An improved purification system for flax resistance proteins M, L6 and L7
H. BURDETT (1), P. Anderson (1), S. Williams (2), P. Sornaraj (3), M. Rahman (1), B. Kobe (4) (1) Flinders University, Australia; (2) Australian National University, Australia; (3) Department of Industry Innovation and Science, Australia; (4) University of Queensland, Australia

The interaction between flax and flax rust is one of the most thoroughly studied plant pathogen interactions. The flax resistance protein M is able to recognise the flax rust effector, AvrM, and induce a hypersensitive response. Biochemical studies of recombinant M, and the related flax proteins L6 and L7, have demonstrated their ability to bind ADP and ATP nucleotides oscillating between an inactive and active form in what has been termed the equilibrium switch model of R protein activation. However, purification methods used so far have yielded partially pure protein, unsuitable for more sensitive biochemical and protein-protein interaction experiments required to further probe the activation model.  Here we present a new method for purifying the M21-1334 protein using a StrepTactin affinity chromatography system. M, with a C-terminal Strep-II tag, is expressed in P. pastoris, and cell lysates are loaded onto a StrepTactin affinity column. Elution with d-Desthiobiotin gives ~90% pure M in a single step, with nucleotide binding properties comparable to that reported previously. This more pure protein enables more sensitive biochemical assays to determine ATP hydrolysis rates and nucleotide dissociation kinetics, as well as in vitro protein-protein interaction studies. For example, surface plasmon resonance will enable the interaction kinetics between the M and AvrM proteins to be measured, rather than the qualitative data comm found in Co-IP and Y2H experiments.

Abstract Number: P17-502
Session Type: Poster