High cell density cultivation of recombinant Escherichia coli for prodrug of recombinant human GLPs production

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• 作者：Ying Zhou ; Xue Ma ; Zheng Hou ; Xiaoyan Xue ; Jingru Meng ; Mingkai Li ; Min Jia ; Xiaoxing Luo
• 关键词：Glucagon-like peptide-1 ; Purification ; Prodrug ; High cell density cultivation
• 刊名：Protein Expression and Purification
• 出版年：2012
• 出版时间：September, 2012
• 年：2012
• 卷：85
• 期：1
• 页码：38-43
• 全文大小：651 K

文摘

Glucagon-like peptide-1 (GLP-1) has been attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical applications are limited by the short half-life in vivo. To overcome this limitation, a new polymer of GLP-1 was developed by prodrug strategy. In this study a recombinant protein, rhGLPs, was successfully constructed, cloned into plasmid pET30a (+) and expressed in Escherichia coli ArcticExpress(DE3)RP in the form of inclusion body. The recombinant fusion protein productivity could be enhanced by high cell density culture of the recombinant strain. As a result, about 40 g wet weight cells per liter were obtained. The protein was purified by size-exclusion chromatography on a Superdex 75 column and refolded using reverse dilution and dialysis methods. SDS-PAGE, HPLC and MALDI-TOF mass spectrometry were undertaken to determine the purity and molecular weight of rhGLPs. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.