The methylotrophic yeast, Pichia pastoris, is a popular protein expression system because of its ability to produce high levels of heterologous gene products either intracellularly or extracellularly. Targetting proteins to the extracellullar medium serves as a major purification step because the yeast secretes only a small amount of native protein. Hundreds of recombinant proteins, ranging from spider silk proteins to human insulin, have been produced in this yeast. Despite success of the P. pastoris system, relatively little is known about the molecular biology of its secretion machinery. In order to better understand the mechanisms by which P. pastoris produces and localizes proteins, we have undertaken an analysis of various elements which affect secretion of proteins. We would like to continue projects currently ongoing in our lab: 1. to characterize the genes from isolated mutant strains which secrete higher levels of beta-galactosidase; 2. to understand the molecular nature of leader peptides typically used for secretion of foreign proteins;and 3. to elucidate the nature of the E. coli maltose-binding protein (MBP) as an escort protein and to determine how this bacterial partner protein enhances secretion of fusion proteins in yeast. These studies will identify cis- and trans-acting factors that dictate the export efficiency of proteins expressed in P. pastoris-- illuminating the basic biology behind this secretory system. The elucidation of secretion mechanisms will have two long term benefits: it will increase the basic understanding of how Pichia pastoris targets its proteins, and it will help create a better host system for the growing number of scientists who employ the Pichia pastoris heterologous protein expression as a tool in their own research.
This research will benefit public health by improving a popular yeast protein factory currently used to produce hundreds of proteins useful for both applied and basic research. These include important products such as human collagen, vaccines, and anti-cancer drugs.
