Staphylococcus protein A variant and application thereof

Abstract

The present invention relates to variants of Staphylococcus Protein A and uses thereof. The variants exhibit better alkali stability compared to wild-type Staphylococcus Protein A.

Description

Technical Field

[0001] The present invention relates to the field of mutant proteins, and more specifically to mutants of Staphylococcus protein A molecules. Compared with the parent molecule, the mutant molecule exhibits better alkaline stability. Background Art

[0002] In biotechnology and pharmaceutical industries, a large number of applications require strict removal of contaminants. These contaminants can be non-eluting molecules adsorbed by the stationary phase or matrix in the chromatographic purification method, such as non-target biomolecules or microorganisms, including, for example, proteins, carbohydrates, lipids, bacteria and viruses. Usually, these contaminants are removed from the matrix after the first elution of the target product, so that the matrix can be regenerated before subsequent applications. Such removal usually includes a method known as cleaning in place (CIP), in which a reagent capable of eluting contaminants from the stationary phase is used, and such reagents commonly used are alkaline solutions flowing through the stationary phase. The most widely used purification and cleaning reagent at present is NaOH, and its concentration can be in the range of 0.1M to 1M according to the degree and nature of the contamination. It is known that NaOH is an effective CIP reagent that can achieve multi-pathway reduction of contaminants such as microorganisms, proteins, lipids and nucleic acids. Another advantage of NaOH is that it can be easily removed without any further treatment. However, this scheme involves exposing the matrix to a pH value exceeding 13. Such alkaline environments are very harsh conditions for many affinity chromatography media containing protein affinity ligands and can result in reduced media performance due to the instability of the ligands towards the high pH values ​​involved.

[0003] Therefore, extensive research has focused on developing protein ligands with increased tolerance to alkaline pH values. For example, Gulich et al. (Susanne Gulich, Martin Linhult, Per- ke Nygren, Mathias Uhlén, Sophia Hober, Journal of Biotechnology 80 (2000), 169-178: Stability towards alkaline conditions can be engineered into a protein ligand) proposed to increase the stability of streptococcal albumin binding structure (ABD) in alkaline environment through protein engineering.

[0004] Affinity chromatography is also used in the process of purifying other molecules such as immunoglobulins for pharmaceutical applications. A particularly interesting class of affinity reagents are proteins that can specifically bind to the invariant portion of an antibody molecule, such interaction being independent of the antigen binding specificity of the antibody. Such reagents are widely used to recover immunoglobulins from different samples such as, but not limited to, serum or plasma preparations or cell cultures obtained from stock species by affinity chromatography. An example of such a protein is Staphylococcal protein A, which contains domains that can bind to the Fc and Fab portions of IgG immunoglobulins from different species.

[0005] Currently, SpA-based affinity chromatography media are probably the most widely used affinity media for the separation of monoclonal antibodies and their fragments from different samples including industrial raw materials from cell cultures.

[0006] Therefore, there is a need in the art to have protein ligands that are able to bind to immunoglobulins, in particular via their Fc-fragment, which are also resistant to one or more cleaning methods utilizing alkaline agents. Summary of the invention

[0007] The present invention relates to a staphylococcal protein A variant. The variant has better alkali resistance than the parent molecule. The affinity chromatography medium prepared by using the staphylococcal protein A variant is more stable in a high pH environment and can maintain a higher ability to bind to immunoglobulins after being washed with a high alkaline solution.

[0008] The present invention uses the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 as the parent molecule, and obtains a variant with improved alkaline stability by inserting one or more amino acids between Q9 and Q10. The inserted amino acids are amino acids other than cysteine, glutamine and asparagine.

[0009] The present invention inserts an amino acid between Q9 and Q10, and optionally glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, methionine, tryptophan, serine, threonine, tyrosine, histidine, aspartic acid, glutamic acid, lysine, or arginine can be inserted.

[0010] The present invention inserts two amino acids between Q9 and Q10, which can be a combination of any two amino acids selected from glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, methionine, tryptophan, serine, threonine, tyrosine, histidine, aspartic acid, glutamic acid, lysine and arginine, or a repeated sequence of any one amino acid.

[0011] The present invention inserts a sequence consisting of three or more amino acids between Q9 and Q10, which can be optionally a sequence consisting of any amino acids selected from glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, methionine, tryptophan, serine, threonine, tyrosine, histidine, aspartic acid, glutamic acid, lysine, and arginine.

