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- W2101159016 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." @default.
- W2101159016 created "2016-06-24" @default.
- W2101159016 creator A5041798258 @default.
- W2101159016 creator A5044648274 @default.
- W2101159016 creator A5045207334 @default.
- W2101159016 creator A5063103444 @default.
- W2101159016 date "2013-03-10" @default.
- W2101159016 modified "2023-10-11" @default.
- W2101159016 title "Optimized sequential purification protocol for in vivo site-specific biotinylated full-length dengue virus capsid protein" @default.
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- W2101159016 doi "https://doi.org/10.1093/protein/gzs107" @default.
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