For biotechnology, N-glycosylation is an important cotranslational modification reaction that allows many of the nascent glycoproteins to fold correctly, to become biologically active and to be secreted efficiently. In this work, it was shown that for yeast invertase that Nglycans were essential for the formation of oligomers. Invertase from secl8 mutant cells and unglycosylated internal invertase were isolated by a novel mild procedure without denaturation or precipitation. Antibodies were

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... rated and used to characterize intermediate states of oligomers that were produced by the action of Endo H under native conditions. Outer chains of N-linked glycans are believed to contribute to stability of invertase oligomers as found by correlating the enzymatic activity with the degree of deglycosylation. When outer chain extension reactions were blocked by 1-deoxynojirimycin, glycosylated invertase activity was drastically reduced as revealed by native gel electrophoresis of total cell extracts. This indicated that outer chains were required for the glycosylated invertase to become fully active or to stabilize the enzymatic activity. In this work, yeast was studied as a tool for expression of foreign proteins. In contrast to mammalian cells, yeast does not add complex type sugars to its glycoproteins, but it extends the core glycans with mannoside residues. Yeast glycoproteins therefore elict an immunogenic response when they are administered to mammals. Endo H, the enzyme that efficiently cleaves high mannose type glycans was purified first by conventional chromatography procedures. Since this was time consuming, a shorter procedure was developed, based on the affinity of the enzyme for its reversibly immobilized substrate. This procedure gave a glycosidase and protease free Endo H preparation. In addition, this protocol was also efficient for the purification of recombinant Endo H expressed in E. coli. Antibodies raised against the E. coli Endo H recognized both, substrate affinity purified S. plicatus Endo H and conventionally purified Endo H from S. plicatus. Sialic acid residues increase the half life of serum glycoproteins. We describe here for the first time the incorporation of sialic acid into a yeast glycoprotein. Yeast invertase was completely deglycosylated with Endo H and the generated GlcNAc residues were modified with galactose moieties using the specific galactosyltransferase. The galactose residues themselves served in a third reaction as acceptors of sialic acid residues, coupled by the specific sialyltransferase. The potential to carry out N-glycosylation is not the sole aspect that makes yeast interesting for biotechnology. Due to its porous, but sturdy cell wall, yeast cells can be immobilized to solid supports and secreted products can be recovered from column reactors. Here, yeast was used as a host for the expression of two heterologous proteins, human somatomedin C and rabbit polymeric immunoglobulin receptor. SMC was directed to the secretory pathway by the glycosylated pre-pro-afactor SMC fusion precursor. Correctly processed, biologically active SMC was secreted from both, batch cultures and immobilized cells to levels of 1.2 to 1.4 mg liter" medium. In order to increase the