Cloning, Expression And Protein Purification Of M₂-type Pyruvate Kinse

Objective:To constract the pET30a (+)-PKM2expression plasmid, amplifiy this plasmid in E.clio DH5a.express and purify the fusion proteion in E.clio BL21,the recombinant6×His fused protein was perified by Ni-NTA purification.Methods:Extract RNA from Hela cell,and then reverse transcriptase the RNA,we can get cDNA.The PKM2gene was amplified by PCR from cDNA template.Use restriction enzyme(BamHⅠ、Sal Ⅰ and NdeⅠ、Sal Ⅰ)digestion PKM2and pET30a (+),get recombination plasmid pET30a (+)-PKM2by T4DNA ligase. Restriction enzyme digestion and sequence recombination plasmid pET30a (+)-PKM2, PKM2sequence is correct.Express and purify the fusion proteion in E.clio BL21,the recombinant6×His fused protein was perified by Ni-NTA purification and dialysis method.The recmbination proteion electrophoresis get through SDS-PAGE,decoration by Coomassie brilliant blue method,analyzed by gel imaging and analysis system.Results:Successed get PK.M2through PCR.The recombination plasmid pET30a (+)-PKM2were identified by restriction enzyme digestion and nucleotide sequencing.The result of the recombination proteion electrophoresis get through SDS-PAGE,show E.clio BL21can express recombination proteion PKM2. After Ni-NTA and dialysis method purification,the molecular weight of the recombination proteion PKM2approximately58KDa. preparations of the recombination proteion PKM2showed a single diffuse band on SDS-PAGE.Conclusion:The successfully constract the pET30a(+)-PKM2expression plasmid. Recmbination proteion PKM2is succeed expressed and purified,which can provide basis for research of tumor vaccine and tumor diagnosis.