title: Optimized sequential purification protocol for in vivo site-specific biotinylated full-length dengue virus capsid protein.
abstract: Dengue virus (DENV) capsid (C) protein is one of the three structural proteins that form a mature virus. The main challenge impeding the study of this protein is to generate pure non-truncated, full-length C proteins for structural and functional studies. This is mainly due to its small molecular weight, highly positively charged, stability and solubility properties. Here, we report a strategy to construct, express, biotinylate and purify non-truncated, full-length DENV C protein. A 6× His tag and a biotin acceptor peptide (BAP) were cloned at the N-terminus of C protein using overlapping extension-polymerase chain reaction method for site-specific biotinylation. The final construct was inserted into pET28a plasmid and BL-21 (CodonPlus) expression competent cell strain was selected as there are 12% rare codons in the C protein sequence. Strikingly, we found that our recombinant proteins with BAP were biotinylated endogenously with high efficiency in Escherichia coli BL-21 strains. To purify this His-tagged C protein, nickel-nitriloacetic acid affinity chromatography was first carried out under denaturing condition. After stepwise dialysis and concurrent refolding, ion exchange-fast protein liquid chromatography was performed to further separate the residual contaminants. To obtain C protein with high purity, a final round of purification with size exclusion chromatography was carried out and a single peak corresponding to C protein was attained. With this optimized sequential purification protocol, we successfully generated pure biotinylated full-length DENV C protein. The functionality of this purified non-truncated DENV C protein was examined and it was suitable for structural and molecular studies.
authors.affiliation: Flavivirology Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.
authors.forename: Mun Keat
authors.initials: MK
authors.lastname: Chong
authors.forename: Krupakar
authors.initials: K
authors.lastname: Parthasarathy
authors.forename: Hui Yu
authors.initials: HY
authors.lastname: Yeo
authors.forename: Mah Lee
authors.initials: ML
authors.lastname: Ng
mesh_terms: D000595:Amino Acid Sequence; D019921:Biotinylation; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003715:Dengue / Q000821:virology; D003716:Dengue Virus / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment
chemical_list: D036022:Capsid Proteins; D011994:Recombinant Proteins