The Construction, Expression, And Activity Assay Of Metase Gene In Highly Efficient Prokaryote Expression System

Objective:To construct two highly efficient prokaryotic expression vectors of L-methionineγ-lyase(Metase) gene,and to establish the best efficient prokaryotic expression system.So that Metase from Pseudomonas putida was highly expressed in E.coli,and lay the foundation for studying anti-tumor effects of Metase.Methods:On the basis of Metase gene from Pseudomonas putida has been synthesized(pBSK-Metase),Metase gene was connected to the highly efficient prokaryotic expression vector pBV220 and pGEX-4T-1 respectively by molecular cloning technology.Then recombinant expression plasmids pBV220-Metase and pGEX-4T-1-Metase were gengrated.The two recombinant plasmids were transformed into the feeling E.coli DH5α,and then were spreaded on the agar plates which included ampicillin resistance.Next.the white monoclonal colonies were selected. The recombinant plasmids were extracted in small quantities and identified by PCR, two-enzyme digestion.positive clones were sent to sequenc.pBV220-Metase was heat-induced to express by 42℃;pGEX-4T-1-Metase was induced to express by IPTG.Then expressive conditions were optimized.Next.bacterial sediment was collected and broken by ultrasonic wave.Crude Metase was collected and Metase activity was measured.Statistical analvsis and comparison were carried out.Results:Two highly efficient prokaryotic expression system pBV220-Metase and pGEX-4T-1-Metase of Metase gene were constructed successfully,and the Metase gene sequence was confirmed to be correct by sequencing.After that recombinant expression plasmid pBV220-Metase and pGEX-4T-1-Metase were transformed into E.coli DH5αand induced,both of the Metase activity can be measured.Conclusion:Metase gene from Pseudomonas putida was highly expressed in Escherichia coli successfully.Recombinant expression plasmid pBV220-Metase had stronger Metase activity than pGEX-4T-1-Metase,and pBV220-Metase could be regarded as the best high-performance prokaryotic expression system of Metase.