title: Optimization of Dengue-3 recombinant NS1 protein expression in E. coli and in vitro refolding for diagnostic applications.
abstract: 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.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
authors.forename: T N
authors.initials: TN
authors.lastname: Athmaram
authors.forename: Shweta
authors.initials: S
authors.lastname: Saraswat
authors.forename: Princi
authors.initials: P
authors.lastname: Misra
authors.forename: Saurabh
authors.initials: S
authors.lastname: Shrivastava
authors.forename: Anil K
authors.initials: AK
authors.lastname: Singh
authors.forename: Shailendra K
authors.initials: SK
authors.lastname: Verma
authors.forename: N
authors.initials: N
authors.lastname: Gopalan
authors.forename: Prativa K
authors.initials: PK
authors.lastname: Behara
authors.forename: P V L
authors.initials: PV
authors.lastname: Rao
mesh_terms: D000595:Amino Acid Sequence; D003715:Dengue / Q000175:diagnosis / Q000821:virology*; D003716:Dengue Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D058849:Protein Refolding; D016415:Sequence Alignment; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: C546113:NS1 protein, Dengue virus type 3; D017361:Viral Nonstructural Proteins