A fluorescent protein-based biological screen of proteinase activity.
A cell-based fluorescent protein reporter assay for proteinase activity amenable to high-throughput applications was developed. This assay is based on Förster resonance energy transfer (FRET) between 2 variants of the green fluorescent protein connected by a short cleavable linker and expressed in Escherichia coli as tagged proteins. A library to assay proteinase specificity was generated by randomizing a portion of the linker using PCR. The library could be grown in microplates, allowing cells to be lysed in situ and substrate cleavage to be monitored through loss of FRET signal using a plate reader. Progress curves were generated to estimate cleavage efficiency, facilitating the identification of well-cleaved substrates. The polyhistidine-tagged fluorescent substrates could then be purified and used for further characterization. To establish the general utility of the screen, it was used to demonstrate that the cysteine proteinase of the hepatitis A virus, 3C(pro), prefers Ile, Val, or Leu at the P(4) position of the cleavage sequence and Gly, Ser, or Ala at the P'(1) position. The assay can also be used to screen small-molecule libraries for inhibitors.
Huitema
Carly
C
Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, Canada.
Eltis
Lindsay D
LD
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics; D030041:Hepatitis A virus / Q000201:enzymology; D057166:High-Throughput Screening Assays; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011506:Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D054852:Small Molecule Libraries; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014169:Transformation, Bacterial; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D011506:Proteins; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D057056:Cysteine Proteases; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases