Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Plasmid. 2011 Jan;65(1):65-9. doi: 10.1016/j.plasmid.2010.08.004. Epub 2010 Aug 31.
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.
我们在此描述了 Gateway 兼容载体 pBGP1-DEST 和 pPICZα-DEST 的构建，用于快速方便地制备毕赤酵母中分泌蛋白表达质粒。这两个载体指导融合蛋白的合成，融合蛋白由酿酒酵母α因子的 N 端信号肽和前肽序列、Kex2 和 Ste13 加工蛋白酶的识别位点、成熟的外源蛋白区域以及分别位于 N 端和 C 端的 attB1 和 attB2 衍生序列组成，在 C 端末端还添加了 myc 和六组氨酸标签。为了测试这些载体的有用性，我们从白蚁共生菌中生产了内切葡聚糖酶和木聚糖酶，以及一种真菌糖醛酸酯酶。在培养上清液中检测到了酶活性，表明设计的嵌合蛋白被合成并分泌。
