重组蛋白酿酒酵母表达载体

概述

我们的酿酒酵母基因表达载体系统是基于广泛使用的pYES2载体而构建的,它是一个可用于在酵母中表达重组蛋白,或者通过在酵母中进行过表达以研究基因功能的强大而有效的系统。目的基因可以克隆在该载体上,通过客户选择的启动子来介导表达。VectorBuilder上有几种可供选择的标准启动子,其中之一是来自酵母半乳糖激酶(GAL1)基因的强诱导型启动子,它是酵母重组蛋白表达系统中最常用的启动子。

在典型的酵母实验菌株(如INVSc1)中,GAL1启动子的转录活性与培养基中的碳源有一定的关系。在葡萄糖存在的情况下,GAL1启动子的的转录受到抑制;半乳糖则会激活该启动子。因此,通过简单地去除葡萄糖的培养基,并用含有半乳糖的培养基替代,可以实现目的基因的诱导表达。

或者,将棉子糖作为碳源。棉子糖既不抑制也不诱导GAL1启动子的转录,即使在棉子糖存在情况下,加入半乳糖也足以激活GAL1启动子。与使用葡萄糖培养基培养的细胞相比,半乳糖对使用含棉子糖培养基培养的细胞诱导更快。然而,由于棉子糖无法抑制GAL1启动子,所以该方法会导致诱导前目的基因的“泄漏”表达。

通常情况下,利用葡萄糖维持培养的细胞经半乳糖诱导后约4小时可检测到重组蛋白的表达,而用棉子糖培养的细胞仅需约2小时即可。我们建议您设置一个时间梯度来优化重组蛋白的表达。

该载体系统的更多信息,请参考以下文献。

参考文献 主题
Science. 127:28-9 (1958)
Mol. Cell. Biol. 4:1985-98 (1984)
Mol. Cell. Biol. 4:2467-78 (1984)
The GAL1 promoter
Cell 40:767-774 (1985) Induction of gene expression using the GAL1 promoter
Methods Enzymol. 194:1-863. (1991) Extensive information about gene expression in yeast

亮点

该载体系统用于在酿酒酵母中组成型或诱导型表达目的基因。将GAL1启动子和目的基因克隆到载体上,然后通过向培养基中加入半乳糖来诱导目的基因的表达。培养基中的葡萄糖会抑制目的基因的表达,可使用棉子糖作为替代碳源,它不会激活和抑制GAL1启动子。

优势

高水平表达:诱导型GAL1启动子可以使目的基因高水平表达。

表达严谨:在GAL1启动子的控制下,葡萄糖会高效抑制目的基因的表达,而半乳糖则会高效激活其表达。

快速诱导:棉子糖培养的细胞诱导后约2小时内可检测到GAL1介导的重组蛋白。

不足之处

潜在泄漏表达:对于由GAL1启动子驱动的蛋白表达,棉子糖可以替代葡萄糖或者半乳糖用作碳源。然而,棉子糖无法抑制GAL1启动子的活性,这会引起目的基因的泄漏表达。葡萄糖可以用于抑制GAL1启动子的活性。

载体关键元件

Promoter: The promoter that drives your gene of interest is placed here. When the inducible GAL1 promoter is used, galactose will induce high-level transcription of the gene of interest, while glucose will strongly repressed expression. Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

CYC1 terminator: Sequence which facilitates transcriptional termination and polyadenylation of mRNA in yeast.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

Marker: A yeast selectable marker is placed here. It allows the yeast cells successfully transformed with the vector to be selected. One commonly used marker is the orotidine-5'-phosphate decarboxylase (URA3) gene, which allows selection of yeast transformants in uracil or uridine deficient medium. Additionally, if 5-Fluoroorotic acid (5-FOA) is added to the media, the URA3 gene product will convert 5-FOA into 5-fluorouracil, which is a toxin that will cause cell death, thereby allowing selection against yeast carrying the plasmid.

2µ ori: Origin of replication which permits high-copy replication and maintenance in S. cerevisiae.