ABSTRACT
The gene coding for the HIV-1 protease was cloned in an Escherichia coli expression vector adding three-histidine codons to the amino and carboxy terminus of the protease sequence. Expression of the protease from this construct led to the accumulation of high amounts of insoluble histidine-linked protease entrapped in inclusion bodies. The histidine-linked protease could be efficiently released from purified inclusion bodies with 6 M guanidine hydrochloride and further purified by metal chelate affinity chromatography. The refolded protease cleaved synthetic peptide substrates and the viral polyprotein p55 with the same specificity as the wild type protease. It displays a specific activity of 4.4 mumol/min/mg.
Subject(s)
Cloning, Molecular , Gene Expression , HIV Protease/genetics , Histidine , Peptides/metabolism , Amino Acid Sequence , Base Sequence , Blotting, Western , Escherichia coli/genetics , Gene Products, gag/metabolism , HIV Core Protein p24/metabolism , HIV Protease/chemistry , HIV Protease/metabolism , HIV-1/enzymology , Hydrogen-Ion Concentration , Molecular Sequence Data , Protein Folding , Protein Precursors/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purificationABSTRACT
Elimination of the protease domain from the polymerase open reading frame (pol) of the human immunodeficiency virus type 1 (HIV-1) leads, in Escherichia coli, to synthesis and accumulation of a non-processed 98-kDa reverse transcriptase/endonuclease (RT/ENDO) polyprotein. A partially purified preparation of this reverse RT/ENDO polyprotein displays little or no RT activity. Introduction of the pol protease domain as a separate transcriptional unit on the same plasmid restores the processing program, generating correctly sized RT and ENDO polypeptides. Concomitant with restoration of processing is the reappearance of RT activity. These results suggest that for HIV-1 RT to be active, it must be matured from the pol polyprotein.
