Purification of human immunodeficiency virus-1 p66 protein expressed at low temperature and long-time induction and the identification of its biological activity

Objective Reverse transcriptase,a heterodimer composed of subunits p66 and p51,is considered as an important target for AIDS therapy.HIV-1 replication can be blocked by inhibition of the reverse transcriptase activity.The obtainment of purified HIV-1 p66 protein will help to study its activity and develop a screening assay for anti-HIV-1 drugs.In the present study,we constructed a p66 prokaryotic expression plasmid,and obtained highly pure and active HIV-1 p66 recombinant protein.Methods The sequence encoding HIV-1 p66 protein was amplified by PCR from the plasmid containing the HIV-1 HXB2 genome,and then cloned into the pET-28a(+) vector by restriction enzyme digestion and ligation to construct the prokaryotic expression plasmid pET-28a(+)-p66.Then it was transformed into E.coli BL21(DE3) strain.IPTG was used to induce the expression of p66.Finally,nickel affinity gel was used to purify the p66 protein.Results A 1680 bp fragment was identified by restriction enzyme digestion.The sequencing result indicated that the HIV-1 p66 gene was cloned into pET-28a(+) correctly.Conclusion The prokaryotic expression plasmid pET-28a(+)-p66 was constructed successfully.Highly expressed and active HIV-1 p66 recombinant protein was obtained.