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Optimization of Dengue-3 recombinant NS1 protein expression in E. coli and in vitro refolding for diagnostic applications.
MedLine Citation:
PMID:  23188193     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Dengue non-structural protein (NS1) is known to be protective antigen and also has immense application for serodiagnosis. Several serodiagnostic assays available for dengue viral infection are dependent on tissue culture-grown viral proteins. This task is unsafe, laborious, more expensive that makes it unsuitable for routine large-scale production. Although bacterial expression is relatively simple and easy for recombinant protein expression, it is more challenging to make NS1 protein with native structural and immunological features using bacterial expression system. We have successfully developed a method leading to the purification and refolding of recombinant dengue virus type 3 (DENV3) NS1. The gene encoding NS1 was amplified and cloned in pET28a (+) vector. In order to increase the purity of the recombinant NS1, the transgene was engineered to carry 6× Histidine tags at both N and C-terminal ends. The recombinant construct (pETNS1) was transformed into E. coli Rosetta-gami cells and the expression conditions viz IPTG concentration, media type, temperature, and harvest time were optimized. The size of the expressed protein was found to be ~45 kDa and the authenticity of the expressed protein was confirmed using anti-His and anti-NS1 monoclonal antibodies. The NS1 protein was purified under denaturing conditions, to attain the native conformation, NS1 protein was in vitro refolded and dialyzed. The refolded NS1 protein was detected by commercial Immuno chromatographic strip and NS1 specific monoclonal antibodies. IgM antibody capture ELISA was performed using refolded recombinant NS1 protein which recognized the IgM antibodies in dengue-positive samples of acute phase of infection. Our result suggests that rNS1 protein has immense diagnostic potential and can be used in developing point of care diagnostic assays.
Authors:
T N Athmaram; Shweta Saraswat; Princi Misra; Saurabh Shrivastava; Anil K Singh; Shailendra K Verma; N Gopalan; Prativa K Behara; P V L Rao
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-11-28
Journal Detail:
Title:  Virus genes     Volume:  -     ISSN:  1572-994X     ISO Abbreviation:  Virus Genes     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-11-28     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8803967     Medline TA:  Virus Genes     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Bio-process Scale Up Facility, Defence Research and Development Establishment, Ministry of Defence (Government of India), Gwalior, MP, 474 002, India, athmabiotech@gmail.com.
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