Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanol-utilizing strain (Mut+) of Pichia pastoris

Cost effective C-13/N-15-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 C-13/N-15-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 (Mut(S)) 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 C-13-isotope labeling can be substantially reduced using the Mut(+) strain compared to the Mut(S) strain, as no C-13(3)-glycerol is required during the methanol-induction phase. Finally, nitrogen limitations were precluded for N-15-labeling by an optimal supply of 10 g/L ((NH4)-N-15)(2)SO4 every 24 h.