Studies On The Expression,Purification And Activity Of Acetaldehyde Dehydrogenase 2

After the human body consumes alcohol,acetaldehyde and acetic acid were produced in the metabolic process.Among them,acetaldehyde was a substance harmful to the human body and was classified as a second-class carcinogen.Excessive accumulation of acetaldehyde in the human body can cause uncomfortable reactions such as nausea and vomiting,and even trigger shock.Studies had shown that acetaldehyde dehydrogenase 2(ALDH2)was a key enzyme involved in the oxidation of acetaldehyde in the body and could oxidize acetaldehyde to acetic acid.However,ALDH2 mutants would significantly reduce the activity.Acetaldehyde that cannot be successfully removed may cause DNA damage,which may lead to many diseases e.g.cancer.The supplementation of highly active ALDH2 could alleviate the damage of acetaldehyde to human body.Therefore,looking for a more optimized method to obtain large amount of highly active ALDH2 protein could provide a scientific basis for the subsequent applied research.Therefore,this thesis has carried out the following three aspects of research work focusing on ALDH2.(1)Screening of alcohol metabolism in healthy peopleMouthwash samples were collected the from healthy persons,and the whole human genome from each sample was obtained by extraction with Easy Pure? Micro Genomic DNA Kit from the samples.The SNP analysis method was used to analyze the genotypes of GABRA2,ADH1 B and ALDH2 genes.Then the tester’s theoretical alcohol metabolism ability can be inferred from the genotypes.The results showed that about 20% of the test population had reduced metabolic capacity of acetaldehyde metabolism due to ALDH2 mutations.(2)Prokaryotic expression and purification of ALDH2Many people need to supplement exogenous ALDH2 because ALDH2 mutations cause a decrease in acetaldehyde metabolism.In order to obtain a large amount of human ALDH2 protein,the ALDH2 gene was constructed on the p ET32 a vector containing the TrxA fusion protein tag.The plasmid was transformed into Escherichia coli,and the expression conditions were optimized to maximize the soluble expression of TrxA-ALDH2.The soluble expression of TrxA-ALDH2 was increased by 97.5% as compared to ALDH2 alone.The renaturation conditions of TrxA-ALDH2 inclusion body were studied.Under the optimal renaturation conditions,the activity of TrxA-ALDH2 was 0.510 IU/mg,which can reach 58.32% of soluble expressed protein.(3)Eukaryotic expression and purification of ALDH2Due to the limitations of the E.coli expression system,which expressed large amount of inclusion body protein,we further explored the expression of human ALDH2 in Saccharomyces cerevisiae.The ALDH2-p YX212 and ALDH2-p YES2 expression vectors were successfully constructed,and could be expressed in S.cerevisiae W303a(ALDH2-YX and ALDH2-YS strains).The results showed that active ALDH2 protein could be obtained in this eukaryotic expression system.In summary,in this thesis,mutations of drinking-related genes were screened in some healthy people,and we found that about 20% of the population reduce the metabolic capacity of acetaldehyde due to ALDH2 mutations and need to supplement exogenous ALDH2.We expressed the human ALDH2 gene in E.coli and S.cerevisiae respectively,and isolated,purified,and studied the activity of target protein in these two expression systems.At present,there is no ALDH2 product with anti-alcohol effect on the market.This thesis provides a scientific basis for the large-scale preparation and application research of human ALDH2 protein.