Codon Optimization And Molecular Cloning Of Hunan Aldehyde Dehydrogenase 2

Aldehyde dehydrogenase?ALDH?is one of the most important enzymes involved in human alcohol metabolism.ALDH is a large family of proteins,and ALDH2 in liver mitochondria plays a major role in alcohol metabolism.ALDH2 can reduce the toxicity of acetaldehyde by converting acetaldehyde produced during alcohol metabolism into acetic acid.However,lots of asian people lack normal ALDH2 and are vulnerable to alcohol poisoning.In recent years,with the improvement of people's health awareness,the demand for antialcoholic and liver-protecting drugs is increasing.Therefore,the study of ALDH2 is meaningful.At present,commercial ALDH2 is mainly extracted from animal liver or hepatocyte mitochondria,which is expensive and difficult to be mass produced.The aim of this study is to construct prokaryotic expression vector by genetic engineering,and then transfer it into E.coli,using microbial fermentation to prepare recombinantALDH2.Meanwhile,codon optimization technology was adopted to improve the expression level of recombinant ALDH2.Specific research is as follows:1.Total RNA was extracted from hepatoma cells and then reverse transcribed to cDNA.The Aldh2 fragment was obtained by PCR amplification and linked to the pMD19-T-simple vector.The recombinant plasmid was transformed into E.coli DH5?.The recombinant plasmid was extracted and identified by double digestion and sequencing to ensure that the target gene was linked to the cloning vector.Homology analysis showed that the sequence homology was 100%.The ALDH2 fragment was subcloned into the pET44b?+?plasmid and then transformed into E.coli BL21?DE3?.The EcoR I and Nde I double digestions of pET44b-ALDH2 showed two bands at 1515 bp and 5600 bp,suggested that the subcloning is correct.Sequencing result also confirmed the successful subcloning.2.The gene sequence of ALDH2?CR45699?was optimized according to the codon bias of E.coli.The rare codons were replaced with the preferred codons,without changing the amino acid sequence,and the codon adaptation index?CAI?and GC content were optimized as well.The optimized sequence was synthesized and named as ALDH2-OP.The results showed that the CAI increased from 0.73 to 0.96 and the GC content decreased from 57%to 49%after optimization,which was more conducive to the expression of target protein in E.coli.Compared with the original sequence,311 bases were altered,but their amino acid sequences were same.3.The synthesized Aldh2-OP fragment had been constructed into the pMD19-T-simple vector.The Aldh2-OP fragment was cloned into the pET44b?+?plasmid and then transformed into E.coli BL21?DE3?.Double enzyme digestion analysis showed ttwo bands at 1515 bp and 5600 bp,indicated that the result was correct.The sequencing result also confirmed that the prokaryotic expression vector pET44b-ALDH2-OP was successfully constructed.4.IPTG was used to induce the expression of recombinant proteins.The optimum conditions were explored by changing the induction time,temperature and IPTG concentrations.Western-blot was adopted to identify the recombinant proteins.This study showed that the optimal expression condition of ALDH2 was induction with 0.75 mM IPTG for 3 h at 37 ?.As for ALDH2-OP,the optimal expression condition was induction with 0.25 mM IPTG for 4 h at 37 ?.Western-blot results showed that the two recombinant proteins were His-tagged target proteins and existed as inclusion bodies.Electrophoretic pictures of ALDH2 and ALDH2-OP were analyzed by Image-Pro Plus software.The results showed that the IOD values of ALDH2 in total protein and precipitation fractions were 1386.5 and 1593.0,respectively.The corresponding IOD values of ALDH2-OP were 2030.7 and 2434.5,respectively.The proportion of ALDH2-OP in total protein?47.6%?was significantly higher than that of ALDH2?32.9%?.It indicated that codon optimization can significantly improve the expression of target protein.5.The recombinant proteins were purified by Ni²⁺-NTA affinity chromatography,and the purification conditions were optimized by changing the imidazole concentrations in the equilibrium buffer.It was found that the total IOD value of the target protein band was 2995.7 and the purity of the target protein reached 96.8%when using the equilibrium buffer containing 35 mM imidazole.The purified proteins were dialyzed overnight at 4 ? to ensure their refolding.The final concentrations of ALDH2 and ALDH2-OP were 194.80 ?g/mL and 300.39 ?g/mL,respectively.6.According to the method of dehydrogenase activity determination,the activity of ALDH2 and ALDH2-OP were 1.43 U/mL and 1.612 U/mL respectively by measuring 340 nm absorbance change.And the specific activity of ALDH2 and ALDH2-OP was 10.83 U/mg and 13.43 U/mg respectively.In addition,the optimum reaction temperature and pH of the enzyme were optimized.The results showed that the optimum pH of the enzyme was 8.5,and the enzyme activity reached the maximum at 30 ?.Acid base stability studies showed that the enzyme activity was higher in alkaline environment than in acidic environment.The thermal stability study showed that the enzyme had good stability at 40 ?.There was no significant difference in stability between ALDH2 and ALDH2-OP.The effect of metal ions on enzyme activity showed that Ca²⁺?K+?Na+?Mg²⁺? Mn²⁺promoted the activities of ALDH2 and ALDH2-OP.The Km?Vmax?Kcat and Kcat/of ALDH2 is 31.23 ?M?0.081 ?M/s?0.037 s-1 and 1.186 s-1mM-1,respectively.The Km?Vmax?Kcat and Kcat/Km of ALDH2-OP is 19.58?M?0.090 ?M/s?0.041 s-1and 2.145 s-1mM-1,respectively.In this study,codon optimization was used to improve the expression of human aldehyde dehydrogenase 2.The results laid a foundation for the realization of soluble expression of ALDH2 by computer-aided design,and provided an important theoretical basis for the research and development of antialcoholic drugs and relevant health products.