Expression and purification of active form of HIV-1 protease from E.coli.

We have subcloned an N-terminal extended protease gene of human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame into expression vector pGEX-KG. A relatively high level of expression of recombinant HIV-1 protease (PR) was achieved with isopropyl beta-D-thiogalactoside (IPTG) induction and glucose supplement. An isolation method consisting of denaturation of protein and followed by refolding was developed for releasing this recombinant HIV-1 PR into the soluble phase since most of the expressed protease was initially present in insoluble inclusion bodies. High purity of this recombinant HIV-1 PR was obtained by sequential purification using Sephadex G-50 gel filtration and CM-23 cellulose cation exchange chromatography, yielding the protease more than 1 mg per liter culture. N-terminal amino acid sequence analysis showed that the recombinant HIV-1 PR underwent autocleavage from the fusion protein during expression. SDS-PAGE indicated that the molecular weight of this recombinant HIV-1 PR is 11 kDa. This recombinant HIV-1 PR showed proteolytic activity for the synthetic peptide substrates corresponding to the sequence at the Gag MA/CA and Pol p6*/PR junctions. The purified enzyme whose specific activity for the heptapeptide SQNYPIV was 848.7 nmol*min-1*mg protease-1 also processed recombinant polyprotein Gag41 as its substrate.