains, which are found predominantly in human and mouse IgG. Thus, protein L-based affinity chromatography is only suitable for the purification of part of the antibodies (Schimek et al, J. Chromatogr. A,2021, 1638:461702). Many Fc binding peptides have also been screened for lack of Fab binding capacity and therefore cannot be used for Fab purification (Choe et al Materials,2016, 9:994). Because of the need to purify Fab antibodies derived from different species, the existing purification means have failed to meet the market demand, and there is a need to develop novel broad-spectrum Fab binding peptides and use them to build Fab purification platforms.
Disclosure of Invention
To solve the above problems, the present invention provides a multivalent Fab binding peptide which is a Fab binding peptide (G4S) 3 A fusion peptide formed by fusing a linker and a trimerization domain Tri;
or: fab binding peptide (G4S) 3 Fusion peptide formed by connecting the connectors in series;
the amino acid sequence of the trimerization domain Tri is shown in SEQ ID NO. 4;
said (G4S) 3 The amino acid sequence of the linker is shown in SEQ ID NO. 6.
Further, the Fab binding peptide is the Fab binding peptide SpG C3Fab RR has an amino acid sequence shown in SEQ ID NO. 2.
Further, the amino acid sequence of the multivalent Fab binding peptide is shown as SEQ ID NO.8 or SEQ ID NO. 14.
The invention also provides an affinity separation matrix which is prepared by taking the multivalent Fab binding peptide as a ligand and coupling the multivalent Fab binding peptide on a water insoluble carrier.
Further, the water insoluble carrier comprises HiTrap NHS-activated HPHiTrap NHS-activated HP affinity chromatography column.
The invention also provides application of the multivalent Fab binding peptide and the affinity separation matrix in separating and purifying Fab and/or protein containing Fab structural domain.
The present invention also provides a gene fragment encoding the multivalent Fab binding peptide described above.
Further, the nucleotide sequence is shown as SEQ ID NO.7 or SEQ ID NO. 13.
The invention also provides a recombinant vector which is a plasmid containing the gene fragment.
The invention finally provides a recombinant cell which is any one of E.coli, yeast, insect, plant or mammalian cells comprising the aforementioned recombinant vector, preferably E.coli cells. .
The multivalent Fab binding peptide is realized by fusing the Fab binding peptide with a trimerization domain or directly expressing the Fab binding peptide in series. The Fab binding peptide is preferably fused with the trimerization domain tri by using a linker, so that the Fab binding peptide is used for separating and purifying Fab, has high specificity and broad spectrum, has high purification efficiency, and the obtained Fab has high purity, and is proved by experiments to be obviously superior to Protein G-HP and Protein L-HP of similar products in the current market, thereby having practical popularization and application values.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1C Fab 、C Fab -tri、Z Fc And Z Fc Molecular design and protein preparation of tri (A: expression plasmid schematic; B: SDS-PAGE gel electrophoresis, wherein M is the protein molecular weight standard; C, D: SEC analysis (triangle shows molecular weight standard)).
FIG. 2C Fab And C Fab-tri Is a combination of hIgG, hFab, hFc of (C)
FIG. 3Z Fc And Z Fc Binding of a pair hIgG, hFab, hFc of tri
FIG. 4C Fab And C Fab-tri (A) Z is as follows Fc And Z Fc-tri (B) Binding sites of (2)
FIG. 5C Fab-tri And Z Fc-tri Binding to IgG (A) and Fab (B) of different species origin
