FEBS Letters · 1993 · 13 citations · 26 references
Life SciencesBiochemistryMolecular BiologyEnzymologyMolecular VirologyVirologyProtein PurificationProtein SynthesisProteolytic EnzymesProteolytic ProcessingViral MaturationViral Protein ProcessingEscherichia Coli Expression SystemsMetal Chelate Affinity ChromatographyProtease Cleavage SpecificityProtease Substrate SpecificityProteolytic Cleavage Of Viral PolyproteinsRefolding Of Inclusion BodiesViral Polyprotein ProcessingViral Proteases
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.
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