重组蛋白G基因工程菌高密度发酵及其分离纯化

Over-expression and Purification of Recombinant SPG from High Cell-density Fermentation of E. coli BL21

蛋白G是链球菌细胞壁蛋白,能和哺乳免疫球蛋白结合,在分离抗体研究方面具有重要的作用。重组蛋白G(SPG)基因重组工程菌高密度发酵工艺和SPG分离纯化研究工艺直接影响了蛋白G的应用。通过一级、二级种子培养,转接到发酵罐及控制补料浓度、加入IPTG等条件,考察了接种量、氧气、pH、培养方式等发酵工艺,以及超声破碎菌体、镍柱亲和层析、Sephadex G-25凝胶过滤层析和DEAE-FF离子交换层析分离提取SPG工艺,并用SDS-PAGE检测分离提取效果。高密度发酵能得到至少80g/L的菌体,最高达到150g/L的菌体,每升发酵液可得到1g的SPG。本生产工艺可得到高浓度和高产量的SPG。

The high cell-density cultural and isolation technology were investigated to obtain the recombinant protein Streptococci protein G （SPG）, one of the antigens from the Streptococci cell wall, via expression in genet-ically modified E. coli BL21. After the first and second grade seed cultured, the engineering bacteria was put into the fermentation bioreactor containing ferment culture medium; utilizing isopropyl-β-D-thiogalactopyranoside （IPTG） as inducer; culturing effluxion of time continually; proceeding supplement material culture and other con-trol technology. After the engineering bacteria was ultrasonicated, the recombinant SPG was separated by Ni-NTA spin columns, Sephadex G25 and DEAE-FF. The concentration of the bacteria was between 80 g/L and 150 g/L, and the interest protein, SPG, was at a concentration of 1 g/L. Large quantities of SPG can be obtained using this high cell-density cultivation and isolation technology.