Human immunodeficiency virus protease. Bacterial expression and characterization of the purified aspartic protease.
The protease of human immunodeficiency virus has been expressed in Escherichia coli and purified to apparent homogeneity. Immunoreactivity toward anti-protease peptide sera copurified with an activity that cleaved the structural polyprotein gag p55 and the peptide corresponding to the sequence gag 128-135. The enzyme expressed as a nonfusion protein exhibits proteolytic activity with a pH optimum of 5.5 and is inhibited by the aspartic protease inhibitor pepstatin with a Ki of 1.1 microM. Replacement of the conserved residue Asp-25 with an Asn residue eliminates proteolytic activity. Analysis of the minimal peptide substrate size indicates that 7 amino acids are required for efficient peptide cleavage. Size exclusion chromatography is consistent with a dimeric enzyme and circular dichroism spectra of the purified enzyme are consistent with a proposed structure of the protease (Pearl, L.H., and Taylor, W.R. (1987) Nature 329, 351-354). These data support the classification of the human immunodeficiency virus protease as an aspartic protease, likely to be structurally homologous with the well characterized family that includes pepsin and renin.
Darke
P L
PL
Department of Molecular Biology, Merck, Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Leu
C T
CT
Davis
L J
LJ
Heimbach
J C
JC
Diehl
R E
RE
Hill
W S
WS
Dixon
R A
RA
Sigal
I S
IS
D016282:Aspartic Acid Endopeptidases; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005814:Genes, Viral; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D015151:Immunoblotting; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D013379:Substrate Specificity
D004274:DNA, Recombinant; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases