CN113512099A

CN113512099A - Staphylococcus protein A, purification preparation method and application thereof

Info

Publication number: CN113512099A
Application number: CN202110378789.4A
Authority: CN
Inventors: 刘龙英; 叶贤龙; 郭志谋; 于伟; 高岩华; 熊京京; 徐思梦; 胡文峰
Original assignee: Ganjiang Traditional Chinese Medicine Innovation Center
Current assignee: Ganjiang Traditional Chinese Medicine Innovation Center
Priority date: 2021-04-08
Filing date: 2021-04-08
Publication date: 2021-10-19
Anticipated expiration: 2041-04-08
Also published as: CN113512099B

Abstract

The invention discloses a staphylococcal protein A, a purification preparation method and application thereof, and mainly relates to a method for constructing recombinant plasmids by connecting a protein ProteinA gene modified by genetic engineering into an expression vector, introducing the recombinant plasmids into prokaryotic host cells for expression, and purifying through Ni affinity and ion exchange chromatography to obtain target protein with the purity of more than 95%. The purification preparation method has the characteristics of simple and convenient purification process, high yield and the like. Meanwhile, the prepared ProteinA has good immunoglobulin IgG binding force, alkali resistance and thermal stability. And the affinity medium prepared by coupling the solid phase carrier and the ProteinA has the advantages of good IgG adsorption effect, high sample loading amount and alkali resistance. The protein ProteinA and the medium prepared by coupling the protein ProteinA play important roles in the field of monoclonal antibody purification and immunodiagnosis research.

Description

Staphylococcus protein A, purification preparation method and application thereof

Technical Field

The invention relates to the technical field of cloning and modifying ProteinA in genetic engineering, in particular to staphylococcal protein A, a purification preparation method and application thereof.

Background

In the middle of the 20 th century, Jensen discovered that a Protein, first named Protein A in 1964, could bind widely to human and rabbit serum antibodies. Protein a is a s.aureus cell wall Protein, 42KDa in molecular weight, that specifically binds to the Fc region of a wide variety of immunoglobulins with little binding to the Fab or light chain. Native ProteinA has 5 IgG binding domains E, D, A, B, C and a non-Fc binding domain of unknown function, 5 different domains that bind the Fc fragment of IgG with strong specific affinity, and at least two IgG molecules per ProteinA molecule. At the same time, IgA, IgM or IgE may also bind to ProteinA. While a large amount of IgG is bound by the protein A with the structure, the non-Fc binding domain of the protein A can be bound with partial hetero-protein, so that the purity of the eluted IgG is not enough, and the purity of a sample of an industrially synthesized antibody can exceed 90 percent after one-step protein A affinity chromatography. However, this purity is often insufficient for monoclonal antibodies. Therefore, the gene of the ProteinA is cloned by selecting a genetic engineering method and is transformed to obtain the recombinant protein A, the specific binding capacity is enhanced, the non-specific binding is reduced, the alkali resistance and other properties are improved, and the method is an important way for preparing an affinity chromatography medium by obtaining the protein A with excellent properties.

With the approval of a batch of monoclonal antibodies on the market, 86% of FDA approvals used protein a in the monoclonal antibody capture step and protein a chromatography was mainly used as the capture step in the purification process, and since 1978, the yield and load of protein a has increased at rates of 4.3% and 5.5% per year, respectively. Just because of the specific adsorption capacity of the protein A, the protein A presents the advantages of magnitude order in the purification of the monoclonal antibody, and the protein A market is huge with the background of the promotion of the industrialization of the monoclonal antibody.

The tolerance of the protein A to NaOH is particularly important, and is an important evaluation index for synthesizing a high-performance protein A affinity chromatography medium, and the main reasons are as follows:

1) for large-scale applications: inactivation of bacteria, as well as removal of pyrogens and preservative management are important issues for large scale purification to ensure that there is no carryover and to avoid cross contamination of the different isolated proteins.

