Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue
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- 作者：Deeba Amraiz deebaamraiz@gmail.com" class="auth_mail" title="E-mail the corresponding author ; Najam-us-Sahar Sadaf Zaidi ; zaidi.sahar@gmail.com" class="auth_mail" title="E-mail the corresponding author ; Munazza Fatima s.munazza.fatima@gmail.com" class="auth_mail" title="E-mail the corresponding author
- 关键词：Dengue virus ; RNA-dependent RNA polymerase ; Expression ; Purification ; Polymerase activity
- 刊名：Enzyme and Microbial Technology
- 出版年：2016
- 出版时间：October 2016
- 年：2016
- 卷：92
- 期：Complete
- 页码：26-30
- 全文大小：824 K
文摘
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Successful expression of recombinant RNA dependent RNA polymerase (RdRp) of DENV-2 in E. coli cells.
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Two step purification of enzymatically active RdRp protein using Ni-NTA and size exclusion columns.
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High level production of RdRp with final yield of 6.5 mg protein from 1 L of culture.
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Demonstration of in vitro polymerase activity of RdRp using homopolymeric RNA template.
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Optimization of RdRp assay conditions such as protein concentration, GTP concentration and incubation time of the reaction.