1. Expression, purification, and bioactivity of (GLP-1)-HSA analogs in Pichia pastoris GS115.
- Author
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Dou, Wenfang, Feng, Junshang, Zhang, Xiaomei, Xu, Hongyu, Shi, Jinsong, and Xu, Zhenghong
- Subjects
SERUM albumin ,GLUCAGON-like peptide 1 ,RECOMBINANT proteins ,FUNGAL gene expression ,BLOOD sugar ,PICHIA pastoris - Abstract
We developed (GLP-1)-HSA (GGH) analogs that are resistant to degradation and also show high serum glucose-reducing activity in vivo. Five analogs with N-terminal extensions were designed based on the protein GGH. Next, we constructed recombinant plasmids capable of expressing the five analogs in methylotrophic yeast Pichia pastoris GS115. Expression reached 150 mg/L in a small-scale incubation. Fusion proteins were successfully purified from the supernatant using ultrafiltration concentration, affinity absorption chromatography, hydrophobic chromatography, ion exchange chromatography and gel filtration. A single band was observed on SDS-PAGE and the purity was 97%. Activity test results suggested that both A-GGH and G-GGH showed better activity in vitro and that their cAMP levels were significantly increased by 10-fold compared to GGH without N-terminal extension. Additionally, A-GGH efficiently enhanced the glucoselowering effect, which was maintained after the administration for 24 h. A-GGH is a potential drug for treating type 2 diabetes. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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