Expression, purification, and C-terminal site-specific PEGylation of cysteine-mutated glucagon-like peptide-1.
Glucagon-like peptide-1 (GLP-1) is attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical application is limited because of short biological half-life. This study was designed to produce a C-terminal site-specific PEGylated analog of cysteine-mutated GLP-1 (cGLP-1) to prolong its action. The gene of cGLP-1 was inserted into pET32a to construct a thioredoxinA fusion protein. After expression in BL21 (DE3) strain, the fusion protein was purified with Ni-affinity chromatography and then was PEGylated with methoxy-polyethylene glycol-maleimide (mPEG(10K)-MAL). The PEGylated fusion protein was purified with anion exchange chromatography and then was cleaved by enterokinase. The digested product was further purified with reverse-phase chromatography. Finally, 8.7 mg mPEG(10K)-cGLP-1 with a purity of up to 98% was obtained from the original 500 ml culture. The circular dichroism spectra indicated that mPEG(10K)-cGLP-1 maintained the secondary structure of native GLP-1. As compared with that of native GLP-1, the plasma glucose lowering activity of mPEG(10K)-cGLP-1 was significantly extended. These results suggest that our method will be useful in obtaining a large quantity of mPEG(10K)-cGLP-1 for further study and mPEG(10K)-cGLP-1 might find a role in the therapy of type 2 diabetes through C-terminal site-specific PEGylation.
Gao
Mingming
M
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, People's Republic of China.
Tian
Hong
H
Ma
Chen
C
Gao
Xiangdong
X
Guo
Wei
W
Yao
Wenbing
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001786:Blood Glucose / Q000378:metabolism; D003545:Cysteine; D015870:Gene Expression; D052216:Glucagon-Like Peptide 1 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011092:Polyethylene Glycols / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D001786:Blood Glucose; D011993:Recombinant Fusion Proteins; D011092:Polyethylene Glycols; D052216:Glucagon-Like Peptide 1; D003545:Cysteine