Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6xHis tag in Pichia pastoris.
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 6xHis tags were subcloned into the plasmid pPIC9K. The recombinant protein was expressed in P. pastoris GS115 and purified using Ni(2+)-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 Ni(2+)-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.
Zhao
YuFeng
Y
State key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agriculture University, Wuhan, 430070, China.
Lei
MingKe
M
Wu
YuanXin
Y
Zhang
ZiSheng
Z
Wang
CunWen
C
D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D019149:Bioreactors / Q000382:microbiology; D002846:Chromatography, Affinity; D003062:Codon / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D015971:Gene Expression Regulation, Enzymologic; D006639:Histidine / Q000235:genetics; D006801:Humans; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*
D003062:Codon; D018076:DNA, Complementary; D011994:Recombinant Proteins; D006639:Histidine; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial