Related Experiment Video
Updated: Oct 4, 2026
Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies
Published on: January 23, 2019
Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanol-utilizing strain (Mut+) of Pichia
H A van den Burg1, P J de Wit, J Vervoort
1Laboratory of Biochemistry, Wageningen University, The Netherlands.
Abstract:
Cost effective 13C/15N-isotope labeling of the avirulence protein AVR4 (10 kDa) of the fungal tomato pathogen Cladosporium fulvum was achieved with the methylotrophic yeast Pichia pastoris in a fermentor. The 13C/15N-labeled AVR4 protein accumulated to 30 mg/L within 48 h in an initial fermentation volume of only 300 mL, while prolonged optimized overexpressions yielded 126 mg/L. These protein yields were 24-fold higher in a fermentor than in flask cultures. In order to achieve these protein expression levels, we used the methanol-utilizing strain (Mut+) of Pichia pastoris which has a high growth rate while growing on methanol as the only carbon source. In contrast, the methanol-sensitive strain (MutS) could intrinsically yield comparable protein expression levels, but at the expense of additional carbon sources. Although both strains are generally used for heterologous protein expression, we show that the costs for 13C-isotope labeling can be substantially reduced using the Mut+ strain compared to the MutS strain, as no 13C3-glycerol is required during the methanol-induction phase. Finally, nitrogen limitations were precluded for 15N-labeling by an optimal supply of 10 g/L (15NH4)2SO4 every 24 h.
Insights
Cost-effective isotope labeling of the AVR4 protein was achieved using Pichia pastoris in a fermentor, yielding significantly higher protein amounts than flask cultures. The methanol-utilizing (Mut+) strain proved more economical for 13C-labeling, reducing costs substantially.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Isotope labeling is crucial for protein structure and function studies.
- Heterologous protein expression in Pichia pastoris is a common method.
- Cost-effective methods for producing labeled proteins are in high demand.
Purpose of the Study:
- To develop a cost-effective method for 13C/15N-isotope labeling of the AVR4 protein from Cladosporium fulvum.
- To compare the efficiency of the methanol-utilizing (Mut+) and methanol-sensitive (MutS) Pichia pastoris strains for isotope labeling.
- To optimize fermentation conditions for high-yield protein production.
Main Methods:
- Utilized Pichia pastoris (Mut+ strain) for heterologous expression of AVR4 protein.
- Employed fermentor cultivation for large-scale protein production.
- Optimized methanol induction and nitrogen supply for maximal protein yield and labeling.
- Compared protein yields and labeling costs between Mut+ and MutS strains.
Main Results:
- Achieved 30 mg/L of 13C/15N-labeled AVR4 protein within 48 h, with optimized yields reaching 126 mg/L.
- Fermentor cultures yielded 24-fold higher protein amounts compared to flask cultures.
- Demonstrated substantial cost reduction for 13C-isotope labeling using the Mut+ strain by eliminating the need for 13C3-glycerol during methanol induction.
- Ensured efficient 15N-labeling through optimal ammonium sulfate supply, preventing nitrogen limitation.
Conclusions:
- Pichia pastoris (Mut+ strain) in a fermentor is a highly effective and cost-efficient system for producing 13C/15N-labeled AVR4 protein.
- The Mut+ strain offers significant economic advantages for 13C-labeling compared to the MutS strain.
- Optimized fermentation parameters are key to achieving high yields of labeled proteins for structural and functional analyses.