[0012] The protein A variant of the present invention has better alkali resistance, and is more stable than the parent molecule in an environment with a pH higher than 10, or even higher than a pH higher than 12-14. In other words, after being maintained in 0.1M sodium hydroxide, 0.5M sodium hydroxide, or even 1M sodium hydroxide for a certain period of time, it can still maintain more immunoglobulin binding activity.

[0013] The domains of the present invention can be further connected in series to form dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers and higher polymers. The polymeric structural units are directly connected or connected by 1-10 amino acids, and the amino acids are selected from alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0014] The present invention can also add a cysteine ​​to the C-terminus of the multimeric protein molecule.

[0015] The immunoglobulin adsorbent was prepared by using the above protein A variant as a ligand. After being soaked in 0.5M sodium hydroxide for 20 hours, the adsorbent prepared by the variant can retain a higher immunoglobulin adsorption ratio.

[0016] In the present invention, SEQ ID NO: 1 is the B domain of Staphylococcus protein A. SEQ ID NO: 2 is the Z domain, wherein the 29th glycine of the B domain of Staphylococcus protein A is replaced with alanine to obtain a variant with improved alkaline stability. SEQ ID NO: 3 is a variant of the Z domain with further improved alkaline stability obtained by mutation of three sites, N3A, N6D and N23T. This variant is disclosed in WO2003 / 080655.

[0017] An example of the present invention is shown in SEQ ID NO: 4. Alanine is inserted between Q9 and Q10 of SEQ ID NO: 3.

[0018] Another embodiment of the present invention is shown in SEQ ID NO: 5. Glycine is inserted between Q9 and Q10 of SEQ ID NO: 3.

[0019] Another embodiment of the present invention is shown in SEQ ID NO: 6. Leucine is inserted between Q9 and Q10 of SEQ ID NO: 3.

[0020] Another embodiment of the present invention is shown in SEQ ID NO: 7. It is a tetramer formed by the amino acid sequence represented by SEQ ID NO: 4, and cysteine ​​is added at the C-terminus.

[0021] The present invention fixes the aforementioned protein A variant on a suitable matrix through covalent bonds to prepare an immunoglobulin adsorbent.

[0022] Specifically, the present invention relates to the following aspects:

[0023] 1. A Staphylococcal protein A variant, characterized in that the Staphylococcal protein A variant uses the polypeptide defined by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 as a parent molecule, and inserts any one amino acid or a sequence consisting of any two or more amino acids except cysteine, asparagine and glutamine between Q9 and Q10.

[0024] 2. The Staphylococcal protein A variant according to claim 1, characterized in that alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine is inserted between Q9 and Q10.

[0025] 3. The Staphylococcal protein A variant according to any one of claims 1-2, characterized in that a sequence consisting of X1X2 is inserted between Q9 and Q10, wherein X1 and X2 are selected from the group consisting of alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine, wherein X1 and X2 may be the same or different.

[0026] 4. The Staphylococcal protein A variant according to any one of claims 1 to 3, characterized in that a sequence consisting of X1X2X3 is inserted between Q9 and Q10, wherein X1, X2 and X3 are selected from the group consisting of alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine, wherein two or three of X1, X2 and X3 are the same or different from each other.

[0027] 5. The Staphylococcal protein A variant according to any one of claims 1 to 4, characterized in that alanine, glycine, leucine, isoleucine, serine, threonine, histidine or valine is inserted between Q9 and Q10.

[0028] 6. The Staphylococcal protein A variant according to any one of claims 1 to 5, characterized in that amino acids represented by two single letters: AA, AG, AL, GG, GL, LL, II, SS, TT, HH, VV, AI, AS, AT, AH, AV, VA, VG, VL, VI, VS, VT or VH are inserted between Q9 and Q10, and preferably the variant is the sequence shown in SEQ ID NO; 18.

[0029] 7. The Staphylococcal protein A variant according to any one of claims 1 to 6, characterized in that three amino acids represented by single letters are inserted between Q9 and Q10: AAA, AGA, ALA, AAG, AAL, ALG, AGL, GGG, GAG, GLG, GGA, GGL, GAL, GLA, LLL, LAL, LGL, LLA, LLG, LGA or LAG, and preferably the variant is the sequence shown in SEQ ID NO: 19.

[0030] 8. The Staphylococcus protein A variant according to any one of claims 1 to 7, characterized in that the amino acid at position 15 of the Staphylococcus protein A variant according to any one of claims 1 to 7 is substituted with lysine.

