High-throughput recombinant gene expression systems in Pichia pastoris using newly developed plasmid vectors.
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.
Sasagawa
Takahiro
T
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Matsui
Makoto
M
Kobayashi
Yuki
Y
Otagiri
Masato
M
Moriya
Shigeharu
S
Sakamoto
Yasuharu
Y
Ito
Yukishige
Y
Lee
Charles C
CC
Kitamoto
Katsuhiko
K
Arioka
Manabu
M
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
D002480:Cellulase; D043364:Endo-1,4-beta Xylanases; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011994:Recombinant Proteins; D000099188:Alternative Oxidase