Recombinant production of mGLP-1 by coupling of refolding and intein-mediated self-cleavage (CRIS).
Glucagon-like peptide-1 as an endogenous glucose-lowering peptide is a promising candidate for anti-diabetic drug development. Here, we developed a convenient method by coupling of refolding and intein-mediated self-cleavage (CRIS) to improve the recombinant production of a mutated glucagon-like peptide-1 (mGLP-1). Bacterial cell culture employing auto-induction was performed at 37 °C to avoid the intracellular self-cleavage of the intein fusion protein. The impacts of urea, pH, and temperature on the efficiency of CRIS were tested, and then, the optimized CRIS was established. Using the optimized method, we obtained the purified mGLP-1 with a yield of 3.41 mg peptide/g bacterial cells which was 5.6-fold higher than before. After that, using chromatography, peptide electrophoresis, and mass spectrometry, we determined the purity and molecular weight of the purified peptide and then confirmed its glucose-lowering activity by performing glucose tolerance test in mice. These results suggest that CRIS is a relatively simple and efficacious method for the recombinant production of mGLP-1, and as a general method, it can also be used for the recombinant preparation of some other proteins and peptides.
State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Tongjiaxiang 24, Nanjing, 210009, China.
Mingming
M
Gao
Yue
Y
Tong
Hong
H
Tian
Xiangdong
X
Gao
Wenbing
W
Yao
D000818:Animals; D001709:Biotechnology / Q000379:methods; D002845:Chromatography; D004586:Electrophoresis; D004926:Escherichia coli / Q000235:genetics; D052216:Glucagon-Like Peptide 1 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005951:Glucose Tolerance Test; D006868:Hydrolysis; D013058:Mass Spectrometry; D051379:Mice; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D052216:Glucagon-Like Peptide 1