[0031] 9. The Staphylococcal protein A variant according to any one of claims 1 to 8, characterized in that its amino acid sequence is shown in SEQ ID NO: 4, 5, 6, 12, 13, 14, 15, 16 or 17.

[0032] 10. A multimer characterized by using the Staphylococcus protein A variant according to any one of claims 1 to 9 as a structural unit.

[0033] The Staphylococcus protein A polymer according to claim 10, characterized in that a cysteine ​​is added to the C-terminus of the Staphylococcus protein A polymer.

[0034] 12. The multimer according to claim 10 or 11, characterized in that the multimer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer or octamer, preferably, the structural units are directly connected to each other, or connected by 1-10 amino acids, and the amino acids are selected from alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine, more preferably, the amino acid sequence of the multimer is as shown in SEQ ID NO:7, 8, 9 or 11.

[0035] 13. An adsorbent or affinity chromatography medium, characterized in that it comprises the protein Staphylococcus protein A variant according to any one of claims 1 to 10 or the multimer according to any one of claims 11 to 12.

[0036] 14. Use of the protein Staphylococcus protein A variant according to any one of claims 1 to 10 or the multimer according to any one of claims 11 to 12 in the preparation of an adsorbent or an affinity chromatography medium.

[0037] 15. Use of the protein Staphylococcus protein A variant according to any one of claims 1 to 10 or the multimer according to any one of claims 11 to 12 in affinity purification.

[0038] The staphylococcal protein A variants of the present invention have improved alkali stability compared to the parent molecule. For example, the immunoglobulin adsorption medium prepared with the variants of the present invention can retain a higher immunoglobulin binding capacity after being washed with 0.5M sodium hydroxide. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 .Results of induced expression of different Staphylococcal protein A variant strains.

[0040] Figure 2 .SDS-PAGE results of different Staphylococcal protein A variants after purification.

[0041] Figure 3 . Alkaline stability results of different Staphylococcal Protein A variants. Specific implementation method:

[0042] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0043] Example 1. Acquisition of sequence;

[0044] The protein sequence of SEQ ID NO:3 was modified by adding an A between amino acids Q9 and Q10 to obtain the sequence Q9Q10insA (SEQ ID NO:4).

[0045] The protein sequence of SEQ ID NO:3 was modified by adding a G between amino acids Q9 and Q10 to obtain the sequence Q9Q10insG (SEQ ID NO:5).

[0046] The protein sequence of SEQ ID NO:3 was modified by adding an L between amino acids Q9 and Q10 to obtain the sequence Q9Q10insL (SEQ ID NO:6).

[0047] Four identical sequences of SEQ ID NO: 3 were concatenated and cysteine ​​(C) was added at the carbon terminus to obtain sequence 4proA (SEQ ID NO: 10).

[0048] The protein sequence of SEQ ID NO:3 was modified by adding an A between amino acids Q9 and Q10, and four identical sequences were connected in series, and cysteine ​​(C) was added at the carbon end to obtain the sequence 4proA-3A (SEQ ID NO:7).

[0049] The protein sequence of SEQ ID NO:3 was modified by adding an A between amino acids Q9 and Q10, and 6 identical sequences were connected in series, and cysteine ​​(C) was added at the carbon end to obtain the sequence 6proA-3A (SEQ ID NO:11).

[0050] The protein sequence of SEQ ID NO:3 was modified by adding a G between amino acids Q9 and Q10, and four identical sequences were connected in series, and cysteine ​​(C) was added at the carbon end to obtain the sequence 4proA-3G (SEQ ID NO:8).

[0051] The protein sequence of SEQ ID NO:3 was modified by adding an L between amino acids Q9 and Q10, and four identical sequences were connected in series, and cysteine ​​(C) was added at the carbon end to obtain the sequence 4proA-3L (SEQ ID NO:9).

[0052] The protein sequence of SEQ ID NO: 1 was modified by adding an A between amino acids Q9 and Q10 to obtain the sequence Q9Q10insA (SEQ ID NO: 12).

[0053] The protein sequence of SEQ ID NO: 1 was modified by adding a G between amino acids Q9 and Q10 to obtain the sequence Q9Q10insG (SEQ ID NO: 13).

[0054] The protein sequence of SEQ ID NO: 1 was modified by adding an L between amino acids Q9 and Q10 to obtain the sequence Q9Q10insL (SEQ ID NO: 14).

[0055] The protein sequence of SEQ ID NO: 2 was modified by adding an A between amino acids Q9 and Q10 to obtain the sequence Q9Q10insA (SEQ ID NO: 15).

