|SASAGAWA, TAKAHIRO - University Of Tokyo|
|MORIYA, SHIGEHARU - Riken Institute|
|KITAMOTO, KATSUHIKO - University Of Tokyo|
|ARIOKA, MANABU - University Of Tokyo|
Submitted to: Plasmid Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/24/2010
Publication Date: 1/1/2011
Citation: Sasagawa, T., Moriya, S., Lee, C.C., Kitamoto, K., Arioka, M. 2011. A high-throughput protein expression system in Pichia pastoris using a newly developed episomal vector. Plasmid Journal. 65(1):65-69.
Interpretive Summary: Expression of recombinant enzymes in heterologous expression hosts is a critical step for protein characterization and engineering. We have created a plasmid that can be used to rapidly create and test the expression of different genes. The plasmid can be utilized in Pichia pastoris, a laboratory strain known for its ability to express and secrete high levels of recombinant enzyme. Five different cellulase genes were expressed from the new vector to demonstrate its utility.
Technical Abstract: We describe here the construction of a Gateway-compatible vector, pBGP1-DEST, for rapid and convenient preparation of expression plasmids for production of secretory proteins in Pichia pastoris. pBGP1-DEST directs the synthesis of a fusion protein consisting of the N-terminal signal and pro-sequences of Saccharomyces cerevisiae a-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 further C-terminus. To test the usefulness of this vector, production of five endo-glucanases (EGs) from the termite symbionts was performed. EG activities were detected in the culture supernatants, indicating that the chimeric proteins were produced and secreted as designed. This vector provides a convenient and efficient way for systematic construction of expression plasmids for production of heterologous secretory proteins.