Efficient System of Artificial Oil Bodies for Functional Expression and Purification of Recombinant Nattokinase in Escherichia coli

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• 作者：Chung-Jen Chiang ; Hong-Chen Chen ; Yun-Peng Chao ; and Jason T. C. Tzen
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2005
• 出版时间：June 15, 2005
• 年：2005
• 卷：53
• 期：12
• 页码：4799 - 4804
• 全文大小：280K
• 年卷期：v.53,no.12(June 15, 2005)
• ISSN：1520-5118

文摘

Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formedinsoluble aggregates without enzymatic activity. For functional expression and purification, nattokinaseor pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to theC terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodieswere reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed.Soluble nattokinase was subsequently released through self-splicing of intein induced by temperaturealteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide ofpronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolyticactivity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase,although the production yields were similar in both cases. Furthermore, active nattokinase could beharvested in the same system by fusing pronattokinase to the N terminus of oleosin via a differentintein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potentialof this system for bacterial expression and purification of functional recombinant proteins.Keywords: Artificial oil body; intein; nattokinase; oleosin; propeptide