Soluble Expression Of Human Aldehyde Dehydrogenase 2(ALDH2)in E.coli

Aldehyde dehydrogenase 2（ALDH2）plays an important role in the human body’s alcohol metabolism.The recombinant ALDH2 expressed in E.coli as host cells is easy to form inclusion bodies and has low activity by genetic engineering.In this study,ALDH2 and NusA label were fused and expressed,Kp-SP and other signal peptides were added for secretion and expression,so as to obtain the recombinant protein with high soluble expression and activity in E.coli.The main research contents are as follows:1.Recombinant expression vector of construction:according to the ALDH2 gene sequence design primers,introducing Eco RⅠand XhoⅠdigestion sites,propagated the ALDH2 gene fragments by PCR,connected to the p MD19-T-simple carrier,and converted to E.coli DH5αstrains.Digestion of p MD19-T-ALDH2 and expression vector p ET44b（+）with Eco RⅠand XhoⅠ,after sequencing right the ligands were transformed into the competent state E.coli BL21（DE3）.At the same time,Kp-SP signal peptide was introduced in Aldh2 gene at the position between NdeⅠand XhoⅠof p ET22b（+）,then transformed into the competent state E.coli BL21（DE3）.2.Expression of recombinant proteins:These recombinant proteins were induced to be expressed by E.coli through IPTG,but no target proteins were detected in the medium supernatant,indicating that ALDH2 was not secreted into extracellular expression.The NusA-ALDH2 fusion protein was expressed in the cells and obtained good solubility,and mainly existed in the supernatant after the bacteria utrasonication.Kp-SP-ALDH2 is highly expressed in cells,but exists as an inclusion body.To explore the optimal expression conditions of NusA-ALDH2 and Kp-SP-ALDH2,both of which can reach a high expression level at 3 h.When the former was induced to express at 37℃,the target protein yield was relatively high,while the optimal expression temperature of Kp-SP-ALDH2 was 30℃.Several IPTG concentrations had little influence on protein expression.Considering certain toxicity of IPTG,0.25m M and 0.5m M were selected as the optimal IPTG concentrations for NusA-ALDH2 and Kp-SP-Al DH2 respectively.Western Blot was used to identify the recombinant proteins of NusA-ALDH2 and Kp-SP-ALDH2.Kp-SP-ALDH2 was successfully identified,while NusA-ALDH2 showed no obvious color development.It may be that the large molecular weight of NusA protein concealed the histidine label that should have been exposed.3.ALDH2 was purified by Ni²⁺-NTA affinity chromatography column.When the imidazole concentration in the equilibrium buffer was 35 m M,the heteroproteins could be effectively removed.The purified protein concentration was 405.507μg/m L.In the case of NusA-ALDH2,the imidazole of 20 m M is added to the equilibrium buffer to remove the heteroprotein,which is then released in the presence of enterokinase with high purity.4.The activity of NusA-ALDH2 supernatant and the purified Kp-SP-ALDH2were detected,NusA-ALDH2 enzyme activity was 1.643 U,and Kp-SP-ALDH2enzyme activity was 1.029 U.The optimal reaction temperature and p H of the two enzymes were optimized,NusA-ALDH2 optimal reaction temperature was 37℃,the optimal was p H 8.0.The optimum temperature of Kp-SP-ALDH2 was 30℃,and the optimum p H was 8.0.Different metal ions such as Ca²⁺,K⁺,Na⁺,Mg²⁺,and Mn²⁺can activate these two enzymes and Mg²⁺has the best effect.In this study,the soluble expression of ALDH2 in E.coli was realized through the introduction of solubilized label NusA for fusion expression,and the recombinant protein has certain biological activity.However,the results of introducing Pel B,Dsb A,Omp A and Kp-SP signal peptides are not satisfactory,and the cloning conditions need to be further optimized.