Keratinocyte Growth Factor-2 Production in an ompT-Deficient Escherichia coli K-12 Mutant

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• 作者：Michael W. Laird ; Karen Cope ; Ronald Atkinson ; Mark Donahoe ; Kelly Johnson ; and Meredith Melick
• 刊名：Biotechnology Progress
• 出版年：2004
• 出版时间：February 2004
• 年：2004
• 卷：20
• 期：1
• 页码：44 - 50
• 全文大小：132K
• 年卷期：v.20,no.1(February 2004)
• ISSN：1520-6033

文摘

A variant form of Keratinocyte growth factor-2 (KGF-2) spanning amino acids A63-S208 was produced in the Escherichia coli K-12 host W3110. When the protein waspurified using a standard process, the first six N-terminal amino acids were rapidlyand specifically removed from the protein. This cleavage resulted in a truncated KGF-2species (S69-S208). To circumvent this problem, guanidine-HCl was used to inhibitthe putative proteolytic activity. This modified process resulted in a massive loss ofprotein product due to precipitation, in addition to the cost and corrosiveness ofguanidine-HCl. To develop an economically feasible, scaleable, and robust process forKGF-2 production, we were tasked with identifying the protease(s) responsible forthe N-terminal degradation. Experimental evidence revealed that OmpT (outermembrane protein T) was the primary protease involved in the N-terminal cleavageof the A63-S208 KGF-2 protein. Moreover, the OmpT-mediated cleavage occurred ata novel site (Arg-Ser). From this work, we show that production of the A63-S208form of KGF-2 in an ompT-deleted E. coli host nearly abolished the N-terminal cleavageissue.