Human cystatin C is a cysteine proteinase inhibitor with potential applications as an anti-viral agent, cancer tumor growth inhibitor, and in prevention of proteolysis during food processing. A glycosylated cystatin C mutant with increased temperature stability was developed for the latter application [Nakamura et al. (1998) FEBS Lett 427:252-254]. A recombinant variant of cystatin C [Nakamura et al. (2000) International patent no. PLTCA99/00717] with two potential sites for N-linked glycosylation was expressed in Pichia pastoris Mut(s). Little of the cystatin C produced was in the glycosylated form under fermentation conditions of pH 6, temperature 28 degrees C, methanol only feed, and ammonium hydroxide as a nitrogen source. Thus, the effect of addition of complex nitrogen sources, peptone and amino acid supplements, on the yield and glycosylation of this mutant cystatin C were investigated. A full factorial design experiment using 2-l fermenters was performed with three factors: ammonium hydroxide, peptone, and an amino acid mix, at two levels, absent or present. Peptone addition was found to have a positive, and the most significant, effect on cell specific cystatin C yield. A maximum mutant cystatin C yield of 0.82 mumol (g-dry cell weight)(-1) min(-1) was obtained when all three nitrogen sources were used together. However, under these conditions only 16% of protein was in the glycosylated form since ammonia was found to have a significant negative effect on glycosylation extent. The maximum extent of glycosylation was 30% when peptone and amino acid mix were the only nitrogen sources added.