Authors: Kai Qian XiaoHai Gong Bo Guan SuPing Wu JingJing Zhang Jing Qian YanFei Cai Yun Chen ZuoYing Duan Xin Ma HuaZhong Li Jian Jin
Publish Date: 2015/09/25
Volume: 20, Issue: 4, Pages: 694-700
Abstract
Glucagonlike peptide1 GLP1 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 GLP1 GLP1A2G 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 αfactor secretion signal sequence and was expressed by the glyceraldehyde3phosphate 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 Fedbatch 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 961 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
Keywords: