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

Map of Galls gene

GALLS-CT

GALLS-FL

Start Codon

• 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.

Approach

Purify Galls full-length protein

GALLS-CT

Met808Ile6-His tag

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

Acknowledgements

• HHMI

• Dr. Kevin Ahern

• Larry Hodges

• Dr. Walt Ream