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Protein Expr Purif. 2012 Sep;85(1):38-43. doi: 10.1016/j.pep.2012.06.016. Epub 2012 Jul 6.

High cell density cultivation of recombinant Escherichia coli for prodrug of recombinant human GLPs production.

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1
Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi'an, Shaanxi 710032, China.

Abstract

Glucagon-like peptide-1 (GLP-1)(2) has been attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical applications are limited by the short half-life in vivo. To overcome this limitation, a new polymer of GLP-1 was developed by prodrug strategy. In this study a recombinant protein, rhGLPs, was successfully constructed, cloned into plasmid pET30a (+) and expressed in Escherichia coli ArcticExpress(DE3)RP in the form of inclusion body. The recombinant fusion protein productivity could be enhanced by high cell density culture of the recombinant strain. As a result, about 40 g wet weight cells per liter were obtained. The protein was purified by size-exclusion chromatography on a Superdex 75 column and refolded using reverse dilution and dialysis methods. SDS-PAGE, HPLC and MALDI-TOF mass spectrometry were undertaken to determine the purity and molecular weight of rhGLPs. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo.

PMID:
22771632
DOI:
10.1016/j.pep.2012.06.016
[Indexed for MEDLINE]

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