Cloning, expression and purification of a recombinant poly-histidine-linked HIV-1 protease.
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.
Leuthardt
A
A
Ciba-Geigy Ltd., Oncology and Virology Research Department, Basel, Switzerland.
Roesel
J L
JL
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015683:Gene Products, gag / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000378:metabolism; D016333:HIV Protease / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D015683:Gene Products, gag; D016655:HIV Core Protein p24; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D016333:HIV Protease