Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation

Heterologous expression in Pichia pastoris has many of the advantages of eu karyotic expression, proper folding and disulfide bond formation, glycosyla tion, and secretion. Contrary to other eukaryotic systems, protein producti on from P. pastoris occurs in simple minimal defined media making this syst em attractive for production of labeled proteins for NMR analysis. P. pasto ris is therefore the expression system of choice for NMR of proteins that c annot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed protei ns from P. pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein y ields by 10-100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly (NH4OH)-N-15, is prohibitively high. We report fermentation methods that allow for both N-15-labeling from ((NH4)-N-15)(2)SO4 and C-13-labeling from C-13-glucose or C-13-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragm ent of thrombomodulin with two N-linked glycosylation sites shows that ferm entation is more cost effective than shake flask growth for isotopic enrich ment.