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KMID : 1094720150200040694
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Biotechnology and Bioprocess Engineering
2015 Volume.20 No. 4 p.694 ~ p.700
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Efficient expression of glucagon-like peptide-1 analogue with human serum albumin fusion protein in Pichia pastoris using the glyceraldehyde-3-phosphate dehydrogenase promoter
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Qian Kai
Gong Xiao Hai
Guan Bo
Wu Su Ping
Zhang Jing Jing
Qian Jing
Cai Yan Fei
Chen Yun
Duan Zuo Ying
Ma Xin
Li Hua Zhong
Jin Jian
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Abstract
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Glucagon-like peptide-1 (GLP-1) was 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, GGH, in which two GLP-1 (GLP-1A2G) mutants were connected in series and then fused to the N terminal of human serum albumin. The fusion gene was inserted into pGAPZaA plasmid with Saccharomyces cerevisiae α-factor secretion signal sequence, and was expressed by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The engineered strain was constructed by integrating the recombinant plasmid pGAPZαA/GGH into the genome of Pichia pastoris GS115. Genome PCR and western blot showed that the recombinant P. pastoris successfully expressed the fusion protein GGH. The yield of GGH reached 78 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 246 mg/L in 52 h. The fusion protein GGH was purified in four steps, and the final purity was 96.1%. The in vitro bioactivity of GGH was the same as that expressed in P. pastoris by the AOX1 promoter. This study described an efficient way to express GGH fusion protein in P. pastoris using GAP promoter, fermentation was easier to control without carbon source change and fermentation time was 20 h less than AOX1 promotercontrolled GGH fermentation.
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KEYWORD
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GAP promoter, GGH fusion protein, constitutive expression, Pichia pastoris, fed-batch fermentation
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