Bioengineered Proteins for Chemical/Biological Defense,Protection, and Decontamination

"Pichia pastoris is a budding yeast used by hundreds of academic and industrial researchers to synthesize a variety of eukaryotic recombinant proteins. P. pastoris is also a model system used to investigate certain areas of eukaryotic cell biology, such asperoxisome biogenesis and degradation, and Golgi organization. The goals of this project are: (1) to determine, with high fidelity, the DNA sequence of the genome of P. pastoris by a combination of "shot-gun" sequencing and "primer-walking" gap closing;(2) to annotate the expected 6,000 plus ORFs; (3) to develop a publicly accessible web site dedicated to the dissemination of information on the sequencing and annotation results; (4) to construct and make available DNA microarrays focused on ~500 P.pastoris genes whose expression is relevant to recombinant protein production; and (5) to construct certain modified P. pastoris strains for improved production of recombinant proteins. The sequencing, assembly, and preliminary gene annotation work willbe performed by Integrated Genomics (IG), an experienced microbial sequencing and analysis company. The hosting of the web site, DNA microarray, and host strain constructions will be performed at the Keck Graduate Institute (KGI). KGI is a new educationaland research institution focusing on bioinformatics and genomics technologies. The availability of the DNA sequence of the P. pastoris genome with associated materials and information as proposed in this application will be of enormous benefit to the manycommercial and academic researchers utilizing this novel but important yeast. Because of the similarity in amino acid sequence between most P. pastoris proteins and their Saccharomyces cerevisiae counterparts, much of the great wealth of informationgenerated over the years for S. cerevisiae genes and products will be transferred directly to those of P. pastoris. Users of P. pastoris will be able to rapidly identify genes of interest and, in conjunction with PCR methodology, have these genes in handfor further study literally overnight. Thus