2) CIP (cleaning-in-place) requires: NaOH is the preferred cleaning agent and has various advantages. Can effectively dissolve lipid, protein and nucleic acid; and desorbing the conjugate from the chromatography matrix; can effectively inactivate microorganisms and destroy endotoxin; moreover, the price is low, and the monitoring and the clearing are easy;

in summary, according to the current research background, the invention aims at the fact that the protein A is taken as a key step of the industrial monoclonal antibody purification, and meanwhile, the protein A is widely applied in the aspects of immune screening, immune diagnosis and the like, and the protein A which has better performance and meets the requirements of industrial practical application is obtained through technical modification such as genetic engineering and the like, so that the protein A is urgently needed at present.

Disclosure of Invention

The invention aims to solve the problems that: the method modifies the protein ProteinA expressed in a prokaryotic system through gene cloning, has high yield, and has simple, high-efficiency and low cost in the purification preparation process. The prepared protein ProteinA has the advantages of high alkali resistance, strong thermal stability, good storage property, strong IgG specific adsorption capacity and the like, and the affinity chromatography medium obtained by coupling the protein ProteinA with a solid phase carrier has the advantages of strong protein binding specificity, high affinity, high purification efficiency and the like.

The technical scheme provided by the invention for solving the problems is as follows:

a staphylococcal protein A, wherein the amino acid sequence of the B functional structural domain of the staphylococcal protein A is shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The invention also discloses a purification preparation method of the staphylococcal protein A, which comprises the steps of

Carrying out partial site mutation on the B functional structural domain of the ProteinA protein derived from staphylococcus to form ProteinA-1 and ProteinA-2;

constructing recombinant plasmid, inducing and expressing prokaryotic system, and separating and purifying the expressed recombinant ProteinA protein through Ni affinity chromatography and ion exchange chromatography to obtain the product with purity higher than 95%.

Preferably, the mutation site of the protein A-1 comprises: A176V, N178H, N181D, Q184A, E190K, N198T, N203G, G204A, N218A, A221S, D228E and A229S are mutated at the same 12, and the amino acid sequence is shown as SEQ ID NO: 1 is shown.

Preferably, the mutation site of the protein A-2 comprises: A176V, Q184H, E190K, N198S, N203G, G204A, D211H, N218A, A221S, D228E and A229S have 11 mutations, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.

Preferably, the expression vector of the recombinant plasmid is pET30a, the host bacterium is Escherichia coli host bacterium BL21(DE3), and when the OD600 is cultured to be 0.3-0.8, IPTG with the final concentration of 0.1-1.0 mM is added, and the induction expression is carried out for 3-8 h at the temperature of 18-37 ℃.

Preferably, the binding buffer of the Ni affinity chromatography process contains 20mM imidazole, the elution buffer contains 40mM imidazole, and the elution buffer contains 500mM imidazole; and (3) further purifying by anion exchange chromatography, wherein the pH of a mobile phase is 7.0-9.0, and eluting the target protein by adopting a salt ion concentration increasing mode.

The invention also discloses an application of the staphylococcal protein A prepared by the purifying preparation method of the staphylococcal protein A in a ligand of an affinity medium.

The invention also discloses application of the staphylococcal protein A prepared by the purifying preparation method of the staphylococcal protein A in antibody purification and immunodiagnosis research.

Compared with the prior art, the invention has the advantages that:

the protein ProteinA constructed and obtained by the invention has simple purification and preparation process and high yield. The affinity chromatography medium obtained by coupling the affinity chromatography medium with a solid phase carrier has the advantages of strong protein binding specificity, high affinity and high purification efficiency. The staphylococcus protein A purification preparation method and the application thereof have important significance for the antibody purification market with great demand on the current market and related immunodiagnosis and screening.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a schematic diagram of a recombinant plasmid constructed from the protein ProteinA of the present invention;

FIG. 2 is a SDS-PAGE analysis chart of protein A-1 and protein A-2 proteins purified and prepared in example 2 of the present invention;

FIG. 3 is a SDS-PAGE analysis chart of the alkali resistance test of Protein A-1 and Protein A-2 proteins purified and prepared in example 2 of the present invention;

FIG. 4 is a graph showing the analysis results of the IgG antibody specifically adsorbed to the affinity medium obtained by coupling protein A-1 and protein A-2 to a solid phase in example 2 of the present invention;

FIG. 5 is a graph showing the results of alkali resistance test of ProteinA-2 solid phase affinity media in example 2 of the present invention.

