Download Agrobacterium tumefaciens and A. rhizogenes

Purification of Agrobacterium
rhizogenes protein (GALLS)
required for gene transfer to plants.
By
Chris Brown
Dr. Walt Ream’s Laboratory
Microbiology Department
Background
• Agrobacterium damages at least 1.4 million
dollars worth of agriculture per year in
California and Oregon alone.
• Agrobacterium is the only known prokaryote
to transfer genes to a eukaryote.
• Agrobacterium is used to transfer DNA into
plant cells
Agrobacterium tumefaciens attached to a plant cell.
Image by Martha Hawes
• Agrobacterium tumefaciens and Agrobacterium
rhizogenes infect wounded plants and transfer
plasmid DNA (T-DNA) and virulence (Vir)
proteins into plant cells.
http://cms.daegu.ac.kr/sgpark/microbiology/agrobacterium.jpg
http://www.nature.com/nature/jour
nal/v433/n7026/images/433583af2.2.jpg
GALLS replaces virE2
GALLS replaces virE2 in Agrobacterium tumafaciens
Uninfected control
virE2-mutant pTi
virE2-mutant pTi + GALLS
Map of Galls gene
Start
Codon
GALLS-CT
GALLS-FL
Understanding GALLS proteins
• GALLS-FL and GALLS-CT interact.
• A mutation in the GALLS-CT start codon from a
Methanine to an Isolucene abolishes translation
of GALLS-CT.
•Methionine to isoleucine mutation on
the 808th codon of GALLS does not
affect synthesis or activity of GALLS-FL
•GALLS-CT is required in some hosts
and not in others.
GALLS Contains ATP-binding, helicase, NLS, and
Secretion Signal Domains
ATP-binding (Walker A)
Wild-type: RASTMVGVAGSAKT
K172E:
RASTMVGVAGSAET
ATP-binding (Walker B)
Wild-type: RTIGKNTIVVIDE
D239N:
RTIGKNTIVVINE
Type IV secretion signal
Consensus:
RxxxxxxxRxRxRxx
GALLS: PKAANDVDRLTRDFDERIRVRGDGRGL
GALLS-CT
Helicase motif III
GALLS:
KLICVGDDRQLPPVGPGDLL
GALLS D: KLIC----------GPGDLL
Nuclear Localization Signal
GALLS: KRKRAAAKEEIDSRKKMAR
TEV:
GKKNQKHKLKM(X)31 KRKG
Research question: Can you build a mutant Galls
gene to better purify GALLS-FL protien?
Hypothesis: Creation of start codon mutation of
GALLS-CT along with a histidine tag will
better purify GALLS-FL.
Approach
Purify Galls full-length protein
6-His tag
Met808Ile
GALLS-CT
Met808Ile
Galls
gene
Bgl
II
6-His-tag
1. Restriction
Digest with Bgl II
pLH 416
pCB 1
pBlueScribe_SK(PLUS)
pBlueScribe_SK(PLUS)
(2964bp)
bp)
(2964
pBluescript_II_KS(PLUS)
pBluescript_II_KS(PLUS)
Bgl
II
Bgl II
II
Bgl
(2961 bp)
(2961 bp)
Bgl
II
Bgl II
II
Bgl
2. Ligation
pLH444
Transformation of plasmid pLH
444 into Escherichia coli DH5α
Cell culture, in
L-broth
E. coli cells were
plated on L-agar
medium with
ampicillin
Transformation of pLH 445 into
Agrobacterium cells
Plasmid DNA
extraction from
E. coli cells
Purification of GALLS-FL with nickel affinity chromatography
Extraction of cells contents through
“French press method”
Add mixture to column. Discard
supernatant solution.
Add wash to column. Discard
supernatant solution.
Add elution buffer to column.
Retain supernatant solution.
Purified GALLS-FL protein in solution.
Results obtained along with Larry
Hodges
• Ligated Bgl II fragment containing Met808Ile
mutation into 6-His tagged Galls.
– Correct orientation of the ligated fragment
• Ligated His-Galls gene into Agrobacterium
plasmid and transformed into Agrobacterium.
• Cultured Agrobacterium cells for purification
of GALLS-FL protein.
Future experiments
• DNA binding assays
– Determine if GALLS-FL binds to single stranded
DNA or double stranded DNA.
• Helicase assay to determine type of DNA used
by GALLS-FL
• ATP binding and hydrolysis assays
Acknowledgements
•
•
•
•
HHMI
Dr. Kevin Ahern
Larry Hodges
Dr. Walt Ream