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

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Abstract

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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Acknowledgments

This work was supported by the China National Nature Science Foundation (30772679) and Hi-Tech Research and Development Program of China-863 Program (2007AA02Z101).

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Authors and Affiliations

  1. School of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China

    Mingming Gao, Hong Tian, Chen Ma, Xiangdong Gao, Wei Guo & Wenbing Yao

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  1. Mingming Gao
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  2. Hong Tian
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  3. Chen Ma
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  4. Xiangdong Gao
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  5. Wei Guo
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  6. Wenbing Yao
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Correspondence to Wenbing Yao.

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Mingming Gao and Hong Tian contributed equally to this article.

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Gao, M., Tian, H., Ma, C. et al. Expression, Purification, and C-terminal Site-Specific PEGylation of Cysteine-Mutated Glucagon-Like Peptide-1. Appl Biochem Biotechnol 162, 155–165 (2010). https://doi.org/10.1007/s12010-009-8725-6

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  • DOI: https://doi.org/10.1007/s12010-009-8725-6

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