Fahnestock S R, Bedzyk L A
Central Science and Engineering Laboratories, E.I. DuPont de Nemours & Co., Wilmington, DE 19880-0328, USA.
Appl Microbiol Biotechnol. 1997 Jan;47(1):33-9. doi: 10.1007/s002530050884.
The methylotrophic yeast Pichia pastoris was tested as a host for the production of long, repetitive protein polymers. Synthetic genes for a designed analog of a spider dragline silk protein were readily expressed at high levels under control of the methanol-inducible AOX1 promoter. Transformants containing multiple gene copies produced elevated levels of silk protein, but of a variety of altered sizes as a result of gene rearrangements at the time of transformation. Genes up to 3000 codons in length or longer could be expressed with no evidence of the prevalent truncated synthesis observed for similar genes in Escherichia coli, though genes longer than 1600 codons were expressed less efficiently than shorter genes. Silk-producing P. pastoris strains were stable without selection for at least 100 doublings.
对甲基营养型酵母巴斯德毕赤酵母作为生产长链重复蛋白聚合物的宿主进行了测试。在甲醇诱导型AOX1启动子的控制下，用于蜘蛛拖丝蛋白设计类似物的合成基因很容易高水平表达。含有多个基因拷贝的转化体产生了更高水平的丝蛋白，但由于转化时的基因重排，产生了各种大小改变的丝蛋白。长度达3000个密码子或更长的基因能够表达，没有观察到在大肠杆菌中类似基因普遍存在的截短合成现象，不过长度超过1600个密码子的基因表达效率低于较短的基因。产丝的巴斯德毕赤酵母菌株在无选择条件下至少传代100次仍保持稳定。
