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Cloning, expression and purification of a recombinant poly‐histidine‐linked HIV‐1 protease
Author(s) -
Leuthardt Andreas,
Roesel Johannes L.
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)81807-c
Subject(s) - protease , histidine , biochemistry , recombinant dna , escherichia coli , affinity chromatography , microbiology and biotechnology , inclusion bodies , expression vector , peptide sequence , biology , cloning (programming) , masp1 , hiv 1 protease , chemistry , amino acid , enzyme , serine protease , gene , computer science , programming language
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 μmol/min/mg.