酿酒酵母蛋白表达载体（Episomal）

概述

S. cerevisiae gene expression vector systems are widely used for the expression of recombinant proteins in yeast as they combine the genetic simplicity of microbial systems with key eukaryotic features such as protein folding and post-translational modifications. Our vectors are designed as shuttle plasmids, which allow for easy manipulation and propagation in E. coli and subsequent transfer into yeast for expression and further studies. 

Yeast shuttle vectors are generally classified into three main types: yeast integrating plasmids (YIp), yeast episomal plasmids (YEp), and yeast centromeric plasmids (YCp). These systems vary in terms of how they are maintained in yeast and consequently their stability and expression levels, catering to a wide range of experimental needs. The table below highlights the main differences between these plasmids and their common applications:

Several standard promoters are available for selection on VectorBuilder. One of them is the strong inducible promoter from the yeast galactokinase (GAL1) gene, which is the most commonly used promoter in yeast recombinant protein expression systems. In typical yeast laboratory strains (e.g. INVSc1), the transcriptional activity of the GAL1 promoter is responsive to the carbon source present in the medium. The presence of glucose represses transcription from the GAL1 promoter, while galactose activates the promoter. Therefore, induction of the gene of interest can be achieved by simply removing the glucose-containing medium from the cells and replacing it with galactose-containing medium.

Alternatively, raffinose may be used as a carbon source. Raffinose neither represses nor induces transcription from the GAL1 promoter, and addition of galactose is sufficient to activate the GAL1 promoter even in the presence of raffinose. Induction of the GAL1 promoter by galactose is more rapid in cells maintained in raffinose-containing medium when compared to cells maintained in glucose-containing medium. However, since raffinose does not repress the GAL1 promoter, this methodology can result in leaky expression of the gene of interest prior to induction.

In general, recombinant proteins can be detected ~4h after induction with galactose in cells that have been maintained in glucose, and after ~2h in cells that have been cultured in raffinose. We recommend that you perform a time course to optimize expression of your recombinant protein.

For further information about this vector system, please refer to the papers below:

亮点

This vector system is designed for constitutive or inducible gene expression in S. cerevisiae. In the case of inducible expression, the gene of interest is cloned into the vector under the control of the GAL1 promoter, which allows induction of the gene of interest by addition of galactose to the medium.

试验验证

Figure 1. EGFP expression in yeast using VectorBuilder’s yeast episomal plasmid (YEp) gene expression vector. (A) An episomal vector expressing a codon-optimized EGFP gene driven by a GPD promoter is transformed into a uracil auxotrophic S. cerevisiae strain. (B) The EGFP expression from the transformants was observed using a fluorescence microscope.

优势

Eukaryotic system: Yeast cells are capable of carrying out moderate post-translational processing of proteins similar to that of mammalian cells. It is particularly suitable for expressing mammalian and other eukaryotic proteins whose function requires post-translational processing not present in prokaryotic expression systems, such as disulfide bond formation.

Technical simplicity and low costs: Protein production is relatively straightforward as yeast is easy to genetically manipulate due to its highly efficient homologous recombination and simple plasmid system. Yeast also grows robustly to high cell densities using standard fermentation processes, resulting in quick and inexpensive large-scale production.

Strong and controlled expression: The inducible GAL1 promoter allows for very high-level expression of genes of interest when activated by galactose and strong repression of genes in the presence of glucose.

Rapid induction: Recombinant proteins expressed under the control of the GAL1 promoter may be detectable ~2 hours after induction in cells that have been cultured in raffinose.

不足之处

Limited post-translational modifications: While yeast possesses the cellular machinery for certain post-translational modifications, it lacks complex glycan structures and utilizes a different method of glycosylation than in humans.

Potential leaky expression: For protein expression driven by the GAL1 promoter, raffinose may be used as a carbon source instead of glucose or galactose. However, raffinose does not repress the GAL1 promoter, which can result in leaky expression of the gene of interest. Glucose must be used in the media for repression of the GAL1 promoter.

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