High-Density Fermentation Of Engineering Bacteria Escherichia Coli BL21(DE3)/pBV-SFH And Purification, Self-Association Of RSFH In Solution And Thrombin-Targeting Assay In Vivo

Objective: The combination therapy with anticoagulant and thrombolytic drugs is the prime choice in the treatment for clinical thrombosis diseases. The recombinant fusion protein rSFH is a polypeptide composed of staphylokinase and hirudin linked by factor Xa, expressed in the engineering bacteria E.coli BL21(DE3)/pBV-SFH and the expression vector is temperature-sentitive pBV220. The results, obtained from the pharmacological experiments in vitro and in vivo, suggested that the rSFH possesses good bifunction anticoagulation and thrombolysis, and the bleeding risk was reduced, even with the thrombus-targeting feature. Thus the rSFH is a promising candidate for efficient and safe clinical thrombolysis therapy. However, the yield of rSFH expressed in E.coli, is less than 100mg/L fermentation broth under the low cell density fermentation, incapable of satisfying the needs of clinical thrombolysis therapy, therefore, how to increase the yiled of rSFH is a urgent problem to be solved as early as possible. The high-density fermentation of engineering bacteria to increasing the volumetric productivity is an effective way to solve this problem.The purification of recombinant protein from the fermentation broth is arduous work, the purification strategies for isolation of the protein are different and need trial-and-error experiments because of the different properties of each protein, such as molecular mass, pI, polarity, hydrophobic and affinity characterization.It is known that HV contains two functional domains bonding to independent site of a-thrombin: a compact N-terminal core domain binds to the catalytic site of thrombim, and a disordered C-terminal tail is complementary to the exosite(fibrinogen recognition site) of the enzyme. The chromogenic thrombin substrate(S-2251) method had suggested that the anticoagulant activity is losing because of the extension of N-terminus of the HV, but the HV's C-terminus complementary to the exosite may exist and needs to be justified. Methods and results: Recombinant fusion protein staphylokinase-hirudin has been produced by adopting the fed-batch of dissolved oxygen feed-back with the high cell density cultivation strategy for the engineering bacteria E.coli BL21(DE3)/pBV-SFH. After...