Enomoto S, Chen G, Berman J
University of Minnesota, St. Paul, USA.
Biotechniques. 1998 May;24(5):782-6, 788. doi: 10.2144/98245st01.
We have constructed a series of vectors (YGALSETs) for the expression of epitope- and affinity-tagged fusion proteins in yeast cells using the regulated GAL10 promoter. Fusion proteins produced from YGALSET plasmids include a leader peptide at the N terminus that encodes both a T7 gene 10 epitope tag and a His6 affinity tag. The YGALSET vector series includes centromere plasmids for low-copy plasmid maintenance and 2 micron episomal plasmids for multicopy plasmid maintenance and four different selectable markers: TRP1, URA3, LEU2 and HIS3. We also provide a convenient approach for transferring cloned genes from a bacterial expression vector into YGALSET vectors by in vivo recombination and a rapid method to screen directly for clones that express the fusion protein of interest.
我们构建了一系列载体（YGALSETs），用于利用受调控的GAL10启动子在酵母细胞中表达表位和亲和标签融合蛋白。由YGALSET质粒产生的融合蛋白在N端包含一个前导肽，该前导肽编码一个T7基因10表位标签和一个His6亲和标签。YGALSET载体系列包括用于低拷贝质粒维持的着丝粒质粒和用于多拷贝质粒维持的2微米附加体质粒，以及四种不同的选择标记：TRP1、URA3、LEU2和HIS3。我们还提供了一种通过体内重组将克隆基因从细菌表达载体转移到YGALSET载体中的便捷方法，以及一种直接筛选表达感兴趣融合蛋白的克隆的快速方法。