[0056] The protein sequence of SEQ ID NO: 2 was modified by adding a G between amino acids Q9 and Q10 to obtain the sequence Q9Q10insG (SEQ ID NO: 16).

[0057] The protein sequence of SEQ ID NO:2 was modified by adding an L between amino acids Q9 and Q10 to obtain the sequence Q9Q10insL (SEQ ID NO:17).

[0058] The protein sequence of SEQ ID NO:3 was modified by adding two As between amino acids Q9 and Q10 to obtain the sequence Q9Q10insAA (SEQ ID NO:18).

[0059] The protein sequence of SEQ ID NO:3 was modified by adding three A's between amino acids Q9 and Q10 to obtain the sequence Q9Q10insAAA (SEQ ID NO:19).

[0060] The corresponding base sequences of the above proteins were synthesized at GenScript Biotech Co., Ltd.

[0061] Example 2. Expression of target protein

[0062] Prokaryotic expression of plasmid BL21 bacteria containing the target protein coding sequence:

[0063] BL21 bacteria are used as competent bacteria to induce expression of BL21 bacteria containing target protein plasmids. After induced expression, the protein expression level is analyzed by SDS-PAGE electrophoresis. The protein expression level is detected and expression is stopped if it meets the preset value. Other types of bacteria can also be used as competent bacteria, such as DH5α, TOP10, etc., if the experimental conditions permit.

[0064] BL21 competent cells were transformed according to conventional methods.

[0065] The cultured bacteria are collected and pre-purified to obtain a solution containing the target protein, and the expression level of the target protein in the solution is analyzed by SDS-PAGE electrophoresis.

[0066] Target protein expression Figure 1 shown.

[0067] Example 3.

[0068] Purification: 8L of E. coli fermentation broth expressing Staphylococcus protein A variant was centrifuged at 6000g for 15 minutes to collect the cells. The cells were suspended in 1L of pH 7.0 phosphate buffer and homogenized three times at 800 bar using a high-pressure homogenizer. Centrifuged at 6000g for 15 minutes and the supernatant was collected.

[0069] Adjust the pH to 3.5 with acetic acid, centrifuge again, and collect the supernatant. Purify with SP Sigarorse column. Equilibrate 3 column volumes of 3 column volumes with equilibration buffer (20mM acetic acid-sodium acetate, pH 3.5) on a chromatography column equipped with 200ml SP Sigarorse (Zhejiang Huachun). Load the sample and allow the slurry containing the Staphylococcus protein A variant to pass through the chromatography column at a flow rate of 50ml / min. Continue to rinse the chromatography column with equilibration buffer until the 280nm light absorption returns to the baseline. Rinse the chromatography column with eluent (20mM acetic acid-sodium acetate, 0.1M sodium chloride, pH3.5) for 2 column volumes. Elute with eluent (20mM acetic acid-sodium acetate, 0.35M sodium chloride, pH 3.5). Collect the eluate.

[0070] The eluate of the SP chromatography column was adjusted to pH 8.0 with 1M Tris, and diluted 3 times with water, and further purified with a Q Sigarorse (Zhejiang Huachun) chromatography column. A chromatography column equipped with 200ml of Q Sigarorse was equilibrated with equilibration buffer (20mM Tris, pH 8.0) for 3 column volumes. Load the sample and allow the slurry containing the Staphylococcus protein A variant to pass through the chromatography column at a flow rate of 50ml / min, and continue to rinse the chromatography column with equilibration buffer until the light absorption at 280nm returns to the baseline. Rinse the chromatography column with eluent (20mM Tris, 0.1M sodium chloride, pH 8.0) for 2 column volumes. Elute with eluent (20mM Tris, 0.35M sodium chloride, pH 8.0). Collect the eluate. The purity of the obtained Staphylococcus protein A variant was analyzed by SDS-PAGE, and the results are as shown below. Figure 2 shown.

[0071] Example 4. Preparation of immunoglobulin adsorption medium:

[0072] Activation of the matrix: Take 100 ml of high-flow agarose gel (Zhejiang Huachun internal product), wash with water, and drain. Add 100 ml of 2M sodium hydroxide solution, add 0.2 g of sodium borohydride, add 40 ml of epichlorohydrin, and react at room temperature for 4 hours. Wash with water and drain.

[0073] Treatment of Staphylococcus protein A variants: Take about 50 mg of each variant, add 1% volume of 0.1M EDTA (pH 8.0) and 0.2% volume of 1M DTT (dithiothreitol) solution based on the volume of the protein solution. Mix well and leave at room temperature for 30 minutes. Use a 30ml G25 desalting column to replace the coupling buffer (0.2M phosphate, 1mM EDTA, pH 8.3), and concentrate to a concentration of 30mg / ml using a 5kD ultrafiltration tube.

