Purification of the M flax-rust resistance protein expressed in Pichia pastoris

Schmidt, S., Williams, S., Wang, C., Sornaraj, P., James, B., Kobe, B., Dodds, P., Ellis, J. and Anderson, P. (2007) Purification of the M flax-rust resistance protein expressed in Pichia pastoris. Plant Journal, 50 6: 1107-1117. doi:10.1111/j.1365-313X.2007.03104.x


Author Schmidt, S.
Williams, S.
Wang, C.
Sornaraj, P.
James, B.
Kobe, B.
Dodds, P.
Ellis, J.
Anderson, P.
Title Purification of the M flax-rust resistance protein expressed in Pichia pastoris
Formatted title
Purification of the M flax-rust resistance protein expressed in Pichia pastoris
Journal name Plant Journal   Check publisher's open access policy
ISSN 0960-7412
Publication date 2007-02-09
Sub-type Article (original research)
DOI 10.1111/j.1365-313X.2007.03104.x
Volume 50
Issue 6
Start page 1107
End page 1117
Total pages 11
Place of publication Oxford
Publisher Blackwell Publishing
Language eng
Subject C1
270103 Protein Targeting and Signal Transduction
780105 Biological sciences
Abstract The M flax-rust resistance (R) gene is predicted to encode a 150-kDa protein of the Toll-interleukin-like receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) class of plant disease resistance proteins and provides resistance against the Melampsora lini (flax rust) fungus carrying the AvrM avirulence gene. The extremely low level of this class of R proteins found in plant tissue has precluded their biochemical and structural analysis, and the study of these proteins has been largely restricted to genetic analyses and in vivo investigations. Here we report the production and purification of the M protein in the methalotrophic yeast, Pichia pastoris. Expression trials with five different constructs reveals optimum levels of soluble native M protein can be obtained as an N-terminally 9x His-tagged protein, in which the first 21 amino acids of the predicted wild-type protein are deleted. Expression was achieved using a high cell density fed-batch bioreactor culture at low temperature. M protein was purified to near homogeneity from whole-cell lysates using cation exchange, immobilised metal ion affinity chromatography and gel filtration with a final yield of approximately 3 mg of protein/1000 g wet weight of yeast cells lysed. The successful expression and purification of soluble M protein opens the way for biochemical and structural analysis of this class of important plant proteins.
Keyword Plant Sciences
disease resistance
flax
recombinant protein
TIR-NBS-LRR
Pichia pastoris
Plant-disease Resistance
Nucleotide-binding Site
Circular-dichroism Spectra
Downy Mildew Resistance
Avirulence Genes
Arabidopsis
Bacterial
Products
Domain
Identification
Q-Index Code C1
Q-Index Status Confirmed Code

 
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Created: Tue, 19 Feb 2008, 03:27:56 EST