High-yield mutant streptococcus protein G and application thereof

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a high-yield mutant streptococcus protein G and application thereof. Compared with a wild type protein G, the mutant recombinant protein G provided by the invention has the advantages that the yield in an expression system in escherichia coli is multiplied, the cost for preparing a G Column filler is greatly reduced, and the problem of high cost of a monoclonal antibody is solved. The mutant recombinant protein G retains the binding function of wild protein G to IgG protein, can greatly reduce the manufacturing cost of a G Column affinity chromatography column, is more stable and not easy to degrade, has more recycling times while reducing the production cost of the protein G affinity chromatography column, and has wide application prospects in an antibody purification process.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to a high-yield mutant streptococcal protein G and an application thereof. Technical Background

[0002] Streptococcal protein G (hereinafter referred to as Protein G) can bind to the Fc segment of human and various mammalian IgG without affecting the antibody activity of IgG, so it is widely used in various recombinant and non-recombinant antibody purification processes. Due to the high cost of Protein G ligands, low binding capacity and limited life cycle of fillers, this affinity-based purification step is usually one of the factors that make monoclonal antibody (mAb) products expensive to manufacture. At present, most domestic antibody production uses imported Protein G affinity chromatography media such as GE and Millipore fillers, which are expensive. Providing a low-cost method for preparing recombinant Protein G is a technical problem that needs to be solved urgently. Summary of the invention

[0003] In order to solve the problems in the prior art, one of the objectives of the present invention is to provide a high-yield mutant streptococcal protein G.

[0004] The present invention adopts the following technical solutions:

[0005] A high-yield mutant streptococcal protein G, wherein the mutant streptococcal protein G is Protein G-2C, whose amino acid sequence is shown in SEQ ID NO:1, and whose nucleotide sequence is shown in SEQ ID NO:2.

[0006] A second object of the present invention is to provide an expression vector comprising the nucleotide sequence described above.

[0007] A third object of the present invention is to provide a host cell comprising the expression vector as described above.

[0008] Preferably, the host cell is a host cell for expressing foreign proteins, which is selected from any one of bacteria, yeast, insect cells or mammalian cells.

[0009] A fourth object of the present invention is to provide a fusion protein comprising the above-mentioned Protein G-2C amino acid sequence.

[0010] A fifth object of the present invention is to provide a use of the mutant streptococcal protein G as described above in the preparation of a GColumn affinity chromatography column.

[0011] A sixth object of the present invention is to provide a G Column affinity chromatography column, which comprises the mutant streptococcal protein G as described above.

[0012] The present invention finally protects the use of the G Column affinity chromatography column as described above in purifying human IgG-Fc antibodies.

[0013] The beneficial effects of the present invention are:

[0014] Compared with the wild-type protein G, the mutant recombinant protein G provided by the present invention has a doubling of the yield in the Escherichia coli expression system, which is nearly ten times the yield of the wild-type protein G, greatly reducing the cost of preparing G Column fillers and solving the problem of high cost of monoclonal antibodies.

[0015] The mutant recombinant protein G provided by the present invention retains the binding function of the wild-type protein G to the IgG protein, which greatly reduces the production cost of the G Column affinity chromatography column, and is more stable and not easy to degrade, thereby reducing the production cost of the protein G affinity chromatography column while having more recycling times, and has broad application prospects in the antibody purification process. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the degradation status of purified Protein G protein.

[0017] Figure 2 It is the molecular weight fragment contained in the degradation band.

[0018] Figure 3 This is the result after Protein G-2C nickel column purification.

[0019] Figure 4 This is the protein G-2C polyacrylamide gel electrophoresis (SDS-PAGE) identification result diagram.

[0020] Figure 5 , 6 They are the protein yields of different batches of wild-type Protein G expression bacteria. Figure A is the elution diagram of wild-type Protein G protein on AKTA, and Figure B is the SDS-PAGE identification result of the purified wild-type Protein G protein.

[0021] Figure 7 The purification results of human IgG-Fc protein using G Column prepared for Protein G-2C.

[0022] Figure 8 These are the circulating stability results of Protein G-2C and wild-type protein. DETAILED DESCRIPTION

[0023] For ease of understanding, the technical solution of the present invention is described in more detail below in conjunction with embodiments.

