title: Enzymatic analysis of recombinant Japanese encephalitis virus NS2B(H)-NS3pro protease with fluorogenic model peptide substrates.
abstract: BACKGROUND
  Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20-30% are fatal, while 30-50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease.
  
  
  RESULTS
  The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, K(m) and k(cat), for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)(4)SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency k(cat)/K(m) was found for Pyr-RTKR-amc (k(cat)/K(m): 1962.96 ± 85.0 M(-1) s(-1)) and the lowest for Boc-LRR-amc (k(cat)/K(m): 3.74±0.3 M(-1) s(-1)). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro.
  
  
  CONCLUSIONS
  A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery.
authors.affiliation: Laboratory of Molecular and Cellular Microbiology, Institute of Molecular Biosciences, Mahidol University, Salaya, Nakornpathom, Thailand.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Junaid
authors.forename: Chakard
authors.initials: C
authors.lastname: Chalayut
authors.forename: Anna
authors.initials: A
authors.lastname: Sehgelmeble Torrejon
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
authors.forename: Iryna
authors.initials: I
authors.lastname: Shutava
authors.forename: Maris
authors.initials: M
authors.lastname: Lapins
authors.forename: Jarl E S
authors.initials: JE
authors.lastname: Wikberg
authors.forename: Gerd
authors.initials: G
authors.lastname: Katzenmeier
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D004664:Encephalitis Virus, Japanese / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D000085242:Viral Proteases
chemical_list: D005456:Fluorescent Dyes; D010447:Peptide Hydrolases; D010455:Peptides; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases