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

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Abstract

Human mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) catalyzes the oxidation of acetaldehyde to acetic acid. Therefore, ALDH2 has therapeutic potential in detoxification of acetaldehyde. Furthermore, 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.

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Authors and Affiliations

  1. State key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agriculture University, Wuhan, 430070, China

    YuFeng Zhao

  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

    YuFeng Zhao, MingKe Lei, YuanXin Wu & CunWen Wang

  3. Department of Chemical Engineering, University of Ottawa, Ottawa, Canada, K1N 6N5

    ZiSheng Zhang

Authors
  1. YuFeng Zhao
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  2. MingKe Lei
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  3. YuanXin Wu
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  4. ZiSheng Zhang
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  5. CunWen Wang
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Corresponding author

Correspondence to YuanXin Wu.

Additional information

Supported by the International Collaboration Key Project of Hubei Province (Grant No. 2006CA013) and Sci-Tech Brainstorm Stress Projects of Hubei Province (Grant Nos. 2007AA201C27 and 2007AA301B24).

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Zhao, Y., Lei, M., Wu, Y. et al. Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6×His tag in Pichia pastoris. SCI CHINA SER C 52, 935–941 (2009). https://doi.org/10.1007/s11427-009-0134-0

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  • DOI: https://doi.org/10.1007/s11427-009-0134-0

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