Departamento de Bioquímica, Microbiología, Biología Celular y Genética, Universidad de La Laguna, 38206, La Laguna (Tenerife), Spain.
Microb Biotechnol. 2019 Nov;12(6):1139-1153. doi: 10.1111/1751-7915.13322. Epub 2018 Oct 5.
Heterologous protein expression in yeast, mostly in Saccharomyces cerevisiae and Pichia pastoris, is a well-established and widely used technique. It typically requires the construction of an expression vector in Escherichia coli containing the foreign gene and its subsequent transformation into yeast. Although simple, this procedure has important limitations for the expression of large numbers of proteins, that is, for the generation of protein libraries. We describe here the development of a novel system for the easy and fast expression of heterologous proteins both in S. cerevisiae and in P. pastoris, under the control of the GAL1 and AOX1 promoters respectively. Expression in S. cerevisiae requires only the transformation of yeast cells with an unpurified PCR product carrying the gene to be expressed, and the expression of the same gene in P. pastoris requires only the isolation of the plasmid generated in S. cerevisiae and its transformation into this second yeast, thus making this system suitable for high-throughput projects. The system has been tested by the extracellular expression of 30 secretory fungal proteins.
酵母中的异源蛋白表达，主要是在酿酒酵母和巴斯德毕赤酵母中，是一种成熟且广泛应用的技术。它通常需要在大肠杆菌中构建一个包含外源基因的表达载体，然后将其转化到酵母中。尽管这种方法很简单，但对于大量蛋白质的表达，即蛋白质文库的构建，它有重要的局限性。我们在这里描述了一种新的系统，用于在酿酒酵母和巴斯德毕赤酵母中分别在 GAL1 和 AOX1 启动子的控制下，轻松、快速地表达异源蛋白。在酿酒酵母中表达只需要用未纯化的携带待表达基因的 PCR 产物转化酵母细胞，而在巴斯德毕赤酵母中表达同样的基因只需要分离在酿酒酵母中生成的质粒并将其转化到第二种酵母中，因此该系统适用于高通量项目。该系统已经通过 30 种分泌真菌蛋白的细胞外表达进行了测试。
