Method for producing protein A by utilizing recombinant pichia pastoris

Abstract

The invention relates to a process for producing staphylococcus aureus protein A by utilizing recombinant pichia pastoris. According to the production process, plasmid pPIC9K is utilized for integrating genes in a protein A antibody-binding fraction into a genome of the pichia pastoris GS115, and screening, expression and optimization of purification conditions are performed to get the active excretion protein A. The invention provides preparation and screening of a recombinant strain, as well as a culture and fermentation method by utilizing the strain to express the protein A, and a purification method of the expressed protein A. The strain is firstly cultured in a BMGY (buffered glycerol-complex medium) culture medium for about 17h and then expressed, screened and optimized in a culture medium BMMY (buffered methanol-complex medium) with the pH of 2.6-5.6 and the methanol content of 0.5%-2.0%. Then the expression of the recombinant protein A is further optimized in a fermentation tank, the expression level of the final protein A can achieve 8-10g per liter of bacterial liquid, accounting for about 80% of the secreted total protein. The high-purity protein A can be obtained by filtering through a desalting column and purifying by ion exchange chromatography, and the yield is about 80%. The process creates favorable conditions for low-cost and large-scale production of the high-purity protein A.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and relates to a method for producing protein A by using recombinant Pichia pastoris. Background technique [0002] Staphylococcus aureus protein A (Staphylococcus aureus Protein A, SPA) is a cell wall-binding protein of certain species of Staphylococcus aureus, which was developed by Danish scientist Klaus Jensen in the 1950s to study the cell wall structure of Staphylococcus aureus discovered when. Its basic structure is composed of the following three parts, namely signal peptide, antibody binding functional region and cell wall binding region. The antibody-binding functional region of protein A contains five homologous single domains E, D, A, B, and C, and each single domain can act independently with immunoglobulin G (IgG). The binding of the protein to IgG is mainly based on the specific binding ability to the Fc region of IgG. It is precisely because of the specific binding abilit...

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Problems solved by technology

Unfortunately, the secretory expression of many foreign proteins is inefficient, especially compared to intracellular expression

[0012] So far, there is only one related paper "Recombinant Expression of Protein A and Optimization of Fermentation Process of Recombinant Bacteria" related to the previous research work of this research group, which mentions the expression of recombinant protein A by Pichia pastoris, but this part of the work has not been explored as a research. In 2008, Tang et al. used Pichia pastoris to secrete and express the fusion protein ZZ-EGFP based on the tandem Z domain of protein A B domain and EGFP, but the expression amount was only 115 mg / mL. limited

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