Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6×His tag in Pichia pastoris

查看全文 作　　者：ZHAO YuFeng1,2, LEI MingKe2, WU YuanXin2, ZHANG ZiSheng3 & WANG CunWen2 1 State key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agriculture University, Wuhan 430070, China;2 Key Laboratory for Green Chemical Processes of the Ministry of Education, Hubei Key Lab of Novel Reactor & Green Chemical Technology, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430074, China;3 Department of Chemical Engineering, University of Ottawa, Ottawa, Canada K1N 6N5

出　　处：《Science China(Life Sciences)》索引2009年第52卷第10期,共7页

基　　金：Supported by the International Collaboration Key Project of Hubei Province (Grant No. 2006CA013);Sci-Tech Brainstorm Stress Projects of Hubei Province (Grant Nos. 2007AA201C27 and 2007AA301B24). 摘　　要：Human mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) catalyzes the oxidation of acetaldehyde to acetic acid. Therefore, ALDH2 has therapeutic potential in detoxification of acetaldehyde. Further-more, ALDH2 catalyzes nitroglycerin to nitrate and 1, 2-glyceryldinitrate during therapy for angina pectoris, myocardial infarction, and heart failure. Large quantities of ALDH2 will be needed for potential clinical practice. In this study, Pichia pastoris was used as a platform for expression of human ALDH2. Based on the ALDH2*1 cDNA sequence, we designed ALDH2 cDNA by choosing the P. pastoris preferred codons and by decreasing the G + C content level. The sequence was synthesized using the overlap extension PCR method. The cDNA and 6×His tags were subcloned into the plasmid pPIC9K. The recombinant protein was expressed in P. pastoris GS115 and purified using Ni2+-Sepharose affinity chromatography. The amount of secreted protein in the culture was 80 mg/L in shake-flask cultivation and 260 mg/L in high-density bioreactor fermentation. Secreted ALDH2 was easily purified from the culture supernatant by using Ni2+-Sepharose affinity chromatography. After purification of the fermentation supernatant, the enzyme had a specific activity of 1.2 U/mg protein. The yield was about 16 mg/L in a shake flask culture of P. pastoris GS115 which contained the original human ALDH2*1 cDNA. 关键词：ACETALDEHYDE DEHYDROGENASE CODON optimization overlap extension PCR PICHIA pastoris.

相关文献

参考文献（27）
引证文献（3）
耦合文献（29）

参考文献(27)

引证文献(3)

耦合文献(29)

互联网出版许可证新出网证(渝)字10号全国400电话 - 免长途话费

Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6×His tag in Pichia pastoris

参考文献(27)

引证文献(3)

耦合文献(29)

用户提示