FIG. 6C Fab The tri-HP column was used for purification of human (a), rat (B), mouse (C) and rabbit (D) Fab: p (P)<0.05，**：P<0.01，***：P<0.001
FIG. 7C Fab -tri-HP column for different substancesPurification of IgG in plasma species
FIG. 8 (C) Fab ) 3 Preparation of proteins and binding and purification of hIgG (A (C) Fab ) 3 Protein SDS-PAGE electrophoresis; b, C, SEC analysis chart; d: SDS-PAGE electrophoresis of hIgG before and after purification from plasma
Detailed Description
The sequence information related to the invention is as follows:
1)C Fab nucleic acid sequence (SEQ ID NO. 1):
5-accacctataaactggttattaatggtaaaaccctgaaaggtgaaaccaccaccaaagcagttgatgcagaaaccgcagcagcagcatttgcacagtatgcacgtcgtaatggtgttgatggtgtttggacctatgatgatgcaaccaaaacctttaccgttaccgaa-3
2)C Fab amino acid sequence (SEQ ID No. 2):
NH2-TTYKLVINGKTLKGETTTKAVDAETAAAAFAQYARRNGVDGVWTYDDATKTFTVTE-COOH
3) Tri nucleic acid sequence (SEQ ID NO. 3):
5-ggtagccgtaatctggttaccgcatttagcaatatggatgatatgctgcagaaagcacatctggttattgaaggtacctttatttatctgcgtgatagcaccgaattttttattcgtgttcgtgatggttggaaaaaactgcagctgggtgaactgattccgattccggca-3
4) Tri amino acid sequence (SEQ ID NO. 4):
NH2-GSRNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPA-COOH
5) (G4S) 3 nucleic acid sequence (SEQ ID NO. 5): 5-ggtggtggtggtagcggtggtggtggtagcggtggtggtggtagc-3
6) (G4S) 3 amino acid sequence: SEQ ID NO.6
NH2-GGGGSGGGGSGGGGS-COOH
7)C Fab -a tri nucleic acid sequence (SEQ ID No. 7):
5-accacctataaactggttattaatggtaaaaccctgaaaggtgaaaccaccaccaaagcagttgatgcagaaaccgcagcagcagcatttgcacagtatgcacgtcgtaatggtgttgatggtgtttggacctatgatgatgcaaccaaaacctttaccgttaccgaaggtggtggtggtagcggtggtggtggtagcggtggtggtggtagcggtagccgtaatctggttaccgcatttagcaatatggatgatatgctgcagaaagcacatctggttattgaaggtacctttatttatctgcgtgatagcaccgaattttttattcgtgttcgtgatggttggaaaaaactgcagctgggtgaactgattccgattccggca-3
8)C Fab -Tri amino acid sequence (SEQ ID No. 8):
NH2-TTYKLVINGKTLKGETTTKAVDAETAAAAFAQYARRNGVDGVWTYDDATKTFTVTEGGGGSGGGGSGGGGSGSRNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPA-COOH
9)Z Fc nucleotide sequence (SEQ ID NO. 9):
5-gtggataacaaattcaacaaagaacaacaaaatgctttctatgaaatcttacatttacctaacttaaacgaagaacaacgcaatgctttcatccaaagcctaaaagatgacccaagccaaagcgctaaccttttagcagaagctaaaaagctaaatgatgctcaagcaccaaaa-3
10)Z Fc amino acid sequence (SEQ ID No. 10):
NH2-VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK-COOH
11)Z Fc the nucleotide sequence of tri (SEQ ID NO. 11)
5-gtggataacaaattcaacaaagaacaacaaaatgctttctatgaaatcttacatttacctaacttaaacgaagaacaacgcaatgctttcatccaaagcctaaaagatgacccaagccaaagcgctaaccttttagcagaagctaaaaagctaaatgatgctcaagcaccaaaaggtggtggtggtagcggtggtggtggtagcggtggtggtggtagcggtagccgtaatctggttaccgcatttagcaatatggatgatatgctgcagaaagcacatctggttattgaaggtacctttatttatctgcgtgatagcaccgaattttttattcgtgttcgtgatggttggaaaaaactgcagctgggtgaactgattccgattccggcatgt-3
12)Z Fc -a tri amino acid sequence (SEQ ID No. 12):
NH2-VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGGGSGGGGSGGGGSGSRNLVTAFSNMDDMLQKAHLVIEGTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPA-COOH
13)(C Fab ) 3 nucleic acid sequence (SEQ ID NO. 13):
5-accacctataaactggttattaatggcaaaaccctgaaaggtgaaaccaccacaaaagcagttgatgcagaaaccgcagcagcagcatttgcacagtatgcacgtcgtaatggtgttgatggtgtttggacctatgatgatgcaaccaaaacctttaccgttaccgaaggtggcggaggcagcggaggcggtggtagcggaggtggcggttcaacaacgtacaaactggtgatcaacggtaaaaccttaaaaggcgaaacaacaaccaaagccgtggatgccgaaacagctgccgctgccttcgcgcaatatgcccgtcgcaacggcgtggatggcgtgtggacatatgacgacgccacaaaaacatttacagtgacggaaggcggtggtggctctggtggtggcggatcaggcggaggtggttcaacgacctataagttagtgattaatggtaagacactgaagggcgaaacgacgacgaaagcggttgacgcagagacagctgcggcagcgttcgcccagtacgctcgccgtaatggcgtagacggcgtatggacgtacgatgacgcgacgaaaaccttcacagttacagaatgt-3
14)(C Fab ) 3 amino acid sequence (SEQ ID No. 14):
