The effect of nitrogen source on yield and glycosy

Authors: Jason Pritchett Susan A Baldwin

Publish Date: 2004/11/18

Volume: 31, Issue: 12, Pages: 553-558

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Abstract

Human cystatin C is a cysteine proteinase inhibitor with potential applications as an antiviral 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 427252–254 A recombinant variant of cystatin C Nakamura et al 2000 International patent no PLTCA99/00717 with two potential sites for Nlinked glycosylation was expressed in Pichia pastoris Muts Little of the cystatin C produced was in the glycosylated form under fermentation conditions of pH 6 temperature 28°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 2l 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 082 μmol gdry 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 addedM Ogawa C Scaman and S Nakai of Food Nutrition and Health Faculty of Agricultural Sciences University of British Columbia are acknowledged for providing us with the original cystatin C DNA Gary Lesnicki of the Biotechnology Laboratory University of British Columbia is acknowledged for helping to optimize the methanol feed rate during induction Ross MacGillivray of Biochemistry University of British Columbia is acknowledged for allowing us to use his laboratory for the SDSPAGE procedures

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