Refolding, purification and characterization of replication-initiator protein from soybean-infecting geminivirus.
The replication-initiator protein (Rep) from a soybean-infecting geminivirus was overexpressed in E. coli as a fusion protein with maltose binding protein (MBP). In spite of the presence of the highly soluble MBP as the fusion partner, the overexpressed MBP-Rep fusion protein formed insoluble inclusion bodies. The protein was solubilized from the inclusion bodies and refolded. The refolded MBP-Rep protein was purified using ion exchange and amylose affinity chromatography. The activity of the purified MBP-Rep was assessed using an in vitro cleavage assay. Soluble and stable MBP-Rep protein was obtained in high abundance, providing the feasibility of large-scale production of active Rep protein for functional characterization and X-ray crystallographic structure determination.
Girish
K R
KR
Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India.
Palanivelu
S
S
Kumar
P D
PD
Usha
R
R
D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D017791:Geminiviridae / Q000235:genetics*; D002479:Inclusion Bodies; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013025:Glycine max / Q000821:virology*; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014764:Viral Proteins; C045264:replication initiator protein; D004265:DNA Helicases