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

Science china(Life Sciences) 2009年第10期52卷 935-941页

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 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

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.

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