NH2-TTYKLVINGKTLKGETTTKAVDAETAAAAFAQYARRNGVDGVWTYDDATKTFTVTEGGGGSGGGGSGGGGSTTYKLVINGKTLKGETTTKAVDAETAAAAFAQYARRNGVDGVWTYDDATKTFTVTEGGGGSGGGGSGGGGSTTYKLVINGKTLKGETTTKAVDAETAAAAFAQYARRNGVDGVWTYDDATKTFTVTE-COOH
EXAMPLE 1 design of multivalent Fab binding peptide and isolation and purification of Fab Effect study
1. Molecular design of Fab binding peptides
There are hundreds of peptides with IgG binding ability, some bind only Fab or Fc, some bind both Fab and Fc, and there is no clear binding site of the polypeptide to IgG. Theoretically, for Fab purification, fab specific binding peptides are the most desirable affinity ligands. However, these peptide monomer molecules are used as such, and Fab may not be purified due to insufficient affinity. Thus, it is necessary to increase its affinity for Fab by altering the molecular form of the Fab binding peptide.
Fab binding peptide (one of the Fab binding peptides SpG described by Unvedorben et al, ploSOne,2015,10:e 0139838) C3Fab RR, abbreviated as C Fab The base acid sequence is shown as SEQ ID NO. 2) and trimerization domain tri (Wirz et al, matrix biol.2011,30:9-15, the amino acid sequence is shown as SEQ ID NO. 4), and a linker [ (G4S) is inserted between the two fragments 3 The amino acid sequence is shown as SEQ ID NO.6]To obtain trivalent Fab binding peptide (C) Fab And-tri, the amino acid sequence is shown in SEQ ID NO. 8).
To determine the Fab specificity and broad spectrum of the multivalent Fab binding peptides designed previously, Z peptides with high affinity for IgG have also been selected (Nilsson et al, protein Eng.,1987,1:107-113, abbreviated as Z Fc (the amino acid sequence is shown as SEQ ID NO. 10) and a trimerization domain tri (the amino acid sequence is shown as SEQ ID NO. 4)) Fusion, inserting linker [ (G4S) between two fragments 3 The amino acid sequence is shown as SEQ ID NO.6]To obtain trivalent Fc binding peptide (Z) Fc Tri, amino acid sequence shown in SEQ ID NO. 12), prepares Z Fc Tri for comparison.
2. Gene cloning, induced expression and separation and purification of trivalent Fab binding peptide
According to C Fab 、C Fab- tri、Z Fc And Z Fc- the amino acid sequence of tri, the corresponding coding gene was designed and BamHI and SalI were added at both ends. The coding gene is synthesized by biotechnology company. The obtained gene was cloned into pQE30 expression plasmid as shown in FIG. 1A. The expression plasmid was then transferred into competent cells of E.coli M15 according to conventional methods of molecular biology and screened with solid LB plates containing ampicillin (100. Mu.g/ml) and kanamycin (30. Mu.g/ml). The obtained positive clone is inoculated in LB liquid medium (ampicillin 100 mug/ml, kanamycin 30 mug/ml) and vibration amplified at 37 ℃ until the expressed bacteria is amplified to logarithmic phase (about A) 600nm Approximately 0.8-1), isopropyl β -d-thiogalactoside (0.05 mM) was added for induction. And (5) centrifugally collecting thalli, homogenizing under high pressure, and breaking bacteria. The target protein was recovered from the supernatant by Ni-NTA affinity chromatography. As shown in FIGS. 1B, C, D, purified C Fab 、C Fab- tri、Z Fc And Z Fc- tri were shown as a single component by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) and size exclusion chromatography (Size exclusion chromatography, SEC), indicating that C was obtained Fab 、C Fab- tri、Z Fc And Z Fc- Pure product of tri.
C Fab And Z Fc The molecular weight shown on the SDS-PAGE gel was consistent with its molecular weight on the SEC gel column, indicating that both were monomeric in solution. However, C Fab- tri and Z Fc- tri has a molecular weight three times that of its molecules on an SEC gel column, indicating C Fab- tri and Z Fc Tri are trimers in solution.
