Cloning, Expression And Activity Analysis Of Artificial Endonuclease For Recognition DNA Methyl-sites

DNA methylation plays an important biology function, and it's the main method to carry on the methylation analysis by methylation sensitive restrictive interior contact enzyme. The research uses the DNA reorganization technology to construct an artificial interior contact enzyme gene which can identify DNA methylation. The reorganization material particle was expressed by E.coli, and tring to purify the target protein and analyzing enzymatic activity.Based on analyzing restriction endonuclease Bmr I catalytic region and CpG methylation of DNA repair protein MBD4 Sequence, synthesizing two clone primers BmrI-linker and linker-MBD4 separately, then BmrI-MBD4 recombinant gene was constructed by splicing overlapping and extension, and the cloned genes of Bmr I-MBD4 were 834bp. BmrI-MBD4 recombinant gene was obtained by PCR and cloned into expression vector pET-21b(+), transformed into E.coli ER2984. After NdeI and BamHI double digestion screening and DNA sequencing, the target rcombinant clone was gained.The reorganization material was transformed into E.coli ER2566 (DE3). After transforming 5h with 16℃, 30℃, 36℃respectively, the target protein was expressed as an inclusion body. The research has indicated that lowering temperature and prolonging induction time could realize soluble expression. After 37℃and 5h cultivating, inclusion body denaturing and refoldeing, the target protein was separated by cation exchange chromatography, and the recombinant protein molecular weight is 330213.91 Da. The refolding protein Bmr I-MBD4 could degrade pACYC-EagI M and pAIT2-CpG M or nicking these plasmids, and which could cause polymerization of plasmids also. The refolding protein also showed lower similar activities with pUC19.