Expression, purification, and C-terminal amidation of recombinant human glucagon-like peptide-1.
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 as expected. The biological activity of rhGLP-1 in a rat model demonstrated that plasma glucose concentrations were significantly lower and insulin concentrations higher after intraperitoneal injection of rhGLP-1 together with glucose compared with glucose alone (P < 0.001).
Zhang
Zhi-Zhen
ZZ
Joint Influenza Research Centre, Department of Microbiology and Immunology, Shantou University Medical College, Guangdong, PR China. zzz@stu.edu.cn
Yang
Sheng-Sheng
SS
Dou
Hong
H
Mao
Ji-Fang
JF
Li
Kang-Sheng
KS
D000818:Animals; D001120:Arginine / Q000737:chemistry*; D043402:Cathepsin A / Q000737:chemistry*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide / Q000737:chemistry*; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005934:Glucagon / Q000008:administration & dosage / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D052216:Glucagon-Like Peptide 1; D005947:Glucose / Q000008:administration & dosage / Q000032:analysis; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000097:blood; D007930:Leucine / Q000737:chemistry; D010446:Peptide Fragments / Q000008:administration & dosage / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011498:Protein Precursors / Q000008:administration & dosage / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D007328:Insulin; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D001120:Arginine; D005982:Glutathione Transferase; D043402:Cathepsin A; D007930:Leucine; D005947:Glucose; D003488:Cyanogen Bromide