Detailed Description

The embodiments of the present invention will be described in detail with reference to the accompanying drawings and examples, so that how to implement the technical means for solving the technical problems and achieving the technical effects of the present invention can be fully understood and implemented.

Example 1

A method for purifying and preparing staphylococcal protein A, which comprises

In this example, the mutation site of the ProteinA-1 comprises: A176V, N178H, N181D, Q184A, E190K, N198T, N203G, G204A, N218A, A221S, D228E and A229S are mutated at the same 12, and the amino acid sequence is shown as SEQ ID NO: 1 is shown.

In this example, the mutation site of the ProteinA-2 comprises: A176V, Q184H, E190K, N198S, N203G, G204A, D211H, N218A, A221S, D228E and A229S have 11 mutations, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.

In the embodiment, the expression vector of the recombinant plasmid is pET30a, the host bacterium is Escherichia coli host bacterium BL21(DE3), and IPTG with the final concentration of 0.1-1.0 mM is added when OD600 is cultured to be 0.3-0.8, and the induction expression is carried out for 3-8 h at the temperature of 18-37 ℃.

In this example, the binding buffer for Ni affinity chromatography contains 20mM imidazole, the elution buffer contains 40mM imidazole, and the elution buffer contains 500mM imidazole; and (3) further purifying by anion exchange chromatography, wherein the pH of a mobile phase is 7.0-9.0, and eluting the target protein by adopting a salt ion concentration increasing mode.

Example 2

1.1protein A protein gene synthesis modification design:

wherein the mutation site comprises: ProteinA-1 protein mutated at 12 positions in total of A176V, N178H, N181D, Q184A, E190K, N198T, N203G, G204A, N218A, A221S, D228E and A229S, and ProteinA-2 protein mutated at 11 positions in total of A176V, Q184H, E190K, N198S, N203G, G204A, D211H, N218A, A221S, D228E and A229S, in order to enhance the stability and alkali resistance of target protein.

1.2 construction of protein recombinant plasmid:

the invention relates to a method for purifying and preparing protein ProteinA and application thereof, which is characterized in that according to the gene synthesis modification design in 1.1, a Kinsley company is entrusted to synthesize a corresponding gene, and a pET30a (Kinsley) vector is connected to construct a recombinant plasmid. To better illustrate the superior performance of the ProteinA of the present invention, wild-type ProteinA (WT) was synthesized by the same consignor as a negative control.

The protein A gene sequences (protein A-1 and protein A-2) and the wild protein A gene sequence (WT) were synthesized by DNA synthesizer. The synthesized 3 protein gene fragments were linked to a prokaryotic expression vector pET30a to construct a recombinant plasmid, as shown in FIG. 1.

1.3 optimization of culture expression conditions of positive bacteria:

transferring the obtained recombinant plasmid into escherichia coli BL21(DE3, CD601-02, all-type gold), selecting a single clone to try a small amount of induced expression attempts, and determining a proper protein expression condition;

(1) taking out a 50 mu L of escherichia coli competent cell BL21(DE3, CD601-02, all-type gold), placing on ice, adding 2 mu L of plasmid (100 ng/mu L) after thawing, lightly flicking and uniformly mixing with fingers, placing on ice for 30min, thermally shocking at 42 ℃ for 90s, placing on ice for 3min, adding 350 mu L of nonresistant LB culture medium, incubating at 37 ℃ and 180rpm for 45 min-1 h, centrifuging at 3000rpm for 2min, discarding 250 mu L of supernatant, lightly flicking and uniformly mixing, coating on an LB solid culture medium containing Kan resistance (100 mu g/mL, Solarbio, K8020-10g), and inverting in a 37 ℃ incubator overnight culture to obtain a single colony.

(2) 2-4 transformed single colonies were picked and inoculated into 5mL of LB liquid medium containing Kan-resistant medium (100. mu.g/mL), and cultured overnight at 37 ℃ with a shaker at 180 rpm. After the preservation of the strain, 1% of the inoculum size was inoculated into 20mL of LB medium containing Kan resistance (100. mu.g/mL), and cultured at 37 ℃ and 180rpm to OD₆₀₀0.4-0.6, and respectively carrying out low-temperature induction at 18 ℃ and induction at 37 ℃.

