Cloning and Fusion Expression of Predigested Human Plasminogen Kringle-5 in Escherichia coli

The predhPK-5 gene was cloned from hominal liver tissue with RT-PCR,and then the predhPK-5 gene was cloned into prokaryotic expression plasmid pGEX-1λT to construct the co-expression plasmid pGEX-1λT/predhPK-5.The pGEX-1λT/predhPK-5 was transformed into E.coli JM109.The positive clone was identified by PCR、restriction enzyme analysis and sequence analysis.The fusion protein GST/predhPK-5 was expressed by IPTG induced and identified by Western-Blotting.The size of RT-PCR product of predhPK-5 is 264bp.The evidences of PCR、enzyme digestion and sequence analysis confirmed that predhPK-5 gene has been correctly recombinant with pGEX-1λT.The recombinant expression plasmid pGEX-1λT/predhPK-5 successfully expressed GST/predhPK-5 fusion protein in E.coli JM109,and the Mr of the fusion protein is about 3.6×10~(4),which is according with the Mr forecasted by Expert Protein Analysis System on internet.And the fusion protein expression quantity occupies 23.6% according to the whole expression protein.