[0074] Variants were covalently bound to the agarose gel matrix: 5 portions of the agarose gel activated in the previous step were taken, 4 ml each, and 2 ml of the variant solution was added to each portion, and then 1.15 g of anhydrous sodium sulfate was added to each portion, and mixed and reacted at room temperature for 4 hours. Wash with water, then add 4 ml of coupling buffer to each portion, and add 0.2 ml of thioglycerol, respectively, mix well, and react at room temperature overnight. Wash with water and drain.

[0075] Example 5. Alkali resistance test:

[0076] Take 2 portions of 50 μl of the immunoglobulin binding medium made of 7 kinds of protein A (4ProA (SEQ ID NO: 10), 4ProA-3A (SEQ ID NO: 7), 6ProA-3A (SEQ ID NO: 11), 4ProA-3G (SEQ ID NO: 8), 4ProA-3L (SEQ ID NO: 9), 1ProA (SEQID NO: 3), 1ProA-3A (SEQ ID NO: 4)) obtained in the previous step, and store them in a refrigerator at 4°C as a control group. Take 2 portions of 50 μl each, add 400 μl of water and 500 μl of 1M sodium hydroxide solution, mix well, and place in a 27°C water bath for 20 hours. The immunoglobulin adsorption amount is measured at the same time as the control group.

[0077] Determination of immunoglobulin adsorption: 50 μl of medium, add 950 μl of binding buffer (20mM Tris, 0.15M sodium chloride, pH 8.0), mix and centrifuge, discard the supernatant. Add 950 μl of binding buffer and repeat once. Add 350 μl of binding buffer and 600 μl of human immunoglobulin at a concentration of 10 mg / ml. Mix for 15 minutes, remove the supernatant and mark it as adsorption supernatant. Add 950 μl of binding buffer and centrifuge, mix and discard the supernatant. Add 950 μl of binding buffer and repeat once. Add 950 μl of elution buffer (0.1M glycine, 10mM NaCl, pH 3.0), mix for 10 minutes, centrifuge, remove the supernatant and mark it as elution. Measure the absorbance of the adsorption supernatant and elution respectively, and calculate the amount of immunoglobulin adsorbed by the medium.

[0078] The percentage of immunoglobulin adsorption capacity retained after alkali treatment was obtained by dividing the amount of immunoglobulin adsorbed by the treated group by the amount of immunoglobulin adsorbed by the control group. The percentage of immunoglobulin adsorption capacity retained by various Staphylococcus protein A variants after alkali treatment is shown in Figure 3 shown.

[0079] sequence:

[0080] SEQ ID NO: 1 (B domain of Staphylococcus protein A)

[0081] ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0082] SEQ ID NO: 2 (Z sequence: variant of B domain)

[0083] VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0084] SEQ ID NO:3 (Zvar)

[0085] VDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0086] SEQ ID NO:4 (Zvar(Q9Q10insA))

[0087] VDAKFDKEQAQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0088] <h2 style=";text-align:left;direction:ltr">SEQ ID NO:5(Zvar(Q9Q10insG))<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0089] <h2 style=";text-align:left;direction:ltr"> VDAKFDKEQGQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0090] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:6(Zvar(Q9Q10insL))<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0091] <h2 style=";text-align:left;direction:ltr"> VDAKFDKEQLQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0092] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:7(4ProA-3A)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0093] <h2 style=";text-align:left;direction:ltr"> VDAKFDKEQAQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQAQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDAKFDKEQAQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQAQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0094] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:8(4ProA-3G)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0095] <h2 style=";text-align:left;direction:ltr"> VDAKFDKEQGQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQGQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK VDAKFDKEQGQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQGQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0096] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:9(4ProA-3L)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0097] VDAKFDKEQLQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQLQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQLQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQLQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKC

[0098] SEQ ID NO:10(4ProA)

[0099] VDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKC

[0100] SEQ ID NO:11(6ProA-3A)

