pYES2/CT釀酒酵母表達載體 pYES2/CT載體大小為6.0 kb左右，用于重組蛋白在釀酒酵母細胞中的誘導型表達。誘導劑為半乳糖。重組蛋白的C-端融合了6XHis標簽，用于重組蛋白的純化和檢測。 pYES2/CT載體含有以下元件： Yeast GAL1 promoter for high level inducible protein expression in yeast by galactose and repression by glucose (Giniger et al., 1985; West et al., 1984)
Multiple cloning site (MCS) with 8 or 9 unique sites (plus two BstX I sites) to facilitate in-frame cloning with the C-terminal peptide
C-terminal peptide encoding the V5 epitope and a polyhistidine (6xHis) tag for detection and purification of your recombinant fusion protein
2μ origin for episomal maintenance and high copy replication or CEN6/ARSH4 sequence for non-integrative centromeric maintenance and low copy replication (pYC2/CT)
URA3 auxotrophic marker for selection of yeast transformants
Ampicillin resistance gene for selection in E. coli 使用pYES2/CT載體表達目的基因的實驗流程如下： 1 Consult the multiple cloning site described on page 7 to determine a strategy to clone your gene in frame with the C-terminal peptide.
2 Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on LB plates containing 50 to 100 μg/mL ampicillin.
3 Analyze your transformants for the presence of insert by restriction digestion.
4 Select a transformant with the correct restriction pattern and sequence to confirm that your gene is cloned in frame with the C-terminal peptide.
5 Transform your construct into competent INVSc1 cells and select for the appropriate amino acid prototrophy.
6 Test for expression of your recombinant protein by western blot analysis or functional assay.
7 Use metal-chelating resin such as ProBond to purify your recombinant protein.
