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Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue

Paper ID Volume ID Publish Year Pages File Format Full-Text
16756 42610 2016 5 PDF Available
Title
Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue
Abstract

•Successful expression of recombinant RNA dependent RNA polymerase (RdRp) of DENV-2 in E. coli cells.•Two step purification of enzymatically active RdRp protein using Ni-NTA and size exclusion columns.•High level production of RdRp with final yield of 6.5 mg protein from 1 L of culture.•Demonstration of in vitro polymerase activity of RdRp using homopolymeric RNA template.•Optimization of RdRp assay conditions such as protein concentration, GTP concentration and incubation time of the reaction.

NS5 is the largest and most conserved protein among the four dengue virus (DENV) serotypes. It has been the target of interest for antiviral drug development due to its major role in replication. NS5 consists of two domains, the N-terminal methyltransferase domain and C-terminal catalytic RNA-dependent RNA polymerase (RdRp) domain. It is an unstable protein and is prone to inactivation upon prolonged incubation at room temperature, thus affecting the inhibitor screening assays. In the current study, we expressed and purified DENV RdRp alone in Esherichia coli (E. coli) cells. The N-terminally His-tagged construct of DENV RdRp was transformed into E. coli expression strain BL-21 (DE3) pLysS cells. Protein expression was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mM. The induced cultures were then grown for 20 h at 18 °C and cells were harvested by centrifugation at 6000 x g for 15 min at 4 °C. The recombinant protein was purified using HisTrap affinity column (Ni-NTA) and then the sample was subjected to size exclusion chromatography, which successfully removed the degradation product obtained during the previous purification step. The in vitro polymerase activity of RdRp was successfully demonstrated using homopolymeric polycytidylic acid (poly(rC)) RNA template. This study describes the high level production of enzymatically active DENV RdRp protein which can be used to develop assays for testing large number of compounds in a high-throughput manner. RdRp has the de novo initiation activity and the in vitro polymerase assays for the protein provide a platform for highly robust and efficient antiviral compound screening systems.

Keywords
Dengue virus; RNA-dependent RNA polymerase; Expression; Purification; Polymerase activity
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Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 92, October 2016, Pages 26–30
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us