A fluorescent protein-based biological screen of proteinase activity.
J Biomol Screen
; 15(2): 224-9, 2010 Feb.
Article
em En
| MEDLINE
| ID: mdl-20086215
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Virais
/
Cisteína Endopeptidases
/
Cisteína Proteases
/
Ensaios de Triagem em Larga Escala
Tipo de estudo:
Clinical_trials
Idioma:
En
Revista:
J Biomol Screen
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Canadá