ANALYSIS OF YEAST GLYCOSYLATION OF A HUMAN GLYCOPROTEIN

WE PROPOSE TO USE THE GENE FOR ALPHA-1-ANTI-TRYPSIN (1-AT), A MODERATELY GLYCOSYLATED HUMAN SERUM PROTEIN, AS A SPECIFIC MODEL TO TEST THE FEASIBILITY OF USING BAKERS' YEAST (S. CEREVISIAE) AS A HOST MICROORGANISM FOR PRODUCTION OF HUMAN GLYCOPROTEINS WITH PHARMACEUTICAL APPLICATIONS. SPECIFICALLY, WE INTEND TO CONSTRUCT YEAST EXPRESSION VECTORS TO BOTH PRODUCE 1-AT IN THE CYTOPLASM AND TO DIRECT IT INTO THE SECRETION PATHWAY WHERE IT MAY BE GLYCOSYLATED. THE EXPERIMENTS WILL BE DESIGNED TO ANSWER THE QUESTION, CAN THE YEAST CELL ADD CARBOHYDRATE TO THE CORRECT ASPARAGINE RECEPTORS OF A MAMMALIAN GLYCOPROTEIN? ASSUMING THAT ANSWER IS AFFIRMATIVE, IN PHASE II THE SERUM STABILITY OF DIFFERENT FORMS OF 1-AT, PRODUCED IN PHASE I BY SPECIFIC YEAST MUTANTS WHICH ADD DIFFERENT PATTERNS OF CARBOHYDRATE, WILL BE COMPARED WITH ONE ANOTHER AND WITH UNGLYCOSYLATED 1-AT BY MEANS OF A RAT MODEL SYSTEM. ALSO IN PHASE II, ADDITIONAL CARBOHYDRATE PATTERNS WILL BE GENERATED BY IN VITRO ENZYMATIC METHODS APPLIED TO A SUITABLE INTERMEDIATE DERIVED FROM A PARTICULAR YEAST MUTANT WHICH ADDS A SHORTENED CARBOHYDRATE CORE TO PROTEINS. THESE EXPERIMENTS ARE AIMED AT DISCOVERING A METHOD TO PRODUCE HUMAN GLYCOPROTEINS IN A MICROORGANISM BY EXPLORING WAYS TO OVERCOME THE POTENTIAL PROBLEMS OF STABILITY AND ANTIGENICITY.