Purification of the M flax-rust resistance protein expressed in Pichia pastoris.
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.
Schmidt
Simon A
SA
The School of Biological Sciences, Flinders University, G.P.O. Box 2100, Adelaide 5001, Australia.
Williams
Simon J
SJ
Wang
Ching-I A
CI
Sornaraj
Pradeep
P
James
Ben
B
Kobe
Bostjan
B
Dodds
Peter N
PN
Ellis
Jeffrey G
JG
Anderson
Peter A
PA
D001487:Basidiomycota; D019597:Flax / Q000235:genetics* / Q000382:microbiology; D015870:Gene Expression; D017343:Genes, Plant; D005822:Genetic Vectors; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology; D010940:Plant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D010940:Plant Proteins; D011994:Recombinant Proteins