Leaf curl disease of tomato (TLCD) caused by Begomoviruses is most severe disease of tomato throughout the world. We have demonstrated earlier the association of a new Begomovirus species, Tomato leaf curl Gujarat virus associated with severe TLCD in India. This virus belongs to geminiviruses which are characterized by circular, single stranded DNA genomes (∼2.7 kb each) encapsidates within geminate virion. Replication initiator protein (Rep) of geminiviruses is the sole protein indispensable for their replication. Rep protein binds to the direct repeat sequences within the common region and induces nick in stem loop of the origin for initiation of DNA replication. In the present study, we have cloned, expressed and purified Rep protein of ToLCGV. ORF AC1 is composed of 1086 nucleotide, coding for 361 amino acids (∼41 kDa).encoding Rep gene and was cloned in prokaryotic ex-pression vector pET28a(+)at BamHI – Hind III sites. Over ex-pression of Rep protein was standardized by varying different concentrations of isopropyl-b -D-thiogalactopyranoside (IPTG), temperature and time of induction. His-tagged protein was enriched on Ni-NTA affinity matrix and the bound protein was eluted with a gradient of 100-200mM imidazole elution buffer. The eluted protein were further purified through DEAE-Sepharose columns (anion exchange chromatography) and finally dialyzed against 50mM Tris-Hcl (pH 8.0), 100mM NaCl, 1mM DTT, 1mM Phenylmethylsulfonyl fluoride (PMSF) and 40% glycerol. Presence of recombinant protein was confirmed by western blotting using anti-His antibodies. Purified Rep-Protein demonstrated ATP hydrolysis activities.
