Study On The Expression Ang Function Of Bsa? Methylases

DNA methylase can transfer the methyl from the donor to the specific recognition site of the target DNA,which is an important part of the realization of DNA methylation modification.M1.BsaI,M2.BsaI and restriction enzymeBsaI constitute a restriction-modification system.The overlapped recognition sequences of M1.BsaI,M2.BsaI and BsaI can be used as a transformable switch for methylation in vivo.By controlling the switch to methylate a specific site within a nucleic acid sequence,the target sequence can be assembled to a specific site as needed.Therefore,detailed characterizations of M1.BsaI and M2.BsaI is particularly important.The expression level and enzyme activity of M1.BsaI and M2.BsaI will directly affect the level of DNA methylation.The study of their expression and function will provide the basis for the establishment of the BsaI-M.BsaI DNA assembly system.This thesis investigated the expression and function of M1.BsaI and M2.BsaI from Bacillus stearothermophilus.The main contents and results are as follows:M1.BsaI and M2.BsaI were constructed into pBR322 vector.His6 tag was added to the N-terminus of the enzyme,and the expression was induced in E.coli.The proteins of M1.BsaI and M2.BsaI were obtained after Ni-TAT and molecular sieve purification.The enzyme activity dynamics of M1.BsaI and M2.BsaI were studied.The optimal reaction temperature of M1.BsaI was determined to be 55?.The maximum reaction rate was 0.24 ?M·s-1.The Michaelis constant was 0.596?M,and the catalytic constant was 8.51 s-1.The specific activity of the enzyme is 12.05 U/mg.The optimal reaction temperature for M2.BsaI is 50?.The maximum reaction rate is 0.33 ?M·s-1.The Michaelis constant is 1.05 ?M,and the catalytic constant is 7.08 s-1.The specific activity of the enzyme is 13.51 U/mg.The structure of M2.BsaI was simulated to obtain the active sites and ligand binding sites.The wild type was modified by site-directed mutation of alanine,and the E.coli expression system of pHisC-M2.BsaI was reconstructed to detect the enzymatic activity in vivo.Detection and comparison of the in vivo enzyme activity of the mutant and the wild type confirmed that the active site is glutamic acid at site 245,and the ligand binding sites are glycine at site 134 and aspartic acid at site 154.