title: Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens.
abstract: Bacteria of the genus Agrobacterium can transfer a portion of their Ti plasmid (T-DNA) in complex with the VirE2 and VirD2 proteins into the plant-cell nucleus and cause it to be integrated in the host-cell chromosomes. The mechanism of T-DNA transfer across the plant-cell membrane and cytoplasm is unknown. The aim of this study was to isolate the virulence protein VirE2 in order to explore its role in T-DNA transfer across the eukaryotic-cell membrane and cytoplasm. To obtain VirE2, we cloned the virE2 gene into plasmid pQE31 in Escherichia coli cells. VirE2 protein was isolated from E. coli XL-1 blue cells containing a recombinant plasmid, pQE31-virE2. The cells were ultrasonically disrupted, and the protein containing six histidine residues at the N-terminal end was isolated by affinity chromatography on Ni-NTA agarose. The purified preparation was tested by immunodot, by using polyclonal rabbit antibodies and miniantibodies produced toward VirE2. The capacity of the recombinant protein VirE2 for interacting with single-stranded DNA was tested by the formation of complexes, recorded by agarose-gel electrophoresis. In summary, A. tumefaciens virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into the plant cell, was isolated, purified, and partially characterized. Anti-VirE2 miniantibodies were obtained, and direct labeling of VirE2 with colloidal gold was done for the first time.
authors.affiliation: Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 13 Prospect Entuziastov, Saratov 410049, Russia.
authors.forename: Irina
authors.initials: I
authors.lastname: Volokhina
authors.forename: Inna
authors.initials: I
authors.lastname: Sazonova
authors.forename: Vladimir
authors.initials: V
authors.lastname: Velikov
authors.forename: Mikhail
authors.initials: M
authors.lastname: Chumakov
mesh_terms: D016960:Agrobacterium tumefaciens / Q000378:metabolism* / Q000472:pathogenicity; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007118:Immunoassay; D007473:Ion Channels / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D036741:Plant Tumor-Inducing Plasmids / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D011817:Rabbits; D014774:Virulence
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D007473:Ion Channels; D008565:Membrane Proteins; C051583:virE2 protein, Agrobacterium