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Recombinant protein A and applications thereof

A technology of recombinant protein and staphylococcal protein, which is applied in the field of new recombinant Staphylococcus aureus protein A and its application in protein separation and purification, can solve the problems of unsatisfactory and unstable, and achieve improved stability and high alkali resistance performance, increase the maximum load effect

Active Publication Date: 2016-04-13
亿一生物制药(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Z fragment was used as a ligand in affinity chromatography. Compared with the B fragment, the Z fragment showed enhanced stability to some chemical reagents except NaOH, but under higher alkaline conditions, still It is unstable and cannot meet the needs of industrial online cleaning (CleaningInPlace, CIP) regeneration

Method used

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  • Recombinant protein A and applications thereof
  • Recombinant protein A and applications thereof
  • Recombinant protein A and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Embodiment 1: Preparation of recombinant protein A polymer

[0108] a. Synthesis of recombinant protein A polymer gene

[0109] The cDNA sequence encoding the recombinant protein A polymer (as shown in SEQIDNo.1) was synthesized using the whole gene. The full length of the sequence is 768bp (entrusted to Shanghai Jierui Bioengineering Co., Ltd.), including the start codon ATG and the stop codon TAA, The synthetic sequence was ligated into the expression vector pET30a(+) to obtain an expression plasmid named pET30a-4Z', such as figure 1 shown.

[0110] b. Sequence identification and expression

[0111] with CaCl 2 The plasmid pET30a-4Z' was transformed into Escherichia coli BL21 (DE3), grown overnight on LB plates containing 50 μg / mL kanamycin (kana for short), picked single-clonal colonies, and inserted into LB liquid medium ( 50 μg / mL kana) at 37°C, shaking at 200 rpm for 8 hours. Take 2 mL of bacterial liquid and add it to 100 mL of LB medium (50 μg / mL kana), cul...

Embodiment 2

[0122] Embodiment 2: load determination

[0123] The recombinant protein A polymer prepared in Example 1 was used as a ligand, and the monodisperse porous copolymer polystyrene-divinylbenzene (PS / DVB) microspheres were used as a carrier. In the PB buffer solution with a pH of 6-8, stirred and reacted at 25°C for 8 hours, the protein and the microsphere carrier were cross-linked through thioether bonds, and the obtained medium was washed several times with PB buffer solution to remove unbound protein, and dried in vacuum to obtain the And chromatography medium (named recombinant protein A polymer medium).

[0124] Use the cell supernatant sample (2.0g / L) expressing human IgG to carry out load determination on the following affinity chromatography medium:

[0125] Recombinant protein A polymer medium;

[0126] MabCapture TM Medium A (purchased from AB Applied Biosystems, Cat. No. 4374730) was used as a contrast medium.

[0127] The loading buffer is 10mMPB+0.2MNaCl, pH7.5. ...

Embodiment 5

[0135] Embodiment 5: Recombinant protein A polymer and recombinant wild-type protein A (rPA) alkali resistance determination

[0136] Add 10mL of 1M NaOH solution to 10mL of the recombinant protein A polymer prepared in Example 1 (6mg / mL) and mix well (the concentration of NaOH in the mixed solution is 0.5M), and then incubate in a 37°C incubator for 4hr, 8hr, 16hr, 24hr sampling, 12% SDS-PAGE electrophoresis analysis of untreated (0hr) and lye-treated samples, the sample load of each time point is 5 μg, the electrophoresis results are as follows Figure 4 In the figure (a) shown.

[0137] Take 0.6mL of recombinant wild-type protein A (Wild-type Staphylococcus aureus protein A recombinantly expressed by E.coli, the concentration is 1.2mg / mL) solution, add 5.4mL10mMPB, pH7.2, mix well, reserve 1mL of the sample, and add Add an equal volume of 1M NaOH solution (5mL) and mix evenly (the concentration of NaOH in the mixed solution is 0.5M), incubate in a 37°C incubator, take samp...

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Abstract

The present invention relates to a recombinant protein A and applications thereof, and particularly discloses a novel recombinant Staphylococcus aureus protein A, which has a structure defined in the specification, wherein the function comprises but is not limited to application as a ligand-specific binding immunoglobulin, and the recombinant protein A can be used as a specific affinity ligand, and has excellent alkali resistance. The invention further relates to a method for constructing the mutant protein.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention relates to a novel recombinant Staphylococcus aureus protein A and its application in protein separation and purification. Background technique [0002] Staphylococcal Protein A (Staphylococcal Protein A, SpA) includes five structural domains, namely E, D, A, B, and C, of ​​which the B domain (B fragment) has the strongest binding ability to immunoglobulins. Wild-type staphylococcal protein A, recombinant staphylococcal protein A (rPA) and improved proteins have been widely used in the field of affinity chromatography, and affinity chromatography media made of staphylococcal protein A are widely used to capture antibodies , to remove host proteins and residual DNA. [0003] Affinity chromatography medium often needs to be washed in order to retain the selectivity and high specificity of the affinity ligand and prevent bacterial contamination. It is usually washed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31C07K19/00C12N15/31C12N15/62B01J20/24B01D15/38C07K16/00C07K1/22
Inventor 先宗树朱庆庆黄予良
Owner 亿一生物制药(北京)有限公司
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