[0024] Example 1

[0025] Preparation of Protein G-2C

[0026] 1. Design of Protein G-2C sequence

[0027] When producing wild-type Protein G protein (Protein G-WT), it was found that the purified Protein G was subject to protein degradation, such as Figure 1 As shown, the extent of protein degradation increases with the storage time at 4°C or -80°C.

[0028] The degraded bands were analyzed by mass spectrometry for molecular weight determination, e.g. Figure 2 As shown, it was found that degradation band 1 mainly contained two fragments with molecular weights of 7226Da and 7424Da, and degradation band 2 mainly contained a fragment with molecular weight of 7666Da. It is speculated that wild-type Protein G will eventually be degraded into three fragments with molecular weights of 7226Da, 7424Da and 7666Da. At present, the protein structure of the full-length sequence of amino acids 185-385 of the wild-type Protein G protein has not been found in the protein structure website PDB. It is speculated that the possible reason is the degradation of the wild-type Protein G protein. Referring to the sequence of the wild-type Protein G protein, there are three segments with high sequence repetition (domain 1, domin 2, domain 3, marked with black underline in the sequence of Protein G-WT). It is speculated that the degradation site exists between these three segments, so the flexible GS Linker (amino acid sequence: GGGGS) commonly used for protein connection is used to replace the connection sequence between the domains of the wild-type protein. The domain 3 with the highest affinity was selected for modification, and the modified sequence was domain 3-2×GS Linker-domain 3 (Protein G-2C). The wild-type Protein G sequence has three domains, while the modified 2C has one domain truncated. On the one hand, the affinity of the domain 3 selected in 2C for the antibody Fc fragment is more than 5 times that of domain 1 and 2, and the affinity of the two tandem domain 3 is basically the same as that of the wild-type Protein G. On the other hand, it is because proteins are usually easier to express after being truncated, which may increase protein production.

[0029] Primers were designed to insert the gene sequence of Protein G-2C into the E. coli expression vector pET30a, a Cys was added to the N-terminus of the Protein G-2C gene sequence for subsequent coupling with agarose, and an 8×His tag was added to the C-terminus. The final obtained Protein G-2C protein amino acid sequence is shown in SEQ ID NO. 1. The constructed plasmid was sequenced and stored.

[0030] 2. Expression and purification of Protein G-2C fusion protein

[0031] The above sequenced vector was transformed into competent Rosetta, and E. coli was cultured on a large scale. The expression of the target protein was induced by IPTG. After the expression was induced overnight, the bacteria were collected and lysed by ultrasonic disruption. Then, the fusion protein in the supernatant was purified by nickel column. The purification results are shown in Figure 2. Figure 3 shown.

[0032] 3. Protein G-2C polyacrylamide gel electrophoresis (SDS-PAGE) identification

[0033] Take the protein purified in step 2, add SDS-loading buffer, heat in a metal bath at 100°C for 5 min, run the sample on a gradient gel, and use Coomassie Brilliant Blue for staining and decolorization. The results are shown in the figure below. Figure 4 As shown in the figure, 1, 2, and 3 are the precipitation, supernatant, and flow-through results, 4 and 5 are elution peaks 1 and 2, and 6 and 7 are tail flows. The results show that the purity of the purified Protein G-2C is above 90%, which meets the requirements for preparing agarose fillers.

[0034] Example 2

[0035] Use the same strain and induction conditions as Protein G-2C to produce wild-type Protein G, such as Figure 5 As shown, Figure A is the elution diagram of the protein on AKTA, the left ordinate A280 (mAu) reflects the concentration of the eluted protein, the right ordinate B% ​​reflects the concentration of the eluate, and Figure B is the SDS-PAGE identification result of the protein. It can be seen that the target band mainly appears at 24kDa in the lane, while the remaining miscellaneous bands are less and lighter, indicating that the obtained protein has high purity and less impurities. The wild-type Protein G expression bacteria cultured in 5L culture medium were collected, broken and purified, and the wild-type Protein G protein was purified by nickel column affinity chromatography. The protein obtained after elution was about 100mg, and the average yield was 20mg / L.

