Expression, purification, and C-terminal amidation of recombinant human glucagon-like peptide-1

Protein Expression and Purification
Zhi-Zhen ZhangKang-Sheng Li

Abstract

Human glucagon-like peptide-1 (hGLP-1) (7-36) amide, a gastrointestinal hormone with a pharmaceutical potential in treating type 2 diabetes mellitus, is composed of 30 amino acid residues as a mature protein. We report here the development of a method for high-level expression and purification of recombinant hGLP-1 (7-36) amide (rhGLP-1) through glutathione S-transferase (GST) fusion expression system. The cDNA of hGLP-1-Leu, the 31st-residue leucine-extended precursor peptide, was prepared by annealing and ligating of artificially synthetic oligonucleotide fragments, inserted into pBluescript SK (+/-) plasmid, and then cloned into pGEX-4T-3 GST fusion vector. The fusion protein GST-hGLP-1-Leu, expressed in Escherichia coli strain BL21 (DE3), was purified by affinity chromatography after high-level culture and sonication of bacteria. Following cleavage of GST-hGLP-1-Leu by cyanogen bromide, the recombinant hGLP-1-Leu was released from fusion protein, and purified using QAE Sepharose ion exchange and RP C(18) chromatography. After purification, the precursor hGLP-1-Leu was transacylated by carboxypeptidase Y, Arg-NH(2) as a nucleophile, to produce rhGLP-1. Electrospray ionization mass spectrometry showed the molecular weight was...Continue Reading

References

Feb 1, 1991·International Journal of Peptide and Protein Research·K BreddamM Meldal
Jan 1, 1997·Experimental and Clinical Endocrinology & Diabetes : Official Journal, German Society of Endocrinology [and] German Diabetes Association·M A NauckW Schmiegel
Feb 15, 2000·Regulatory Peptides·B GallwitzW E Schmidt
May 8, 2000·Biotechnology and Bioengineering·L DominguesJ A Teixeira
Apr 25, 2001·Metabolism: Clinical and Experimental·D G ParkesA Young
May 20, 2003·Srpski arhiv za celokupno lekarstvo·Tatjana RadosavljevićBranka Sikić
Sep 17, 2003·Current Diabetes Reports·Bo Ahrén
Dec 12, 2003·Drug News & Perspectives·Michael A NauckWerner Creutzfeldt

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Citations

Sep 4, 2009·Applied Biochemistry and Biotechnology·Mingming GaoWenbing Yao
Dec 11, 2013·PloS One·Isabelle SermadirasPeter Ravn
Mar 23, 2011·Protein Expression and Purification·Sung-Gun KimJin-Ho Seo
Jul 25, 2013·Bioorganic & Medicinal Chemistry Letters·Chengzao SunBruce Forood
May 4, 2005·Protein Expression and Purification·Xiaopu YinYushu Ma
Oct 12, 2010·Protein Science : a Publication of the Protein Society·Shruti N Patel, Steffen P Graether

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