3. Trivalent Fab binding peptide C Fab-tri Identification of binding sites
1. SEC method for detecting binding of Fab binding peptide to IgG, fab and Fc
C is C Fab 、C Fab- tri、Z Fc And Z Fc- tri were mixed with human IgG (hIgG), human Fab (hFab) and human Fc (hFc) in a molecular ratio of 1:1, respectively, and incubated at room temperature for 0.5 hours, and then separated by gel filtration column. The results are shown in FIG. 2A, C Fab The mixture with hIgG shows molecular weight and pure hIgG and C on gel filtration column Fab The same two protein peaks, indicating monomer C Fab No higher molecular weight complex was formed with hIgG, suggesting that the two did not bind Fab The mixture of tri and hIgG showed a single protein peak on the gel filtration column, with a molecular weight greater than IgG, indicating the formation of a larger complex, suggesting C Fab Tri is able to bind to hIgG (FIG. 2B). C in monomeric form Fab C in trimeric form without binding IgG Fab Tri to bind IgG, suggesting that trimerization enhances C Fab Is used for the binding capacity of the polymer. Further analysis found that C Fab The mixture of tri and hFab showed a single protein peak on the gel filtration column (FIG. 2C), but C Fab The mixture of tri and hFc shows a molecular weight per se and pure hFc and C on a gel filtration column Fab Tri the same protein peak (FIG. 2D), description C Fab Tri is able to specifically bind Fab without binding Fc. Analysis of Z by the same method Fc And Z Fc Binding of tri to hIgG, hFab and hFc, results show that Z Fc And Z Fc- tri is able to bind Fc instead of Fab, whose binding to IgG is achieved by Fc (fig. 3).
2. ELISA method for detecting binding of Fab binding peptide to IgG, fab, fc
To test binding of different concentrations of Fab binding peptides to hIgG, hibs and hfcs, the same amount of IgG and fragments thereof were first added to the high affinity elisa plate. After coating overnight at 4 ℃, blocking with 5% bovine serum albumin. C (C) Fab 、C Fab -tri、Z Fc And Z Fc- tri was labeled with Biotin and then added to an ELISA plate and incubated at 37℃for 1h with phosphate buffer (PBS, 10mM Na 2 HPO 4 ,137mM NaCl,2.68mM KCl,and 2mM KH 2 PO 4 pH 7.4) for 3 times, and then using horseradish peroxidase (Horseradish Peroxidase, HRP) marked strepitavidin and its substrate for color development, and measuring A on an enzyme-labeled instrument 450nm 。A 450nm The higher the amount of label binding, the more. The results are shown in FIG. 4A, C Fab Binding to IgG and fragments thereof followed by C Fab The increase in (2) is not significant. But C is Fab- tri showed dose-dependent binding to hIgG and hifab, but not significantly to hFc. C (C) Fab Binding to IgG and fragments thereof, while C Fab- tri showed strong binding to both hIgG and hFab, indicating that trimerization significantly improved C Fab Is used for the binding capacity of the polymer. C (C) Fab- tri binds hIgG and hFab but not hFc, indicating C Fab- the binding site of tri is mainly Fab. Using the same analytical method, Z was found Fc And Z Fc Tri showed strong binding to hFc and hIgG (fig. 4B).
3. ELISA method for detecting binding spectrum of Fab binding peptide
The foregoing experiments demonstrate that C Fab Tri is capable of binding to human Fab (hFab), in order to examine whether the Fab binding peptide has a broad spectrum, C was further analyzed by ELISA Fab -tri binding to mouse Fab (mFab), rat Fab (rFab), rabbit Fab (rbFab) and rhesus Fab (rhFab). The results are shown in FIG. 5A, C Fab Tri shows dose-dependent binding to Fab of the 4 species sources mentioned above, C at the same concentration Fab The amount of binding of tri is significantly greater than that of the control peptide Z Fc -tri. This is described as C Fab Tri has broad spectrum of binding to Fab. C due to the Fab binding ability Fab Tri is therefore also able to bind IgG derived from the above species (fig. 5B).
