Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8359225 | Protein Expression and Purification | 2018 | 6 Pages |
Abstract
Optimal conditions for a high cell-density fermentation of Escherichia coli 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 10â¯g/L, yeast extract 30â¯g/L, trypton 10â¯g/L, NaCl 5â¯g/L. The late-stage induction with 0.05â¯mM isopropyl-β-d- thiogalactopyranoside showed the highest productivity after 28â¯h of the fed-batch fermentation. This optimized process yielded about 150â¯mg of purified rAT-INS from 1â¯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.
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Authors
Jian Jing, Yaoling Chen, Li Sheng, Mi Wu,