Substitution mutations of the highly conserved arginine 87 of HIV-1 protease result in loss of proteolytic activity.
The 297bp gene coding for the HIV-1 protease was chemically synthesized and expressed in E. coli. Single amino acid substitutions (Arg 87 - greater than Lys; Arg 87 - greater than Glu) were introduced in the C-terminally located conserved region GlyArgAsn of the protease gene in the wild-type clone. The products of the mutant and the wild-type clones were expressed at approximately similar levels at 30 minutes of induction but the mutant protease proteins accumulated as a function of time of induction unlike the wild-type protease which declined after 60 minutes. The mutants were completely devoid of proteolytic activity as determined in assays employing as substrates a synthetic nonapeptide and a gag related recombinant polyprotein.
Louis
J M
JM
Division of Cancer Biology and Diagnosis, National Cancer Institute, Bethesda, MD 20892.
Smith
C A
CA
Wondrak
E M
EM
Mora
P T
PT
Oroszlan
S
S
D001120:Arginine / Q000235:genetics*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016333:HIV Protease; D015497:HIV-1 / Q000201:enzymology*; D006868:Hydrolysis; D009154:Mutation
D001120:Arginine; D010450:Endopeptidases; D016333:HIV Protease