Construction of prokaryotic expression plasmid PET-28a(+)-Ha-Ras and expression and purification of p21ras

Objective To construct the prokaryotic expression plasmid PET-28a(+)-Ha-ras and to obtain purified protein of p21ras. Methods Using RT-PCR, Ha-ras cDNA was generated from amplification of the total RNA extracted from human hepatocellular carcinoma cell line 7703. The purified product of Ha-ras cDNA was then inserted into the pMD18-T vector to form a recombinant pMD18-T vector-Ha-ras. The recombinant plasmid was cut by restriction enzymes BamH/Hind and then purified to become Ha-ras cDNA with viscous ends, as was PET-28a(+)to become a linear plasmid fragment with the same viscous ends as Ha-ras cDNA. The Ha-ras cDNA was combined with the PET-28a(+) that had been cut by the enzymes to construct the recombinant plasmid PET-28a(+)-Ha-ras. This plasmid was identified via restriction enzymes and was finally confirmed by the sequencing of the nucleotides. The correct PET-28a(+)-Ha-ras was transformed into BL21(DE3) to induce expression. The p21ras was purified by immobilized metal ion affinity chromatography with a histidine label (His-Tag). Results Sequence analysis showed that the Ha-ras cDNA sequence of PET-28a(+)-Ha-Ras had the identical sequence as the Ha-ras cDNA sequence printed in GenBank (accession NO.NM～005343). SDS-PAGE and Western-blot confirmed PET-28a(+)-Ha-ras was highly expressed in E.coli and the purified p21ras protein was generated by using Ni-NTA affinity chromatography. Conclusions The prokaryotic expression plasmid PET-28a(+)-Ha-ras has been constructed successfully. The successful production of active p21ras protein lays the foundation for exploring the effect of p21ras on the genesis of neoplasms and for the research on the intracellular antibody of p21ras against neoplasms.