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://www.nature.com/nature/journal/v433/n7026/images/433583a-f2.2.jpg
http://cms.daegu.ac.kr/sgpark/microbiology/agrobacterium.jpg
GALLS replaces virE2
Uninfected control virE2-mutant pTi virE2-mutant pTi + GALLS
GALLS replaces virE2 in Agrobacterium tumafaciens
• 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.
Understanding GALLS proteins
•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: RASTMVGVAGSAKTK172E: RASTMVGVAGSAET
ATP-binding (Walker B)Wild-type: RTIGKNTIVVIDED239N: RTIGKNTIVVINE
Helicase motif IIIGALLS: KLICVGDDRQLPPVGPGDLLGALLS : KLIC----------GPGDLL
Nuclear Localization Signal GALLS: KRKRAAAKEEIDSRKKMARTEV: GKKNQKHKLKM(X)31 KRKG
Type IV secretion signal Consensus: RxxxxxxxRxRxRxx GALLS: PKAANDVDRLTRDFDERIRVRGDGRGL
GALLS-CT
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.
1. Restriction Digest with Bgl II
2. Ligation
pBlueScribe_SK(PLUS)(2964 bp)
6-His-tag
pCB 1pBlueScribe_SK(PLUS)
(2964 bp)
pBluescript_II_KS(PLUS)(2961 bp)
Bgl II Bgl II
Bgl II
pLH444
pBluescript_II_KS(PLUS)(2961 bp)
Met808Ile
pLH 416
Bgl II
Bgl II
Galls gene
Transformation of plasmid pLH 444 into Escherichia coli DH5α
E. coli cells were plated on L-agar medium with ampicillin
Cell culture, in L-broth
Plasmid DNA extraction from E. coli cells
Transformation of pLH 445 into Agrobacterium cells
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.
Purification of GALLS-FL with nickel affinity chromatography
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