Site-specific proteolysis is essential in many fundamental cellular and viral processes. It has been previously shown that the Escherichia coli β-galactosidase can be useful for the high-throughput screening of human immunodeficiency virus type 1 protease inhibitors. Here, by using crystallographic and functional data of the bacterial enzyme, we have identified a new accommodation site between amino acids 581 and 582, in a solvent-exposed and flexible β-turn of domain III. The placement of the model peptide reproducing the matrix-capsid (p17/p24) gag cleavage sequence renders a highly active and efficiently digested chimeric construct. The use of this insertion site, that increases the cleavage potential of this reporter enzyme, can improve the sensitivity and dynamic range of the antiviral drug assay. This simple and highly specific analytical test may also be extended to the screening of other specific protease inhibitors by a convenient colorimetric assay. © 2005 Elsevier Inc. All rights reserved.
|Journal||Biochemical and Biophysical Research Communications|
|Publication status||Published - 8 Apr 2005|
- Antiviral drug
- Protein engineering