Mj. Wood et Ea. Komives, Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation, J BIOM NMR, 13(2), 1999, pp. 149-159
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.