Preparation and PEGylation of exendin-4 peptide secreted from yeast  Pichia pastoris.
Exendin-4, a peptide analogue of glucagon-like peptide (GLP-1), has been developed for treatment of type 2 diabetes. Herein, the secretive exendin-4 peptide, expressed by methanol induction in Pichia pastoris, was purified to near homogeneity by Ni-NTA agarose chromatography. 103.6 mg of protein was obtained from 1 L of the supernatant and its purity was 96.1%. Subsequently, the PEGylated exendin-4 was prepared. The bioactivity of exendin-4 was determined by examining the glucose-lowering and insulin-releasing ability in plasma. Then, a safety evaluation was performed by histological examination of the main organs (liver, kidney and pancreas). PEGylated exendin-4 displayed glucose-lowering and insulin-stimulating action in vivo without obvious damage to the above organs. The results suggest that the P. pastoris expression could be used to produce large quantities of exendin-4, and PEGylation is a useful tool to maintain and enhance bioactivity of the peptide.
Zhou
Jin
J
Life Sciences Division, Tsinghua University, Shenzhen, PR China.
Cai
Zhong-Hua
ZH
Li
Lei
L
Kou
Chuang
C
Gao
Yun-Feng
YF
D000595:Amino Acid Sequence; D000818:Animals; D001786:Blood Glucose / Q000032:analysis; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D000077270:Exenatide; D007328:Insulin / Q000097:blood; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics*; D011092:Polyethylene Glycols / Q000737:chemistry*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D014688:Venoms / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D001786:Blood Glucose; D007328:Insulin; D010455:Peptides; D014688:Venoms; D011092:Polyethylene Glycols; D000077270:Exenatide