IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Appl Microbiol Biotechnol. 2010 Jul;87(4):1437-46. doi: 10.1007/s00253-010-2610-7. Epub 2010 Apr 27.
To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-beta-D: -lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.
为了探索棉铃象鼻虫（Ashbya gossypii）作为表达重组蛋白宿主的潜力，并评估其蛋白分泌是否更类似于亲缘关系密切的酿酒酵母（Saccharomyces cerevisiae），还是类似于其他丝状真菌，我们成功地从含有酿酒酵母两个微卫星序列的质粒中，在酿酒酵母 PGK1 启动子的作用下，在棉铃象鼻虫中表达了真菌里氏木霉（Trichoderma reesei）的内切葡聚糖酶 I（EGI）和纤维二糖水解酶 I（CBHI）。EGI 和 CBHI 的天然信号序列能够将 EGI 和 CBHI 分别定向分泌到棉铃象鼻虫的培养基中。虽然不能用 4-甲基伞形酮-β-D：-乳酰-β-D-吡喃糖苷作为底物检测到 CBHI 的活性，但通过使用单克隆抗体的 Western blot 可以检测到 CBHI 蛋白。可以检测到 EGI 的活性，其比活与具有相似 EGI 的酿酒酵母构建体产生的比活相当。与 CBHI 相比，EGI 分泌量更多，或产生的活性蛋白更多。与天然的里氏木霉蛋白相比，在棉铃象鼻虫中表达的 CBHI 和 EGI 经过部分表征后发现过度糖基化，但糖基化程度不如在酿酒酵母中表达的纤维素酶严重。
