title: 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.
abstract: 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.
authors.forename: Baicheng
authors.initials: B
authors.lastname: Ma
authors.forename: Peipei
authors.initials: P
authors.lastname: Tu
authors.forename: Xingyu
authors.initials: X
authors.lastname: Zhao
authors.forename: Yaofang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Chao
authors.initials: C
authors.lastname: Ma
authors.forename: Yanli
authors.initials: Y
authors.lastname: Ji
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Li
authors.forename: Syed A
authors.initials: SA
authors.lastname: Abbas
authors.affiliation: Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, Tianjin 300071, P.R. China. mgl@nankai.edu.cn.
authors.forename: Minggang
authors.initials: M
authors.lastname: Li
mesh_terms: D000818:Animals; D001786:Blood Glucose / Q000378:metabolism*; D003001:Cloning, Molecular; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D052216:Glucagon-Like Peptide 1 / Q000008:administration & dosage* / Q000302:isolation & purification / Q000378:metabolism*; D007333:Insulin Resistance; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D016896:Treatment Outcome
chemical_list: D001786:Blood Glucose; D052216:Glucagon-Like Peptide 1