초록
<P><B>Abstract</B></P> <P>Optimal conditions for a high cell-density fermentation of <I>Escherichia coli</I> strain harboring a recombinant anti-thrombosis insulin variant (named rAT-INS) were investigated by using fed-batch culture employing pH-stat method. The optimized main medium composition were glycerol 10 g/L, yeast extract 30 g/L, trypton 10 g/L, NaCl 5 g/L. The late-stage induction with 0.05 mM isopropyl-β-<SMALL>D</SMALL>- thiogalactopyranoside showed the highest productivity after 28 h of the fed-batch fermentation. This optimized process yielded about 150 mg of purified rAT-INS from 1 L of wet cell mass with high-homogeneity. The amino acid compositions and mass data of the purified rAT-INS were in good agreement with those as expected. Purified rAT-INS exhibited potent inhibitory activity of platelet aggregation. The in vivo assay showed that rAT-INS had a higher activity in prolonging the bleeding time in mice than native-insulin. The purified rAT-INS had almost no insulin receptor binding activity. Our study demonstrates the promise for mass production of novel recombinant antiplatelet agents.</P> <P><B>Highlights</B></P> <P> <UL> <LI> RGD motif was introduced to construct anti-thrombosis insulin variant rAT-INS. </LI> <LI> Medium composition and induction process were optimized for rAT-INS expression. </LI> <LI> High recovery of rAT-INS as cytoplasmic inclusion bodies was realized. </LI> <LI> rAT-INS inhibited platelet aggregation and prolonged bleeding time in mice. </LI> </UL> </P>