title: Split Ssp DnaB mini-intein-mediated production of recombinant human glucagon-like peptide-1/7-36.
abstract: 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.
authors: Institute of Molecular Medicine, Nanjing University, Nanjing, People's Republic of China.
authors: Aiqin
authors: A
authors: Jiang
authors: Institute of Molecular Medicine, Nanjing University, Nanjing, People's Republic of China.
authors: Wenbo
authors: W
authors: Jin
authors: Institute of Molecular Medicine, Nanjing University, Nanjing, People's Republic of China.
authors: Feng
authors: F
authors: Zhao
authors: Institute of Molecular Medicine, Nanjing University, Nanjing, People's Republic of China.
authors: Yanchun
authors: Y
authors: Tang
authors: Institute of Molecular Medicine, Nanjing University, Nanjing, People's Republic of China.
authors: Ziyong
authors: Z
authors: Sun
authors: Institute of Molecular Medicine, Nanjing University, Nanjing, People's Republic of China.
authors: Jian-Ning
authors: JN
authors: Liu
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D053488:DnaB Helicases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D052216:Glucagon-Like Peptide 1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D047668:Inteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D052216:Glucagon-Like Peptide 1; D053488:DnaB Helicases
keywords: E. coli; diabetes; fusion; intein; peptide; rhGLP-1