A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease.
Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, Km, kcat, and kcat/Km for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters Km, kcat, and kcat/Km for Ac-nKRR-amc substrate were 100 µM, 0.112 s(-1), and 1120 M(-1)·s(-1), respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.
Department of Pharmacy, University of Malakand, Chakdara, 18550 Pakistan. juniphdr@gmail.com.
M
M
Junaid
C
C
Angsuthanasombat
J E S
JE
Wikberg
N
N
Ali
G
G
Katzenmeier
D002846:Chromatography, Affinity; D003716:Dengue Virus / Q000201:enzymology*; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D058849:Protein Refolding; D020365:RNA Helicases / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification; D014760:Viral Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D010450:Endopeptidases; D020365:RNA Helicases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D014760:Viral Fusion Proteins; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases