Showing papers on "Pichia pastoris published in 1985"
TL;DR: A methylotrophic yeast, Pichia pastoris, is developed as a host for DNA transformations based on an auxotrophic mutant host of P. pastoris which is defective in histidinol dehydrogenase.
Abstract: We developed a methylotrophic yeast, Pichia pastoris, as a host for DNA transformations. The system is based on an auxotrophic mutant host of P. pastoris which is defective in histidinol dehydrogenase. As a selectable marker, we isolated and characterized the P. pastoris HIS4 gene. Plasmid vectors which contained either the P. pastoris or the Saccharomyces cerevisiae HIS4 gene transformed the P. pastoris mutant host. DNA transfer was accomplished by a modified version of the spheroplast generation (CaCl2-polyethylene glycol)-fusion procedure developed for S. cerevisiae. In addition, we report the isolation and characterization of P. pastoris DNA fragments with autonomous replication sequence activity. Two fragments, PARS1 and PARS2, when present on plasmids increased transformation frequencies to 10(5)/micrograms and maintained the plasmids as autonomous elements in P. pastoris cells.
569 citations
TL;DR: The identification of alcohol oxidase as one of the cloned, methanol-regulated genes has been made by enzymatic, immunological, and sequence analyses and Methanol-regulated expression of each of these three isolated genes can be demonstrated to occur at the level of transcription.
Abstract: The oxidation of methanol follows a well-defined pathway and is similar for several methylotrophic yeasts. The use of methanol as the sole carbon source for the growth of Pichia pastoris stimulates the expression of a family of genes. Three methanol-responsive genes have been isolated; cDNA copies have been made from mRNAs of these genes, and the protein products from in vitro translations have been examined. The identification of alcohol oxidase as one of the cloned, methanol-regulated genes has been made by enzymatic, immunological, and sequence analyses. Methanol-regulated expression of each of these three isolated genes can be demonstrated to occur at the level of transcription. Finally, DNA subfragments of two of the methanol-responsive genomic clones from P. pastoris have been isolated and tentatively identified as containing the control regions involved in methanol regulation.
326 citations
Patent•
10 Jun 1985TL;DR: A process to recover proteins, such as enzymes, from yeast cells is described in this article, which comprises forming an admixture of yeast cells, water, and a minor effective amount of a polychloro aliphatic hydrocarbon, at a suitable pH, and incubating for a suitable time and temperature, such at room temperature, of about 16 to 90 hours.
Abstract: A process to recover proteins, such as enzymes, from yeast cells, which comprises forming an admixture of yeast cells, water, and a minor effective amount of a polychloro aliphatic hydrocarbon, at a suitable pH, and incubating for a suitable time and temperature, such as at room temperature, of about 16 to 90 hours. The resulting supernatant is separated as an aqueous liquid containing a high enzyme activity. Enzymes can be recovered, if desired. Typical applications include Kluyveromyces fragilis for lactase, Pichia pastoris for alcohol oxidase. Typical solvents include methylene dichloride, 1,1,1-trichloroethane, and chloroform.
4 citations