(3) Low-temperature induction at 18 ℃: mu.L of IPTG (1mol/L, Solarbio, I8070-5g) inducer was added and induced at 18 ℃ and 70rpm for 12 h.

Induction at 37 ℃: mu.L of IPTG (1mol/L) inducer was added and induced at 37 ℃ for 3h at 180 rpm.

(4) And collecting the induced thallus for ultrasonic disruption, collecting samples in each step for SDS-PAGE electrophoresis detection, and analyzing the expression condition of the fusion protein induced at different temperatures. The induction result was detected by 12% SDS-PAGE electrophoresis.

1.4 protein expression and purification:

the thalli is subjected to amplification culture under the optimal expression condition, a large amount of target protein expression is induced, and the expressed thalli is subjected to the unit operation preparation processes of ultrasonic disruption, nickel column affinity chromatography, anion exchange chromatography, ultrafiltration concentration and the like, so that the target protein with the electrophoretic purity of more than 95% is finally obtained.

The specific experimental method is as follows:

(1) protein expression: after the recombinant plasmid is transformed into Escherichia coli BL21, a single colony coated with a plate is placed in 20mL LB culture medium containing 100. mu.g/mL Kan, cultured at 37 ℃ and 180rpm overnight, and inoculated in 500mL LB culture medium containing 100. mu.g/mL Kan according to the proportion of 1%, cultured at 37 ℃ and 180rpm for 3h, and then IPTG is added to the final concentration of 0.25mmol/L for induction (125. mu.L of 1M IPTG), and cultured at 37 ℃ and 100rpm for 3 h. The cells were collected by centrifugation at 4700rpm for 20min at 4 ℃.

(2) And thallus crushing treatment: adding lysozyme (Solarbio, L8120-50g final concentration 1mg/mL) into 1 tube (30mL) of the bacterial liquid in the step (1), performing ultrasonic disruption on ice (30% power, 2s for 2s, 3min), and performing disruption for 2 times. 80. mu.L of the disrupted solution was taken, 20. mu.L of 5 Xloading buffer was added, and denaturation was carried out at 95 ℃ for 10min, followed by 12% SDS-PAGE analysis.

(3) And nickel column affinity chromatography: after disruption, the cells were centrifuged at 12000rpm at 4 ℃ for 5min and then passed through a 0.45 μm filter, 80 μ L of the supernatant was collected, 20 μ L of 5 Xloading buffer was added, and the mixture was denatured at 95 ℃ for 10min and subjected to 12% SDS-PAGE. The remaining supernatant was pumped, an AKTApure protein purifier, 40Ni column (16 × 70mM), 15mL column, mobile phase Binding buffer (20mM Na3PO4, 20mM imidazole, pH 7.4); washing buffer (20mM Na3PO4, 40mM imidazole, 500mM NaCl pH 7.4); elution buffer (20mM Na3PO4, 500mM imidazole, pH 7.4); flow rate: 5 mL/min; detection wavelength: 280 nm; balancing: 3 column volumes Binding buffer; leaching: 3 column volumes Binding buffer; elution 1: 3 column volumes Washing buffer; and (3) elution 2: 3 column volumes 60% Elution buffer; fractions are collected and purified by a first nickel column, target protein is effectively captured, and samples are collected for 12% SDS-PAGE electrophoresis detection. Since anion exchange chromatography is needed in the next step to remove more impurities and obtain the pure target protein, the eluted sample is diluted by about 10 times by using sterile pure water.

