The methylotrophic yeast, Pichia pastoris, is a popular host system for heterologous gene expression. This yeast has been reported to produce over 500 recombinant proteins ranging from vaccine antigens to anti-tumorigenesis chemotherapeutics. P. pastoris possesses many qualities enabling it to produce high levels of foreign gene products either intracellularly or extracellularly. Additionally, because the yeast secrete only a small amount of native protein into its growth medium, targeting heterologous protein to the ? extracellullar medium serves as a major purification step. ? ? Despite the 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, modifies and localizes proteins, alterations will be made to optimize the secretion of heterologous proteins. To meet this goal, three reporter gene systems have been developed in our lab. These reporters will be used 1) in a genetic screen to isolate supersecreting strains-mutants which secrete higher levels of recombinant proteins, 2) in site-directed mutagenesis experiments to optimize the signal sequence typically used for secretion of foreign proteins and 3) in fusion constructs to the E. coli maltose-binding protein (MBP) to determine if this bacterial partner protein can enhance secretion of fusion proteins. ? ? These studies will identify cis- and trans-acting factors that dictate the secretory efficiency of proteins expressed in P. pastoris, illuminating ways in which the system can be improved. The elucidation of this ? secretory mechanism 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 heterologous protein expression as a tool in their own research. ? ? This research will benefit public health by optimizing a popular microbial protein factory currently used to produce hundreds of proteins useful for industrial, basic research, and pharmaceutical applications. ? ?
