Expression, purification, and bioactivity of (GLP-1˄A2G)˄2-HSA analogs in Pichia pastoris GS115

• Published in: Biotechnology and bioprocess engineering Vol. 18; no. 6; pp. 1076 - 1082
• Main Authors: Dou, W.F., Jiangnan University, Wuxi, China, Feng, J.S., Jiangnan University, Wuxi, China, Zhang, X.M., Jiangnan University, Wuxi, China, Xu, H.Y., Jiangnan University, Wuxi, China, Shi, J.S., Jiangnan University, Wuxi, China, Xu, Z.H., Jiangnan University, Wuxi, China
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.12.2013; Springer Berlin Heidelberg; 한국생물공학회
• Subjects: absorption; activity,analog; Biotechnology; Chemistry; Chemistry and Materials Science; drugs; hydrophobicity; Industrial and Production Engineering; ion exchange chromatography; noninsulin-dependent diabetes mellitus; PICHIA PASTORIS; plasmids; polyacrylamide gel electrophoresis; proteins; PURIFICACION; PURIFICATION; Research Paper; size exclusion chromatography; ultrafiltration; yeasts; 생물공학; analog; GGH; GLP-1; activity; purification
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-013-0356-7

We developed (GLP-1˄A2G)˄2-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.