A heterologous substrate assay for the HIV-1 protease engineered in Escherichia coli.
A heterologous substrate assay for the human immunodeficiency virus type 1 (HIV-1) protease has been engineered in Escherichia coli. This assay detects the activity of the HIV-1 protease within intact bacterial cells and does not require biochemical purification of either the enzyme or the substrate. For this assay, nine HIV-1 protease specificity sites were genetically engineered into a heterologous protein (galactokinase) and the relative processing of these substrates by the wild-type and a substituted HIV-1 protease was determined. The results from these experiments revealed that the activity of the HIV-1 protease in the engineered heterologous substrate assay is consistent with previously reported in vitro assays and in vivo observations as well as a proposed catalytic specificity model.
Stebbins
J
J
Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Deckman
I C
IC
Richardson
S B
SB
Debouck
C
C
D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005686:Galactokinase / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D016333:HIV Protease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology; D010957:Plasmids; D013379:Substrate Specificity / Q000235:genetics
D005686:Galactokinase; D016333:HIV Protease