(4) And anion exchange chromatography: column 40Q (16 x 100mm, 20mL) was selected using an AKTApure protein purifier; mobile phase a was BufferA (20mM Tris, pH 8.0) and phase B was Buffer B (20mM Tris +1 mnaacl, pH 8.0); the detection wavelength is 280 nm; the flow rate is set to 4 mL/min; the sample is obtained after Buffer displacement in the previous step; the experimental method is as follows: a) balancing: using 2 times of column volume to balance the mobile phase A until the response value is stable, adjusting zero, and keeping the base lines parallel; b) loading: sample loading by a pump; c) leaching: washing the mobile phase A with 2 times of the column volume, and washing an ultraviolet curve to a stable baseline; d) and (3) elution: 3% of mobile phase B isocratic elution, 12% of mobile phase B isocratic elution, 20% of mobile phase B isocratic elution, 30% of mobile phase B isocratic elution and 100% of mobile phase B isocratic elution, collecting ultraviolet peaks, and carrying out 12% SDS-PAGE electrophoresis detection.

(5) And ultrafiltration concentration: and combining fractions according to the electrophoresis result, and selecting a 10KDa interception Millipore ultrafiltration centrifugal tube for ultrafiltration concentration to obtain the fusion protein with qualified purity. The 12% SDS-PAGE electrophoresis detection is shown in FIG. 2.

The obtained protein ProteinA has high yield, can obtain the target protein with high purity and high concentration by a simple two-step purification process, and has simple and efficient purification process and low cost. As shown in the gel image of electrophoresis, the target band is obvious, the appearance of the miscellaneous band is difficult to observe by naked eyes, and the purity of the target protein is more than 95 percent.

1.5 alkaline resistance test on purified ProteinA protein:

in order to research the alkali resistance of protein ProteinA-1 and ProteinA-2, NaOH solution with final concentration of 0.3M is respectively added into protein samples, 0h of control group is set, ultrapure water with corresponding volume is added into protein, after fully mixing, standing and incubating for 12h, sampling and carrying out 12% SDS-PAGE electrophoresis detection, an electrophoresis chart is shown in figure 3, after 0.3M NaOH treatment for 12h, the WT has a severe dispersion phenomenon, the protein ProteinA-1 and ProteinA-2 are relatively stable, and a main band is still visible.

1.6protein A-1 and protein A-2 solid phase coupling affinity medium specific adsorption IgG antibody test:

and (3) coupling the protein ProteinA-1 and ProteinA-2 with a solid phase agarose filler to prepare an affinity medium, washing for 3 times by using a PBS buffer solution, adding the same amount of BSA and IgG protein, supplementing PBS to 1.0mL, and uniformly mixing. After incubating for 2h at room temperature by shaking, centrifuging and taking the supernatant to carry out SDS-PAGE electrophoresis detection, and the electrophoresis result is shown in figure 4. The results show that before and after incubation, the affinity media obtained by coupling Protein A-1 and Protein A-2 with solid-phase agarose packing can specifically and selectively adsorb IgG, but does not adsorb BSA Protein. The affinity introduction was further tested using rabbit serum samples, which were purified with Protein A-1 and Protein A-2 solid phase affinity media to obtain IgG antibodies of higher purity, as shown in FIG. 4.

1.7ProteinA-1 and ProteinA-2 solid phase affinity media alkali resistance test:

in order to test the alkali resistance of the ProteinA-1 and ProteinA-2 solid phase affinity media, the solid phase affinity media are filled into a pre-packed column with the column volume of 1mL, the pre-packed column is washed by 0.1M NaOH for the cycle times, IgG (2.5mg/mL) antibodies are loaded in 8 column volumes after 50 column volumes and 100 column volumes are circulated, and the condition of the IgG adsorption capacity after alkali resistance is monitored. The samples were subjected to SDS-PAGE, and the results are shown in FIG. 5. Gel plot results show that when the pre-packed column was subjected to 50 column volumes of alkaline treatment, the capacity to adsorb IgG was comparable to that without the alkaline treatment. After 100 column volumes of alkaline treatment, the capacity to adsorb IgG tended to decrease significantly. Thus, the ProteinA-2 packing does not affect its ability to adsorb IgG when treated with 50 column volumes of 0.1M NaOH, but only when treated with 100 column volumes of 0.1M NaOH.

