Werten M W, van den Bosch T J, Wind R D, Mooibroek H, de Wolf F A
Agrotechnological Research Institute (ATO-DLO), Bornsesteeg 59, 6708 PD Wageningen, The Netherlands.
Yeast. 1999 Aug;15(11):1087-96. doi: 10.1002/(SICI)1097-0061(199908)15:11<1087::AID-YEA436>3.0.CO;2-F.
Recombinant non-hydroxylated gelatins based on mouse type I and rat type III collagen sequences were secreted from the methylotrophic yeast Pichia pastoris, using the Saccharomyces cerevisiae alpha-mating factor prepro signal. Proteolytic degradation could be minimized to a large extent by performing fermentations at pH 3.0 and by adding casamino acids to the medium, even though gelatin is extremely susceptible to proteolysis due to its open, unfolded structure. Proteolytic cleavage at specific mono-arginylic sites, by a putative Kex2-like protease, could be successfully abolished by site-directed mutagenesis of these sites. Production levels as high as 14.8 g/l clarified both were obtained, using multicopy tranformants. To our knowledge, this represents the highest level of heterologous protein secretion reported to date for P. pastoris.
基于小鼠I型和大鼠III型胶原序列的重组非羟基化明胶，利用酿酒酵母α-交配因子前体信号，从甲基营养型酵母巴斯德毕赤酵母中分泌出来。尽管明胶由于其开放的、未折叠的结构极易受到蛋白水解作用，但通过在pH 3.0下进行发酵并向培养基中添加酪蛋白氨基酸，蛋白水解降解在很大程度上可以降至最低。通过对这些位点进行定点诱变，可以成功消除假定的类Kex2蛋白酶在特定单精氨酸位点的蛋白水解切割。使用多拷贝转化体，获得了高达14.8 g/l的澄清产量。据我们所知，这代表了迄今为止报道的巴斯德毕赤酵母异源蛋白分泌的最高水平。
