Research Project
3497544
We propose to use the gene for alpha-1-anti-trypsin (Yield1-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 application. Specifically, we intend to construct yeast expression vectors to both produce Yield1-AT in the cytoplasm and to direct it into the secretion pathway where it may glycosylated. The ecperiments 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 Yeild1-AT, produce in Phase I by specific yeast mutants which add different patterns of carbyhydrate, will be compared with one another and with unglycosylated Yield1-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 carbyhydrate core to proteins. These exeperiments are aimed at discovering a method to produce human glycoproteins in a microroganism by exploring ways to overcome the potential problems of stability and antigenicity.
