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Anal Biochem. 1996 Nov 1;242(1):90-4.

A heterologous substrate assay for the HIV-1 protease engineered in Escherichia coli.

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Department of Molecular Genetics, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.


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.

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