According to the method and the results of the embodiment, the recombinant plasmid obtained by the gene cloning modification design of the invention can be used for preparing the protein ProteinA with high efficiency through a relatively simple and convenient purification preparation process through expression induction, volume exclusion chromatography analysis of the protein ProteinA obtained by the invention is detected to show that the purity is higher than 95 percent, and experimental tests show that the protein ProteinA obtained by the invention has the advantages of strong alkali resistance, good thermal stability, good storage property, strong IgG adsorption capacity and the like, and an affinity chromatography medium obtained by coupling the protein with a solid phase carrier has the advantages of strong protein binding specificity, high affinity and high purification efficiency. The invention relates to a novel method for purifying and preparing staphylococcal protein A and application thereof, which have profound significance for the antibody purification market with great demand at present and relevant immunodiagnosis and screening. The detection data of the protein A obtained by the gene cloning modification design show that the protein A has certain advantages of alkali resistance, thermal stability, storage performance and IgG specific adsorption performance, and the affinity chromatography medium obtained by coupling the protein A with a solid phase carrier has the advantages of IgG selective specific adsorption, high dynamic sample loading and stable alkali resistance. The invention relates to a method for expressing, purifying and preparing ProteinA, which can provide a material basis for the antibody purification market with great demand at present and relevant immunodiagnosis.

The foregoing is merely illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the claims. The present invention is not limited to the above embodiments, and the specific structure thereof is allowed to vary. All changes which come within the scope of the invention as defined by the independent claims are intended to be embraced therein.

Sequence listing

<110> center of science of traditional Chinese medicine of the institute of continental materialization of the Chinese academy of sciences in Jiangxi province

<120> staphylococcal protein A, purification preparation method and application thereof

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

115 120 125

Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg

130 135 140

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

145 150 155 160

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

165 170 175

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180 185 190

His Leu Pro Asn Leu Thr Glu Glu Gln Arg Gly Ala Phe Ile Gln Ser

195 200 205

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

210 215 220

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

225 230 235 240

Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr

245 250 255

Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser

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Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln

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35 40 45

Ala Pro Lys Ala Asp Ala Gln Gln Asn Lys Phe Asn Lys Asp Gln Gln

50 55 60

Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln

65 70 75 80

Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr

85 90 95

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

100 105 110

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

115 120 125

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130 135 140

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

145 150 155 160

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165 170 175

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180 185 190

Ser Glu Glu Gln Arg Gly Ala Phe Ile Gln Ser Leu Lys His Asp Pro

195 200 205

Ser Gln Ser Ala Ala Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser

210 215 220

Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala

225 230 235 240

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

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Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile

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Claims

1. A staphylococcal protein a which is characterized by: the amino acid sequence of the B functional structural domain of the staphylococcal protein A is shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

2. A process for the purification of staphylococcal protein a according to claim 1 wherein: the method comprises the following steps

3. The method for purifying and preparing staphylococcal protein A according to claim 1, wherein: the mutation site of the Protein A-1 comprises: A176V, N178H, N181D, Q184A, E190K, N198T, N203G, G204A, N218A, A221S, D228E and A229S are mutated at the same 12, and the amino acid sequence is shown as SEQ ID NO: 1 is shown.

4. The method for purifying and preparing staphylococcal protein A according to claim 2 or 3, wherein: the mutation site of the ProteinA-2 comprises: A176V, Q184H, E190K, N198S, N203G, G204A, D211H, N218A, A221S, D228E and A229S have 11 mutations, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.

5. The method for purifying and preparing staphylococcal protein A according to claim 1, wherein: the expression vector of the recombinant plasmid is pET30a, the host bacterium is Escherichia coli host bacterium BL21(DE3), and when OD600 is cultured to be 0.3-0.8, IPTG with the final concentration of 0.1-1.0 mM is added, and induced expression is carried out for 3-8 h at the temperature of 18-37 ℃.

6. The method for purifying and preparing staphylococcal protein A according to claim 1, wherein: the binding buffer of the Ni affinity chromatography process contains 20mM imidazole, the elution buffer contains 40mM imidazole, and the elution buffer contains 500mM imidazole; and (3) further purifying by anion exchange chromatography, wherein the pH of a mobile phase is 7.0-9.0, and eluting the target protein by adopting a salt ion concentration increasing mode.

7. Use of a staphylococcal protein a according to claim 1 in a ligand of an affinity medium.

8. Use of the staphylococcal protein a of claim 1 in antibody purification and immunodiagnostic studies.