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Expression, Purification, and Bioactivity of (GLP-1A2G)2-HSA Analogs in Pichia pastoris GS115
Wenfang Dou,Junshang Feng,Xiaomei Zhang,Hongyu Xu,Jinsong Shi,Zhenghong Xu 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.6
We developed (GLP-1A2G)2-HSA (GGH) analogsthat are resistant to degradation and also show high serumglucose-reducing activity in vivo. Five analogs with Nterminalextensions were designed based on the proteinGGH. Next, we constructed recombinant plasmids capableof expressing the five analogs in methylotrophic yeastPichia pastoris GS115. Expression reached 150 mg/L in asmall-scale incubation. Fusion proteins were successfullypurified from the supernatant using ultrafiltration concentration,affinity absorption chromatography, hydrophobicchromatography, ion exchange chromatography and gelfiltration. A single band was observed on SDS-PAGE andthe purity was 97%. Activity test results suggested thatboth A-GGH and G-GGH showed better activity in vitroand that their cAMP levels were significantly increased by10-fold compared to GGH without N-terminal extension. Additionally, A-GGH efficiently enhanced the glucoseloweringeffect, which was maintained after the administrationfor 24 h. A-GGH is a potential drug for treating type 2diabetes.
Hui Li,Zhenzhen Fu,Heng Li,Wenfang Dou,Jinsong Shi,Zhenghong Xu 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.3
This study investigated the effects of hydroxylase cyptochrome P450 inducers on the efficiency of the biotransformation of dehydroepiandrosterone (DHEA) to 3β, 7α, 15α-trihydroxy-5-androsten-17-one (7α,15α-diOHDHEA)by Colletotrichum lini ST-1. Special attention was given to the substrate DHEA being the best inducer and the fact that it could improve the yield of 7α, 15α-diOHDHEA. Based on the effects of the DHEA pre-induction phases and additional concentrations on 7α, 15α-diOHDHEA production, a substrate pre-induction process was established as follows: 2 g/L DHEA was added for the first time after 12 h of inoculation, followed by the second addition of 6 g/L DHEA after 12 h later. The results showed that this substrate pre-induction process improved the content of cytochrome P450 and that the 7α, 15α-diOH-DHEA yield reached 90.1%, which was 26.9%higher than that obtained in the original process.