Molecular Cloning And Protein Expression Of Aldehyde Dehydrogenase 2 Variant(E487K)

Aldehyde dehydrogenase(ALDH)is a key enzyme that exists in the liver mitochondria to decompose acetaldehyde,an intermediate product of alcohol metabolism.Aldehyde dehydrogenase 2(Aldh2),which is in the liver mitochondria,is an important ALDH involved in human alcohol metabolism.However,the percentage of site-directed mutagenesis at position 487 of Aldh2(E487K)in East Asian population,which changes from glutamate to lysine,is significantly higher than that in European and American population.Eastern Asians carrying the mutant Aldh2(E487K)are very sensitive to ethanol and can lead to blush and dizziness after drink a little.What's more,the mutagenesis and carcinogenesis of acetaldehyde are very harmful to human body.The aim of this study is to clone Aldh2(E487K)gene by genetic engineering,and then transfer it into E.coli expression system to prepare recombinant Aldh2(E487K).The main results were as follows:1.First of all,extract total RNA from human hepatoma cells,and then reverse transcribed to cDNA.According to the gene sequence data registered in GenBank,designing specific primers with site-directed mutagenesis at position 487.Aldh2(E487K)gene was amplificated by PCR and linked to the pMD19-T-simple vector.The plasmid pMD19-T-ALDH2(E487K)was identified by EcoR I and Nde I digestions.After sequencing,the plasmid sequencing result showed that the sequence homology was 100%.The target fragment was subcloned into the pET44b(+)plasmid and then transformed into E.coli BL21(DE3).Electrophoretic results of double digestions showed that there were twobands at 1500bp and 5600bp,suggested that the results of subcloning is correct.The sequencing results also showed that Aldh2(E487K)gene was successfully inserted.2.The optimum conditions were explored by changing the IPTG concentrations,induction time and temperature.The experimental results showed that the optimal expression condition of ALDH2(E487K)was induced with 0.5mM IPTG for 4 hours at 37 ?.Western-blot results showed the specific reaction bands,which means the recombinant proteins was His-tagged target protein and existed as inclusion bodies.3.The recombinant protein was renatured and purified by Ni2+-NTA affinity chromatography.The purification conditions were optimized by changing the imidazole concentrations in the equilibrium buffer.The results showed that the content of impurity proteins was significantly reduced when using the equilibrium buffer containing 30 mM imidazole.The total IOD value of the target protein was about 2192.39 and the purity of the target protein reached 98%.The final concentration of ALDH2(E487K)was 107.7 ?g/mL,which proved that the ALDH2(E487K)was refolded as soluble state.4.According to the method of dehydrogenase activity determination,the enzymatic activity of ALDH2(E487K)was 0.71U/mL,about half of ALDH2.The optimum reaction temperature and pH were optimized.It was found that the optimum reaction temperature of ALDH2(E487K)was 35?,and the optimum pH was 8.The optimum reaction condition didn't appear to be much different from ALDH2.In addition,metal ions can promote the activities of ALDH2(E487K)and ALDH2 with varying degrees,thereinto Mn2+ and Zn2+ showed more significant promote effect.Through calculating by Lineweaver-Burk,the Km values of ALDH2(E487K)and ALDH2 to acetaldehyde were 104.17?M and 30.45 ?M,respectively.The Kcat/Km ratios were 0.192 s-1mM-1 and 1.17 s-1mM-1,respectively.It can be seen that ALDH2 has better affinity and higher catalytic efficiency for acetaldehyde than ALDH2(E487K).What's more,the thermal stability studies showed that temperature had greater influence on the enzyme stability,and ALDH2(E487K)had good stability at 50 ?.Acid base stability studies showed that the stability of the ALDH2(E487K)in alkaline environment was worse than ALDH2,while there was no significant difference between them in acidic environment.In this study,ALDH2(E487K)was prepared by genetic engineering and microbial fermentation.By optimizing its expression and purification conditions,the recombinant protein with higher expression and purity was obtained.Our study fills in the gaps of home and provides a reference and theoretical basis for the follow-up mutant design in our laboratory.Meanwhile,it also provides a new idea for the development of aldehyde dehydrogenase antialcoholic products.