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Purification of a Secreted Agrobacterium rhizogenes Protein(GALLS) Required for
Gene Transfer to Plants
Josh Cuperus, Larry Hodges, Dr. Walt Ream
Department of MicrobiologyOregon State University
Agrobacterium Infects plants by introducing new DNA Integrates DNA into plant genome Several species that have this ability Very useful for genetic modification of plants
Crown Gall Disease
Caused by Agrobacterium tumefaciens
Agrobacterium tumefaciens plant infection and transformation
Ti plasmid T-DNA Region
T-DNA integrated into plant genome
Bacterial Cell
Plant Cell
Plant
D2E2
E2E2
E2
D2
E2
E2
E2
E2
Nucleus
E2
Agrobacterium
E2
D2
E1
E1
E2
E1
Agrobacterium rhizogenes Causes hairy root disease instead of crown
gall. DNA transfer occurs without two essential
proteins (VirE1 and VirE2) found in Agrobacterium tumefaciens.
Arrangement of Virulence Operons in Ti & Ri Plasmids
Ri & Ti Plasmid Maps
Ti Plasmid Ri Plasmid
D2
plantA. tumefaciens
D2 D2
nucleus
GALLS Replaces VirE2 by Mixed Infection
Agrobacterium rhizogenes
GALLSGALLSVirB/D4
VirB/D4
Domains in the GALLS Protein
NTP-Binding TraA-Like T4SS
Similarities between GALLS and VirE2
Both have a type four secretion signal. Both contain a nuclear localization signal,
however they are not the same amino acid sequence.
Differences between GALLS and VirE2
Nucleotide sequences show no homology. Only GALLS has a nucleoside triphosphate
binding motif. GALLS has a molecular weight more than
three times that of VirE2. There is no evidence of a chaperone for
GALLS.
Purpose Protein purification Use in creating antibodies that will work on
normal GALLS protein Antibodies will allow recognition of protein
in other studies
Polyhistidine affinity tags Series of 6 histidine amino acid residues
allows for purification because of its affinity for a nickel column, allowing most other proteins to be removed.
Retaining function Created two his-tagged proteins, one at each
end of the DNA sequence encoding for the protein.
Hopefully one or the other will retain function.
Purification of functional protein will allow us to study: ATP binding and hydrolysis, DNA binding, and other functions.
Acknowledgements
HHMI program, Chris Mathews, Kevin Ahern. Ream Laboratory; Dr. Walt Ream, Larry Hodges,
Jodi Humann, Jen Pitrak. National Science Foundation Special thanks to Kevin Ahern for help and support.