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| Constituted expression and purification of glucagon-like peptide-1 analogue in Pichia pastoris using GAP promoter |
| QIAN Kai1, ZHANG Jing-jing2, WU Su-ping2, CAI Yan-fei2, CHEN Yun2, JIN Jian2 |
1. School of Biotechnology, Jiangnan University, Wuxi 214122, China;
2. School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, China |
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Abstract Glucagon-like peptide-1 (GLP-1) is a potential therapeutic drug for type II diabetes, mainly because of the stimulatory effect on insulin secretion under condition of high blood glucose. We used PCR to obtain a recombination gene AGGH, two GLP-1 (GLP-1A2G) mutants were connected in series and then fused to the N terminal of human serum albumin,and alanine was added at the N terminal of GGH. The fusion gene was inserted into pGAPZaA plasmid and expressed by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The engineered strain was constructed and the recombinant P. pastoris successfully expressed the fusion protein AGGH. The yield of AGGH reached 68 mg/L after 72 h fermentation in a flask, using glucose as the optimal carbon source. Fed-batch fermentation was investigated in a 5 L bioreactor, and the expression level of GGH reached 238 mg/L in 52 h. The fusion protein AGGH was purified in four steps, and the final purity was 95.8%. The bioactivity of AGGH was the same as that expressed in P. pastoris by the AOX1 promoter. This study described an efficient way to express AGGH fusion protein in P.pastoris using GAP promoter, fermentation was easy way to control without carbon source change than AOX1 promoter-controlled GGH expression and fermentation time was 20 h less than AOX1 promoter-controlled GGH expression.
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Received: 13 January 2015
Published: 25 May 2015
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