title: High-throughput recombinant gene expression systems in Pichia pastoris using newly developed plasmid vectors.
abstract: We describe here the construction of Gateway-compatible vectors, pBGP1-DEST and pPICZα-DEST, for rapid and convenient preparation of expression plasmids for production of secretory proteins in Pichia pastoris. Both vectors direct the synthesis of fusion proteins consisting of the N-terminal signal and pro-sequences of Saccharomyces cerevisiae α-factor, the recognition sites for Kex2 and Ste13 processing proteases, the mature region of a foreign protein flanked by attB1- and attB2-derived sequences at N- and C-termini, respectively, and myc plus hexahistidine tags added at the extreme C-terminus. To test the usefulness of these vectors, production of endo-glucanases and xylanases from termite symbionts, as well as a fungal glucuronoyl esterase, was performed. Enzyme activities were detected in the culture supernatants, indicating that the chimeric proteins were synthesized and secreted as designed.
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Sasagawa
authors.forename: Makoto
authors.initials: M
authors.lastname: Matsui
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Masato
authors.initials: M
authors.lastname: Otagiri
authors.forename: Shigeharu
authors.initials: S
authors.lastname: Moriya
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Sakamoto
authors.forename: Yukishige
authors.initials: Y
authors.lastname: Ito
authors.forename: Charles C
authors.initials: CC
authors.lastname: Lee
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Kitamoto
authors.forename: Manabu
authors.initials: M
authors.lastname: Arioka
mesh_terms: D002480:Cellulase / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000378:metabolism; D057075:Enzyme Assays; D015870:Gene Expression; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics*; D024101:Mitochondrial Proteins; D010088:Oxidoreductases / Q000235:genetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D010940:Plant Proteins; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*; D000099188:Alternative Oxidase
chemical_list: D002480:Cellulase; D043364:Endo-1,4-beta Xylanases; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011994:Recombinant Proteins; D000099188:Alternative Oxidase