4. Trivalent Fab binding peptide C Fab Preparation of a tri affinity chromatography column
Aforementioned demonstration C Fab- tri is capable of binding Fab of multiple species origin, possibly as an affinity ligand for Fab purification. To build based on C Fab- the Fab affinity chromatography purification method of tri first requires C Fab- tri is coupled to a solid phase substrate. The solid phase matrix selected by the invention is 1ml HiTrap NHS-a provided by GE companyThe ctified HP pre-packed column (GE Healthcare, uppsala, sweden). With 2M Na 2 CO 3 C is C Fab- the pH of the tri protein solution (containing 10mg protein) was adjusted to about 8.3. After washing off the isopropanol in the pre-packed column with 6ml of pre-chilled 1mM HCl, C was removed Fab- the tri protein solution was injected into the pre-packed column, the effluent protein solution was re-injected, the column was sealed after repeating 3 times, and the coupling was performed at room temperature for 25 minutes. Then, the washing was repeated 3 times in sequence with 6ml of Tris-HCl buffer (0.1M, pH 8.5) containing 0.5M NaCl and 6ml of sodium acetate buffer (0.1M, pH 4.0) containing 0.5M NaCl. Finally, the mixture was washed with PBS and stored in 20% ethanol at 4 ℃. We will couple C Fab The pre-packed column of tri is called C Fab -a tri HP column. For comparison, we used the same method to compare Z Fc Coupling of tri to 1ml HiTrap NHS-activated HP pre-packed column, Z was prepared Fc -a tri HP column.
5. Trivalent Fab binding peptide C Fab Use of a tri affinity chromatography column for the purification of Fab
To evaluate C Fab- Purification efficiency of the tri-HP column, where the C was loaded using high performance liquid chromatography Fab-tri The HP column was used for purification of human, rat, mouse and rabbit Fab, simultaneously with the homemade Z Fc The tri-HP column was compared with a commercial Protein G-HP column and a Protein L-HP column. Fab is generated by cleavage of IgG with papain. The column was equilibrated first with PBS containing 0.3M NaCl, then the same amount of Fab was loaded, the column was washed with PBS and the protein efflux was measured at 280nm with an ultraviolet monitor. After all unbound proteins were washed, the proteins were eluted with glycine-hydrochloric acid buffer at pH 2.5 and collected. Protein concentration was determined using a commercial kit Protein DC and total Protein was calculated and Protein purity was identified using SDS-PAGE gel electrophoresis. The relative yields of the other columns were calculated with 100% Fab yield obtained from Protein G purification. The results are shown in FIG. 6, where the four crude Fab products were subjected to C Fab After the tri-HP column, the next sharp protein peak can be eluted from the column. C under the condition of the same loading amount Fab The elution seal of the tri-HP column is higher than that of the Protein G-HP and Protein L-HP columns. SDS-PAGE identifies eluted proteins consistent with Fab molecular weight, demonstrated by C Fab tri-HP is able to purify Fab.C calculated by taking the yield of Protein G-HP as 100 percent Fab The relative yields of the four Fab isolated and purified by tri-HP were 193.+ -. 10.4% (human), 185.+ -. 13.3% (rat), 517.+ -. 65.9% (mouse) and 301.+ -. 6.2% (rabbit), respectively, 2-5 times that of Protein G. C (C) Fab The efficiency of purification of human Fab by tri-HP was comparable to that of Protein L-HP (193.+ -. 10.4% vs 197.+ -. 18.7%). But C is Fab The yield of purified rat Fab from tri-HP was 3-fold (185.+ -. 13.3% vs. 58.2.+ -. 1.8%) and 2.5-fold (517.+ -. 65.9% vs. 215.2.+ -. 2.5%) and 35-fold (301.+ -. 6.2% vs. 8.1.+ -. 2.6%) higher than that from Protein L-HP. And Z is Fc The purification yields of the tri-HP column for all four Fab's are low. These results demonstrate that C prepared in accordance with the present invention Fab The tri-HP has high efficiency and broad spectrum on the purification of Fab, and is obviously superior to Protein G-HP and Protein L-HP which are similar products on the market.
