Split Ssp DnaB mini-intein-mediated production of recombinant human glucagon-like peptide-1/7-36

Autor: Yanchun Tang, Ziyong Sun, Wenbo Jin, Aiqin Jiang, Jian-Ning Liu, Feng Zhao
Rok vydání: 2015
Předmět:
Zdroj: Biotechnology and Applied Biochemistry. 62:309-315
ISSN: 0885-4513
DOI: 10.1002/bab.1274
Popis: Glucagon-like peptide-1 (GLP-1) plays an important role in the regulation of postprandial insulin release. Here, we used the split DnaB mini-intein system to produce recombinant human GLP-1/7-36 (rhGLP-1) in Escherichia coli. The C-terminal domain of DnaB mini-intein (IntC) was genetically fused at the N-terminus of rhGLP-1 to produce IntC-GLP-1. IntC-GLP-1 and N-terminal domain of DnaB mini-intein (IntN) protein were prepared in a denatured buffer of pH 8.0. IntC-GLP-1 was diluted 1:8 into the phosphate buffer of pH 6.6. IntN was added into the diluted solution of IntC-GLP-1 at the molar ratio of 1:2. Then, rhGLP-1 was released from IntC-GLP-1 via inducible C-terminal peptide-bond cleavage by shifting pH from 8.0 to 6.6 at 25 °C for 24-H incubation. Then, the supernatant was applied to a Ni-Sepharose column, and the pass through fraction was collected. About 5.34 mg of rhGLP-1 with the purity of 97% was obtained from 1 L of culture medium. Mass spectrometry showed the molecular weight of 3,300.45 Da, which was equal to the theoretical value of GLP-1/7-36. The glucose-lowering activity of rhGLP-1 was confirmed by the glucose tolerance test in mice. In conclusion, the reported method was an efficient strategy to produce rhGLP-1 without using enzyme or chemical reagents, which could also be used for other similar peptides.
Databáze: OpenAIRE
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