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Updated: Jul 15, 2026
Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
Purification of the M flax-rust resistance protein expressed in Pichia pastoris
Simon A Schmidt1, Simon J Williams, Ching-I A Wang
1The School of Biological Sciences, Flinders University, G.P.O. Box 2100, Adelaide 5001, Australia.
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.
Insights
Researchers successfully produced and purified the flax rust resistance (M) protein using Pichia pastoris yeast. This breakthrough enables crucial biochemical and structural studies of this important plant disease resistance protein.
Area of Science:
- Plant pathology
- Molecular biology
- Biochemistry
Background:
- The M gene encodes a Toll-interleukin-like receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) protein conferring resistance to flax rust (Melampsora lini).
- Low expression levels of TIR-NBS-LRR proteins in plant tissues have hindered detailed biochemical and structural analyses, limiting studies to genetic and in vivo investigations.
Purpose of the Study:
- To develop a method for producing and purifying the M flax rust resistance protein for further analysis.
- To enable biochemical and structural characterization of TIR-NBS-LRR class proteins.
Main Methods:
- Expression of the M protein in Pichia pastoris using a modified N-terminally 9x His-tagged construct (deletion of first 21 amino acids).
- High cell density fed-batch bioreactor culture at low temperature for optimal soluble protein production.
- Purification using cation exchange, immobilized metal ion affinity chromatography, and gel filtration.
Main Results:
- Optimized expression yielded soluble native M protein.
- Purification achieved near homogeneity, with a final yield of approximately 3 mg/1000 g wet weight of yeast cells.
- A robust protocol for producing and purifying the M protein was established.
Conclusions:
- The successful expression and purification of soluble M protein in Pichia pastoris overcomes previous limitations.
- This work paves the way for detailed biochemical and structural studies of the M protein and related plant disease resistance proteins.
