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Current Pharmaceutical Biotechnology


ISSN (Print): 1389-2010
ISSN (Online): 1873-4316

Expression and Purification of Optimized rolGLP-1, A Novel GLP-1 Analog, in Escherichia Coli BL21(DE3) and its Good Glucoregulatory Effect on Type 2 Diabetic Mice

Author(s): Baicheng Ma, Peipei Tu, Xingyu Zhao, Yaofang Zhang, Yu Wang, Chao Ma, Yanli Ji, Xiaodan Li, Syed A. Abbas and Minggang Li

Volume 14 , Issue 11 , 2013

Page: [985 - 994] Pages: 10

DOI: 10.2174/1389201014666131226155553

Price: $65


Glucagon-like peptide-1 (GLP-1) is an incretin hormone that decreases postprandial glycemic excursions by enhancing insulin secretion but with short half-life due to rapid inactivation by enzymatic N-terminal truncation. Therefore, efforts are being made to improve the stability of GLP-1 via modifying its structure or inhibiting dipeptidylpeptidase IV (DPP IV), which is responsible for its degradation. GLP-M, consisting of 10 tandem repeated rolGLP-1 (GLP-1 analog), has been expressed in Pichia pastoris by our laboratory. Although it had a long effect of maintaining glucose homeostasis, redundant amino acids and purification tag limited its application. Here, optimized rolGLP-1(GLPO) with no redundant amino acids and purification tag was constructed by molecular cloning and site-directed mutagenesis, which was expressed efficiently in Escherichia coli BL21(DE3) with the production of 81.5 mg/L, and confirmed by the results of SDS-PAGE electrophoresis and Western Blotting. Then GLP-O was purified via ion exchange chromatography and gel filtration chromatography. The purity of GLP-O was close to 100%. GLP-O could be cut into single rolGLP-1 by trypsin in vitro, and rolGLP-1 had anti-trypsin activity. After oral administration of GLP-O for 4 weeks, the level of blood glucose in type 2 diabetic mice was lowered effectively, and the oral glucose tolerance of mice was improved significantly. These results settled the foundation for further clinical application of GLP-O.

Keywords: Diabetes, expression, GLP-1, Oral glucose tolerance, Purification, Western Blotting.

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