Expression of Cardamom mosaic virus coat protein in Escherichia coli and its assembly into filamentous aggregates.
Cardamom mosaic virus (CdMV), a member of the genus Macluravirus of Potyviridae, causes a mosaic disease in cardamom. A polyclonal antiserum was raised against the purified virus and IgG was prepared. Electron microscopic studies on the purified virus showed flexuous filamentous particles of approximately 800 nm in length, typical of members of Potyviridae. The coat protein (CP) encoding sequence of the virus was expressed in Escherichia coli and the protein purified by affinity chromatography under denaturing conditions. The viral nature of the expressed CP was confirmed by positive reaction with anti CdMV IgG in a Western blot. The expressed CP aggregated irreversibly upon renaturation at concentrations above 0.07 mg/ml. The expression of the CP led to the formation of filamentous aggregates in E. coli as observed by immuno-gold electron microscopy. The filamentous aggregates were of 100-150 nm in length. Immuno-capture RT-PCR confirmed the absence of coat protein mRNA in the filamentous aggregates. Deletion mutations, which were expected to inhibit virus assembly, were introduced in the core region of the coat protein. However, these mutations did not improve the solubility of the CP in non-denaturing buffers.
Jacob
Thomas
T
Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, India.
Usha
R
R
D015153:Blotting, Western; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D028523:Elettaria / Q000821:virology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017800:Potyvirus / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure*; D011994:Recombinant Proteins / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology*
D036022:Capsid Proteins; D011994:Recombinant Proteins