pYES2/CT/α-factor

产品简介：

 

pYES2/CT/α-factor is a 5.9 kb vector designed for inducible expression of recombinant proteins in Saccharomyces cerevisiae. Features of the vector alow easy cloning of your gene of interest and selection of transformants by uracil prototrophy.The vector contains yeast GAL1 promoter for high level inducible protein expression in yeast by galactose and repression by glucose,and the URA3 gene for selection of transformants in yeast host strains with a ura3 genotype.Proteins expressed fr om pYES2/CT/α-factor are fused at the N-terminus to the a-factor sequence for protein secretion and at the C-terminus to a peptide containing the V5 epitope and six tandem histidine residues for detection and purification.

 

 

pPIC9 was used as a template and α-factor gene of Saccharomyces cerevisiae was amplified by PCR. The gene was cloned in the intracellular expression vector pYES2/CT for Saccharomyces cerevisiae and the secreting expression vector named pYES2/CT/α-factor(pYCα) was constructed. The gene of mannase(man) from recombinant vector of pKLAC1-man (pKLman) was cut by restriction enzymes and linked with pYCα. This recombinant vector pYCα-man was used to determine the secretory ability and stability of pYCα. The excellent secretory ability of pYCα was proved by two experiments. One showed that INVSc1/pYCα-man clones formed the clear rings around the clones on the medium contained trypan blue, while INVSc1/pYCα clones had no rings.Further analysis of mannase activity of extracellular supernatant and intracellular extracts showed that both extracellular and intracellular mannase activities of INVSc1/pYCα were not detected,while INVScl/pYCα-man had evident extracellular mannase activities and no intracellular mannase activities. The stability of pYCα was also very good proved by continuous cultivation for about 150h.

 

以pPIC9为模板,通过PCR扩增获得酿酒酵母的α-交配因子（α-factor）,并克隆至酿酒酵母胞内表达载体pYES2/CT中,构建了一种新型酿酒酵母附加型分泌表达载体pYES2/CT/α-factor（pYCα）。

 

 

 

产品参数：

 

质粒类型：酿酒酵母表达载体

 

出品公司：Invitrogen

 

诱导方法：半乳糖

 

启动子：GAL1

 

5' 测序引物及序列：GAL1-F:AATATACCTCTATACTTTAACGTC

 

3' 测序引物及序列：CYC1-R:GCGTGAATGTAAGCGTGAC

 

载体标签：C-His, C-V5

 

载体抗性：Ampicillin (氨苄青霉素)

 

筛选标记：URA3

 

备注：利用半乳糖诱导蛋白在酿酒酵母中表达。