[0036] like Figure 6As shown, in another batch of wild-type Protein G production, 20 L of culture medium was used to culture the wild-type Protein G expression bacteria. After the eluted protein was collected, the concentration of the protein solution was measured. After multiplying it by the volume of the protein solution, it was calculated that the eluted protein was approximately 400 mg, and the average yield was 20 mg / L.

[0037] The average yield of wild-type Protein G in multiple batches of production is around 10-20 mg / L.

[0038] The average yield of Protein G-2C after sequence optimization was 150 mg / L in multiple batches of production. Figure 3 , after culturing Protein G-2C expression bacteria in 10L medium, about 1.5g of protein was obtained by nickel column purification, and about 2.2g of protein was obtained after culturing in 15L medium. The inventors calculated that after culturing Protein G-2C expression bacteria in 400L medium, about 60g of protein was obtained by nickel column purification. At present, it has been connected to agarose to make Protein G-2C agarose adhesive, which has been successfully applied to the purification of antibody proteins.

[0039] Example 3

[0040] Protein G-2C Effect Verification

[0041] The Protein G-2C protein is linked to agarose gel through a covalent bond to prepare a Protein G-2C agarose filler, which is then packed into a column to prepare a G Column.

[0042] Human IgG-Fc protein was used to test whether G Column can be used to purify IgG antibody protein. The results are shown in the figure below. Figure 7 shown. Figure 7 Figures A and B in the middle are the results of human IgG-Fc protein purification using the G Column. The results show that each milliliter of Protein G-2C agarose filler can bind approximately 4.04 mg of human IgG-Fc protein.

[0043] Verify protein stability:

[0044] The proof that the mutant protein is more stable is that there is still no degradation band after being stored at 4°C for 6 months. Compared with the wild-type protein, more than 30% of the protein is degraded after being stored at 4°C for one week, which proves that the stability of the mutant Protein G protein is greatly enhanced. The mutant Protein G affinity chromatography column has more cycles, which is based on the extremely high stability of the mutant protein. The cycle experiment proves that Figure 8As shown, a 1 mL Protein G-2C affinity column that had been stored for about 4 months was used, and an excess of human IgG-Fc protein was used as the detection object. The initial loading was 4.15 mg. Figure 3 A Figure 4 The loading capacity of 4.04 mg tested a month ago remained basically unchanged, indicating that time has little effect on the loading capacity of the affinity column. After 10 cycles of protein loading and elution, the loading capacity was tested again and was 4.00 mg, which became 96.4% of the initial loading capacity. Through the iterative loss calculation of the loading capacity, it was found that the Protein G-2C affinity column can be reused at least 60 times before the loading capacity drops to 80% of the initial capacity.

[0045] In the present invention, the biggest advantage of the mutant Protein G protein is that the protein itself is not easily degraded, because the two fragments constituting the G protein use GS Linker to replace the original wild-type sequence that is prone to breakage. On the other hand, because the mutant Protein G protein itself has only two domains, compared with the wild-type Protein G protein, which has three domains, it is less likely to break the domain, i.e., protein degradation. Therefore, the affinity column made of the mutant Protein G protein is more durable.

[0046] The above implementation modes are only used to illustrate the technical solutions of the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the above implementation modes, those skilled in the art should understand that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A high-yield mutant streptococcal protein G, characterized in that: The mutant streptococcal protein G is ProteinG-2C, whose amino acid sequence is shown in SEQ ID NO:1, and whose nucleotide sequence is shown in SEQ ID NO:

2.

2. An expression vector comprising the nucleotide sequence according to claim 1. A host cell comprising the expression vector according to claim 2.

4. The host cell according to claim 3, characterized in that The host cell is a host cell for expressing foreign proteins, which is selected from any one of bacteria, yeast, insect cells or mammalian cells. A fusion protein comprising the Protein G-2C amino acid sequence according to claim 1 .

6. Use of the mutant streptococcal protein G according to claim 1 in preparing a G Column affinity chromatography column.

7. A G Column affinity chromatography column, characterized in that: The method comprises the mutant streptococcal protein G as claimed in claim 1.

8. Use of the G Column affinity chromatography column as claimed in claim 7 in purifying human IgG-Fc antibodies.

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