关键词: 生物分离, 蛋白质复性, 二硫键, 正交实验设计, 瑞替普酶

Abstract:

The vector with the gene of recombinant reteplase (rt-PA) was cloned into E. coli BL21(DE3) cells. Over-expression of rt-PA as inclusion bodies was obtained, and renaturation of rt-PA in vitro was investigated. Firstly, single factor experiments were conducted to optimize refolding conditions, including refolding buffer pH, GSH concentration, ratio of GSH to GSSG, rt-PA concentration. On this basis, high concentration protein refolding was further investigated by orthogonal experimental design. Fermentation broth with 1.7 g crude inclusion bodies per liter was obtained after using 0.2 mmol·L^-1 IPTG as inducer and culturing at 33℃ for 6 h. The refolding yield of rt-PA was up to 87.2% under optimal condition: 50 mg·ml^-1 denatured rt-PA, pH 10.0, 1 mmol·L^-1 GSH and ratio of GSH to GSSG 8. The key factors affecting refolding of high concentration protein were initial pH and GSH concentration, and specific bioactivity of rt-PA could reach 7.54×10⁴ IU·mg^-1 after refolding at protein concentration of 800 mg·ml^-1. Fluorescence spectra indicated that structural conformation of refolded reteplase was identical with its native state.

Key words: bio-separation, protein refolding, disulfide bond, orthogonal experimental design, reteplase