NACALAI TESQUE, INC.

ProAssay HCV Protease Kit & HIV-1 Protease Kit
Fret assay for protease quantifying

ProAssay Kits use FRET principle and are for not only measuring the activity of proteases but also high-throughput screening of protease inhibitors.

HCV Protease Assay Kit

The HCV NS3/4A protease is responsible for cleavage at four sites within the HCV polyprotein to generate the N termini of the NS4A, NS4B, NS5A, and NS5B proteins (1-4). The order and kinetics of cleavage as well as the extent of precursor processing appear to be critical steps in the generation of fully infectious, appropriately assembled viral particles. Therefore, inhibition of HCV NS3/4A protease represents an important avenue for antiviral therapy (2).

ProAssay HCV Protease Assay Kit provides a convenient method for high throughput screening of HCV NS3/4A protease inhibitors and continuous quantification of HCV protease activity using FRET peptide substrate. The sequence of this FRET peptide is derived from NS3-dependent cleavage site:Asp/GluXaa4Cys/Thr-Ser/Ala (5). In the FRET peptide, the green fluorescence is quenched by appropriate fluorescence quencher until this peptide is cleaved into two separate fragments by HCV NS3/4A protease at the cleavage site. Upon cleavage, the green fluorescence is recovered and can be monitored at excitation/emission = 490 nm/530 nm.

HIV-1 protease Assay Kit

HIV-1 protease (HIV-1 PR) is responsible for cleaving up to 12 sites in the Gag and Gag-Pol precursor polypeptides (6, 7). The order and kinetics of cleavage as well as the extent of precursor processing appear to be critical steps in the generation of fully infectious, appropriately assembled viral particles (8-11). Therefore, inhibition of HIV-1 protease represents an important avenue for antiviral therapy (11).

ProAssay HIV-1 Protease FRET Assay Kit provides a convenient method for high throughput screening of HIV-1 protease inhibitors and continuous quantification of HIV-1 protease activity using a fluorescence resonance energy transfer (FRET) peptide. The sequence of this FRET peptide is derived from the native p17/p24 cleavage site on Prgag for HIV-1 protease. In the FRET peptide, the green fluorescence is quenched by appropriate fluorescence quencher until this peptide is cleaved into two separate fragments by HIV-1 protease at the cleavage site. Upon cleavage, the green fluorescence is recovered and can be monitored at excitation/emission = 490 nm/530 nm.

Principle of the detection

The cleavage site locates between the quencher and fluorophore on the substrate peptide, and the fluorescence of fluorophore is quenched by the quencher. When proteases cut the cleavage site, the peptide cleaved into 2 flagment, so that the fluorescence is recovered and can be monitored (ex / em = 490 nm / 530 nm).

Kit Components

References

1. Grakoui, A. et al. J Virol. 67, 2832-2843. (1993)
2. Hijikata, M. et al. Proc Natl Acad Sci U S A. 90, 10773-10777. (1993)
3. Tomei, L. et al. J Virol. 67, 4017-4026. (1993)
4. Lamarre, D. et al. Nature 426, 186-189. (2003)
5. Talianie, M. et al. Anal Biochem. 240, 60-67. (1996)
6. Chou, KC. et al. Proteins 24, 51-72. (1996)
7. Henderson, LE. et al. In: Bolognesi, D (ed.), Human Retroviruses, Cancer and AIDS: Approaches to Prevention and Therapy. Alan R. Liss, New York, 135-147. (1988)
8. Krausslich, HG. et al. J Virol. 69, 3407-3419. (1995)
9. Mervis, RJ. et al. J Virol. 62, 3993-4002. (1988)
10. Pettit, SC. et al. J Virol. 68, 8017-8027. (1994)
11. Temesgen, Z. Expert Opin Pharmacother. 2, 1239-1246. (2001)

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