Expression, Purification, and C-terminal Site-Specific PEGylation of Cysteine-Mutated Glucagon-Like Peptide-1

Mingming Gao; Hong Tian; Chen Ma; Xiangdong Gao; Wei Guo; Wenbing Yao
September 2010
Applied Biochemistry & Biotechnology;Sep2010, Vol. 162 Issue 1, p155
Academic Journal
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 (mPEG10K-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 mPEG10K–cGLP-1 with a purity of up to 98% was obtained from the original 500 ml culture. The circular dichroism spectra indicated that mPEG10K–cGLP-1 maintained the secondary structure of native GLP-1. As compared with that of native GLP-1, the plasma glucose lowering activity of mPEG10K–cGLP-1 was significantly extended. These results suggest that our method will be useful in obtaining a large quantity of mPEG10K–cGLP-1 for further study and mPEG10K–cGLP-1 might find a role in the therapy of type 2 diabetes through C-terminal site-specific PEGylation.


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