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Preparation method of recombinant protein A

A technology of recombinant proteins and recombinant strains, applied in the biological field, can solve the problems of low affinity and low purification efficiency, and achieve the effects of high affinity, high expression efficiency and low production cost

Inactive Publication Date: 2012-01-25
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When using the affinity chromatography technology containing proteinA, there are problems such as low affinity and low purification efficiency. Therefore, when using the affinity chromatography technology, an improved ProteinA is needed to achieve higher affinity and improve the efficiency of purification. Effect

Method used

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  • Preparation method of recombinant protein A
  • Preparation method of recombinant protein A
  • Preparation method of recombinant protein A

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, the synthesis of recombinant protein A gene monomer

[0026] By means of chemical synthesis, the sequence of a repetitive fragment of the recombinant protein A gene was designed and synthesized. The sequence included a length of 168 bp, and the NcoI restriction site connected to the expression vector was added to the front end, and 6 His (for affinity chromatography, and Nickel column combination, reducing purification steps) and EK restriction site, and AccI restriction site for ligation of multiple repeat fragments. In addition, it also includes the stop codon TAA, and a total of 9 bp of BamH I restriction endonuclease linker for cloning. It is also necessary to design and synthesize a sequence of a repeat segment of the recombinant protein A gene by chemical synthesis, which has a length of 168 bp and AccI restriction sites at both ends for constructing a sequence containing multiple repeat segments.

Embodiment 2

[0027] Embodiment 2, the construction of the pET32a vector containing a recombinant protein A gene monomer

[0028] Cut out the above-mentioned recombinant protein A gene monomer with restriction endonucleases NcoI and BamHI, connect with the vector pET32a digested with NcoI and BamHI, transform Escherichia coli, screen the transformant with ampicillin resistance, and extract the plasmid , After identification by enzyme digestion, it was proved that the recombinant protein A gene monomer had been cloned into pET32a.

Embodiment 3

[0029] Embodiment 3, the construction of the pET32a-P vector containing recombinant protein A gene

[0030] The above pET32a vector containing a recombinant protein A gene monomer and the recombinant protein A gene monomer were digested with AccI, the corresponding fragments were recovered and ligated, transformed into Escherichia coli, and the transformants with ampicillin resistance were screened, extracted from the plasmid, and enzymatically The pET32a-P vector of recombinant protein A containing 3 protein A gene monomers was obtained by cleavage screening.

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Abstract

The invention provides a preparation method of a recombinant protein A which has an amino acid sequence shown as SEQ ID NO:1. The recombinant protein A prepared with the method provided by the invention can be applied to antibody detection, separation and purification, and can constitute an affinity chromatography medium together with a chromatography medium carrier. The recombinant protein A produced with the method provided by the invention has the advantages of high protein bonding specificity, high affinity and greatly-increased purifying efficiency. Compared with commercially available Protein A Sepharose 4Fast Flow, the recombinant protein A has remarkably-increased dynamic adsorption capacity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a new preparation method of recombinant protein A. Background technique [0002] Staphylococcal Protein A (SPA) is a protein isolated from the cell wall of Staphylococcus aureus. In 1940, Vevwey found that some Staphylococcus aureus contained a substance that could form a precipitate with normal human serum in a two-way diffusion test. Jensen (1959) also discovered a similar phenomenon and named it the A antigen. [0003] In 1963, Lofkvist et al. isolated the A antigen, and proved that it is a protein and is different from sugar; Grov (1960) named it staphylococcal protein A, referred to as SPA protein A (ProteinA). The gene encoding SPA was cloned and expressed in Escherichia coli in 1983 (Duggleby, C.J and Jones, SA: cloning and expression of the Staphylococcus aureus protein A gene in Escherichia coli. Nucl Acids Res. 11(1983) 3065-3076; Lofdahl, S., Guss, B., et al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12N15/31C12N15/63C12N1/21C12R1/19
Inventor 王皓谈珉胡辉
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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