6. Application of trivalent Fab binding peptide affinity chromatography column in IgG separation and purification
The foregoing demonstrates C Fab Tri can bind IgG via Fab. Also prove C Fab The tri-HP column allows efficient purification of Fab's from humans, rats, mice and rabbits. These results suggest C Fab The tri-HP column is also possible for the purification of IgG from these species. To prove this fact, C is used again Fab The tri-HP column separated and purified IgG from human, rat, mouse and rabbit plasma. Loading and elution methods and the foregoing "trivalent Fab binding peptide C Fab Preparation of a tri affinity chromatography column "is the same as described. The results are shown in FIG. 7, where human, rat, mouse and rabbit plasma passed C Fab After the tri-HP column, a distinct protein peak can be eluted from the column, indicating C Fab The tri-HP can also be used for the purification of IgG from a plurality of species. Except for the use of trimerization domains tri and C Fab Fusion construction of trivalent C Fab Tri, the invention also provides three C' s Fab Through connector (G4S) 3 Direct tandem fusion, another trivalent Fab binding peptide (C Fab ) 3 (the amino acid sequence is shown as SEQ ID NO. 14). According to the above clone preparation method, fusion protein (C Fab ) 3 . As shown in fig. 8a, b, purified (C Fab ) 3 Analysis by SDS-PAGE and SEC shows that the molecular weight of the single component accords with three C Fab The tandem amino acid composition was expected, indicating that this approach also allows for higher purity trivalent Fab binding peptides (C Fab ) 3 . In the same manner as described above, (C Fab ) 3 Incubation with human hIgG at a 1:1 ratio and detection by SEC (C Fab ) 3 Binding to hIgG. The results are shown in FIG. 8C, (C) Fab ) 3 The mixture with hIgG showed a single protein peak on the gel filtration column, and the molecular weight was larger than that of IgG, and (C Fab ) 3 No significant protein peak was found at the elution position of (C) Fab ) 3 Binding to hIgG forms a complex. The results indicate that compared to C Fab Monomers (FIG. 2A), three serially fused (C Fab ) 3 Binding to Fab fragments in IgG can also be significantly enhanced. By means of (C) Fab ) 3 Affinity purification of hIgG was also achieved by coupling the prepared affinity column (FIG. 8D), suggesting tandem fusion of multivalent C Fab Can also be used for affinity purification of IgG antibodies or fragments thereof.
In conclusion, the invention will be described in C Fab C from monovalent to multivalent Fab Either (C) Fab ) 3 After that, the binding capacity of the modified antibody to Fab can be obviously improved. Will be polyvalent C Fab After coupling to solid medium, tri shows higher purification efficiency for Fab and IgG including human, rat, mouse and rabbit, and the yield is significantly higher than that of the same kind of commercial ligands Protein G and Protein L, thus having a larger commercial prospect.
Claims (10)
1. A multivalent Fab binding peptide, characterized by: it is Fab binding peptide pass through (G4S) 3 A fusion peptide formed by fusing a linker and a trimerization domain Tri;
or: fab binding peptide (G4S) 3 Fusion peptide formed by connecting the connectors in series;
the amino acid sequence of the trimerization domain Tri is shown in SEQ ID NO. 4;
said (G4S) 3 The amino acid sequence of the linker is shown in SEQ ID NO. 6.
2. The multivalent Fab binding peptide of claim 1, wherein: the Fab binding peptide is Fab binding peptide SpG C3Fab RR has an amino acid sequence shown in SEQ ID NO. 2.
3. The multivalent Fab binding peptide of claim 2, wherein: the amino acid sequence of the multivalent Fab binding peptide is shown as SEQ ID NO.8 or SEQ ID NO. 14.
4. An affinity separation matrix, characterized in that: a substrate prepared by coupling the multivalent Fab binding peptide according to any one of claims 1 to 4 to a water insoluble carrier.
5. The affinity separation matrix of claim 4, wherein: the water insoluble carrier comprises a HiTrap NHS-activated HP affinity chromatography column.
6. Use of the multivalent Fab binding peptide of claim 1, the affinity separation matrix of claim 4 for the isolation and purification of Fab and/or Fab domain containing proteins.
7. A gene fragment characterized in that: encoding a multivalent Fab binding peptide according to any one of claims 1 to 3.
8. The gene segment of claim 7, wherein: the nucleotide sequence is shown as SEQ ID NO.7 or SEQ ID NO. 13.
9. A recombinant vector, characterized in that: which is a plasmid comprising the gene fragment of claim 8.
10. A recombinant cell, characterized in that: it is any one of E.coli, yeast, insect, plant or mammalian cells, preferably E.coli cells, comprising the recombinant vector of claim 9.
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