Construction, expression, and purification of prokaryotic expression vector for PML-RARα fusion gene related proteins

Objective To construct, express, and purify the prokaryotic expression vector for PML-RARα fusion gene related proteins. Methods PML-RARα-pET32a(+) plasmid was used as a template and 1 200 bp sequences of PML and RARα were amplified by polymerase chain reaction and subcloned into expression vector pET32a(+) to construct recombinant plasmid; then the ligated products were transformed into DH5α. The positive clone was propagated and the recombinant plasmids were extracted for further identification and sequencing. The correct recombinant plasmids were named after PML-Flag-pET32a(+) and Flag-RARα-pET32a(+) respectively. E.coli BL21(DE3) were transformed with PML-Flag-pET32a(+) and Flag-RARα-pET32a(+), respectively, and induced with IPTG for protein expression. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography. Purified protein was identified by SDS-PAGE and Western blotting. Results The purpose gene was got by PCR amplification; it was confirmed by EcoRI/HindIII double enzyme digestion and DNA sequencing that the recombinant plasmids PML-Flag-pET32a(+) and Flag-RARα-pET32a(+) were constructed successfully. PML and RARα proteins were expressed efficiently after the recombinant plasmids had been transformed and induced in E.coli BL21(DE3); SDS-PAGE and Western blotting demonstrated that the purified proteins were interest proteins. Conclusions Construction of recombinant plasmids and expression and purification of the PML and RARα proteins laid a solid foundation for antibody preparation. Key words: PML-RARα fusion gene; Recombinant plasmid; Protein expression; Protein purification; Acute promyelocytic leukemia (APL); Antibody preparation.