title: Optimized expression, solubilization and purification of nuclear inclusion protein b of cardamom mosaic virus.
abstract: All RNA viruses encode an RNA-dependent RNA polymerase (RdRP) that is required for replication of the viral genome. Nuclear inclusion b (NIb) gene codes for the RdRp in Potyviridae viruses. In this study, expression, solubilization and purification of NIb protein of Cardamom mosaic virus (CdMV) is reported. The objective of the present study was to express and purify the NIb protein of CdMV on a large scale for structural characterization, as the structure of the RdRp from a plant virus is yet to be determined. However, the expression of NIb protein with hexa-histidine tag in Escherichia coli led to insoluble aggregates. Out of all the approaches [making truncated versions to reduce the size of protein; replacing an amino acid residue likely to be involved in hydrophobic intermolecular interactions with a hydrophilic one; expressing the protein along with chaperones; expression in Origami cells for proper disulphide bond formation, in E. coli as a fusion with maltose-binding protein (MBP) and in Nicotiana tabacum] to obtain the RdRp in a soluble form, only expression in E. coli as a fusion with MBP and its expression in N. tabacum were successful. The NIb expressed in plant or as a fusion with MBP in E. coli can be scaled up for further work.
authors.affiliation: School of Biotechnology, Madurai Kamaraj University, Madurai-625021 I, Tamil Nadu, India.
authors.forename: T
authors.initials: T
authors.lastname: Jebasingh
authors.forename: T
authors.initials: T
authors.lastname: Jacob
authors.forename: M
authors.initials: M
authors.lastname: Shah
authors.forename: D
authors.initials: D
authors.lastname: Das
authors.forename: S
authors.initials: S
authors.lastname: Krishnaswamy
authors.forename: R
authors.initials: R
authors.lastname: Usha
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D014764:Viral Proteins; C061396:nuclear inclusion protein b, mosaic viruses; D012321:DNA-Directed RNA Polymerases