Soluble glypican3 gene cloning and expression in Pichia pastoris

Objective To clone the soluble phosphatidyl glypican 3(sGPC3) gene and express this recombinant protein.Methods GPC3 cDNA was cloned by RT-PCR from human hepatoma cells hepG2.The cDNA encoded GPC3 lacking the signal and GPI-anchored domain was inserted into the pPICZαA vector to construct an expression plasmid,named pPICZαA-sGPC3.Then the plasmid was transformed into a Pichia pastoris strain X-33,and the positive strains were screened on the YPD plates with Zeozin and identified by PCR.The selected strain was induced by methanol and the supernatants were analyzed by SDS-PAGE and western blotting.Results The DNA fragment about 1 600 bp was amplified by RT-PCR.The recombinant plasmid pPICZαA-sGPC3 was obtained successfully and the reading frame was correct.The supernatant of X-33/pPICZaA-sGPC3 had a specific 60 000 band as anticipated,and bound with mouse anti-6×His antibody.Conclusion sGPC3 has been successfully cloned and expressed,which lay the foundation for further functional and mechanism study.