Engineering the E. coli beta-galactosidase for the screening of antiviral protease inhibitors.
Site-specific proteolysis is essential in many fundamental cellular and viral processes. It has been previously shown that the Escherichia coli beta-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 beta-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.
Vera
Andrea
A
Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Spain.
Arís
Anna
A
Daura
Xavier
X
Martínez
Miguel Angel
MA
Villaverde
Antonio
A
D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods; D011480:Protease Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D013329:Structure-Activity Relationship; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*
D000998:Antiviral Agents; D011480:Protease Inhibitors; D001616:beta-Galactosidase