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Kinetic characterization of trans-proteolytic activity of Chikungunya virus capsid protease and development of a FRET-based HTS assay.

Aggarwal M, Sharma R, Kumar P, Parida M, Tomar S - Sci Rep (2015)

Bottom Line: The assay with a Z' factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors.Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 10(3) M(-1) sec(-1) respectively.The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667, India.

ABSTRACT
Chikungunya virus (CHIKV) capsid protein (CVCP) is a serine protease that possesses cis-proteolytic activity essential for the structural polyprotein processing and plays a key role in the virus life cycle. CHIKV being an emerging arthropod-borne pathogenic virus, is a public health concern worldwide. No vaccines or specific antiviral treatment is currently available for chikungunya disease. Thus, it is important to develop inhibitors against CHIKV enzymes to block key steps in viral reproduction. In view of this, CVCP was produced recombinantly and purified to homogeneity. A fluorescence resonance energy transfer (FRET)-based proteolytic assay was developed for high throughput screening (HTS). A FRET peptide substrate (DABCYL-GAEEWSLAIE-EDANS) derived from the cleavage site present in the structural polyprotein of CVCP was used. The assay with a Z' factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors. Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 10(3) M(-1) sec(-1) respectively. The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.

No MeSH data available.


Related in: MedlinePlus

SDS-PAGE and gel filtration profile of CVCP.Both (A) inactive and (B) active CVCP shows the protein purified to homogeneity. The size-exclusion chromatography results suggest the monomeric nature of both the proteins. Lane 1, molecular-weight markers (kDa); lane 2, pellet containing insoluble protein fraction; lane 3, supernatant containing soluble protein fraction; lane 4–6, purified CVCP.
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f2: SDS-PAGE and gel filtration profile of CVCP.Both (A) inactive and (B) active CVCP shows the protein purified to homogeneity. The size-exclusion chromatography results suggest the monomeric nature of both the proteins. Lane 1, molecular-weight markers (kDa); lane 2, pellet containing insoluble protein fraction; lane 3, supernatant containing soluble protein fraction; lane 4–6, purified CVCP.

Mentions: The soluble proteins were purified by IMAC (immobilized metal assisted chromatography) and size exclusion chromatography. In Ni-NTA column chromatography, the protein was eluted by increasing concentration of imidazole. A single band was observed on SDS-PAGE for both the purified proteins confirming the protein homogeneity (Fig. 2). The protein band was visible at ~17 kDa on SDS-PAGE which was also confirmed by gel filtration chromatography by comparing and calculating the molecular weight using standard molecular weight markers. Thus, it is confirmed that both active and inactive CVCP exist as monomer in solution.


Kinetic characterization of trans-proteolytic activity of Chikungunya virus capsid protease and development of a FRET-based HTS assay.

Aggarwal M, Sharma R, Kumar P, Parida M, Tomar S - Sci Rep (2015)

SDS-PAGE and gel filtration profile of CVCP.Both (A) inactive and (B) active CVCP shows the protein purified to homogeneity. The size-exclusion chromatography results suggest the monomeric nature of both the proteins. Lane 1, molecular-weight markers (kDa); lane 2, pellet containing insoluble protein fraction; lane 3, supernatant containing soluble protein fraction; lane 4–6, purified CVCP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4593962&req=5

f2: SDS-PAGE and gel filtration profile of CVCP.Both (A) inactive and (B) active CVCP shows the protein purified to homogeneity. The size-exclusion chromatography results suggest the monomeric nature of both the proteins. Lane 1, molecular-weight markers (kDa); lane 2, pellet containing insoluble protein fraction; lane 3, supernatant containing soluble protein fraction; lane 4–6, purified CVCP.
Mentions: The soluble proteins were purified by IMAC (immobilized metal assisted chromatography) and size exclusion chromatography. In Ni-NTA column chromatography, the protein was eluted by increasing concentration of imidazole. A single band was observed on SDS-PAGE for both the purified proteins confirming the protein homogeneity (Fig. 2). The protein band was visible at ~17 kDa on SDS-PAGE which was also confirmed by gel filtration chromatography by comparing and calculating the molecular weight using standard molecular weight markers. Thus, it is confirmed that both active and inactive CVCP exist as monomer in solution.

Bottom Line: The assay with a Z' factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors.Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 10(3) M(-1) sec(-1) respectively.The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247667, India.

ABSTRACT
Chikungunya virus (CHIKV) capsid protein (CVCP) is a serine protease that possesses cis-proteolytic activity essential for the structural polyprotein processing and plays a key role in the virus life cycle. CHIKV being an emerging arthropod-borne pathogenic virus, is a public health concern worldwide. No vaccines or specific antiviral treatment is currently available for chikungunya disease. Thus, it is important to develop inhibitors against CHIKV enzymes to block key steps in viral reproduction. In view of this, CVCP was produced recombinantly and purified to homogeneity. A fluorescence resonance energy transfer (FRET)-based proteolytic assay was developed for high throughput screening (HTS). A FRET peptide substrate (DABCYL-GAEEWSLAIE-EDANS) derived from the cleavage site present in the structural polyprotein of CVCP was used. The assay with a Z' factor of 0.64 and coefficient of variation (CV) is 8.68% can be adapted to high throughput format for automated screening of chemical libraries to identify CVCP specific protease inhibitors. Kinetic parameters Km and kcat/Km estimated using FRET assay were 1.26 ± 0.34 μM and 1.11 × 10(3) M(-1) sec(-1) respectively. The availability of active recombinant CVCP and cost effective fluorogenic peptide based in vitro FRET assay may serve as the basis for therapeutics development against CHIKV.

No MeSH data available.


Related in: MedlinePlus