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.