[0101] VDAKFDKEQAQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKF

[0102] DKEQAQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQA

[0103] QNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQAQNAFY

[0104] EILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQAQNAFYEILHLP

[0105] <h2 style=";text-align:left;direction:ltr">NLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDAKFDKEQAQNAFYEILHLPNLTEE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0106] <h2 style=";text-align:left;direction:ltr"> QRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0107] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:12(B(Q9Q10insA))<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0108] <h2 style=";text-align:left;direction:ltr"> ADNKFNKEQAQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0109] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:13(B(Q9Q10insG))<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0110] <h2 style=";text-align:left;direction:ltr"> ADNKFNKEQGQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0111] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:14(B(Q9Q10insL))<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0112] <h2 style=";text-align:left;direction:ltr"> ADNKFNKEQLQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0113] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:15(Z(Q9Q10insA))<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0114] <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQAQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0115] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:16(Z(Q9Q10insG))<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0116] <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQGQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0117] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:17(Z(Q9Q10insL))<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0118] <h2 style=";text-align:left;direction:ltr"> VDNKFNKEQLQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0119] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:18<h2 style=";text-align:left;direction:ltr">

[0120] VDAKFDKEQAAQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0121] SEQ ID NO:19

[0122] VDAKFDKEQAAAQNAFYEILHLPNLTEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK。

Claims

1. Staphylococcal protein A variant, characterized in that The Staphylococcus protein A variant uses the polypeptide defined by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 as a parent molecule, and inserts any one amino acid or a sequence consisting of any two or more amino acids except cysteine, asparagine and glutamine between Q9 and Q10.

2. The Staphylococcal protein A variant according to claim 1, characterized in that Insert alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine between Q9 and Q10.

3. The Staphylococcal protein A variant according to any one of claims 1 to 2, characterized in that A sequence consisting of X1X2 is inserted between Q9 and Q10, wherein X1 and X2 are selected from the group consisting of alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine, wherein X1 and X2 may be the same or different.

4. The Staphylococcal protein A variant according to any one of claims 1 to 3, characterized in that A sequence consisting of X1X2X3 is inserted between Q9 and Q10, wherein X1, X2 and X3 are selected from the group consisting of alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine, wherein two or three of X1, X2 and X3 are the same or different from each other.

5. The Staphylococcal protein A variant according to any one of claims 1 to 4, characterized in that Insert alanine, glycine, leucine, isoleucine, serine, threonine, histidine or valine between Q9 and Q10.

6. The Staphylococcal protein A variant according to any one of claims 1 to 5, characterized in that Amino acids represented by two single letters: AA, AG, AL, GG, GL, LL, II, SS, TT, HH, VV, AI, AS, AT, AH, AV, VA, VG, VL, VI, VS, VT or VH are inserted between Q9 and Q10, and preferably the variant is the sequence shown in SEQ ID NO:

18.

7. The Staphylococcal protein A variant according to any one of claims 1 to 6, characterized in that Three amino acids represented by single letters are inserted between Q9 and Q10: AAA, AGA, ALA, AAG, AAL, ALG, AGL, GGG, GAG, GLG, GGA, GGL, GAL, GLA, LLL, LAL, LGL, LLA, LLG, LGA or LAG, and preferably the variant is the sequence shown in SEQ ID NO:

19.

8. The Staphylococcal protein A variant according to any one of claims 1 to 7, characterized in that The 15th amino acid of the Staphylococcus protein A variant according to any one of items 1 to 7 is substituted with lysine.

9. The Staphylococcal protein A variant according to any one of claims 1 to 8, characterized in that Its amino acid sequence is shown in SEQ ID NO:4, 5, 6, 12, 13, 14, 15, 16 or 17.

10. A polymer characterized in that The Staphylococcus protein A variant according to any one of claims 1 to 9 is used as a structural unit.

11. The Staphylococcus protein A polymer according to claim 10, characterized in that A cysteine ​​is added to the C-terminus of the Staphylococcal protein A polymer.

12. The polymer according to claim 10 or 11, characterized in that The polymer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer or octamer. Preferably, the structural units are directly connected to each other or connected by 1-10 amino acids, and the amino acids are selected from alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. More preferably, the amino acid sequence of the polymer is as shown in SEQ ID NO:7, 8, 9 or 11.

13. An adsorbent or affinity chromatography medium, characterized in that It comprises the protein Staphylococcus protein A variant according to any one of claims 1-10 or the multimer according to any one of claims 11-12.

14. Use of the protein Staphylococcus protein A variant according to any one of claims 1 to 10 or the multimer according to any one of claims 11 to 12 in the preparation of an adsorbent or an affinity chromatography medium.

15. Use of the protein Staphylococcus protein A variant according to any one of claims 1 to 10 or the multimer according to any one of claims 11 to 12 in affinity purification.

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