- Oral Presentation
- Open Access
A novel yeast expression system based on a hormone-induced transcriptional cascade
© Quintero et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Heterologous Protein Production
- Heterologous Protein Expression
- Human Estrogen Receptor
- Yeast Expression System
- Transcriptional Cascade
The yeast Saccharomyces cerevisiae is widely utilized in gene expression projects, both as a model eukaryotic organism and as a host for heterologous protein production. Much of this research and biotechnological activity demands the use of highly regulated systems, able to provide accurate control of the expression in gene function analysis, and timely recombinant protein synthesis during fermentative production. Different yeast expression systems have been developed that can be controlled at the transcriptional level. Among these systems, those based on the potent, tightly regulated GAL1-10 promoter and its cognate transcriptional activator Gal4 are most commonly used . However, induction of the GAL system requires the presence of galactose and the absence of glucose in the culture media , a major disadvantage when the metabolic changes associated to this switch in carbon source are relevant to the study. In addition, the high cost of the inducer can preclude scaling up production of a commercially valuable protein using this system. A good alternative to regulate transcription driven by GAL promoters is the incorporation of the hybrid protein developed by D. Picard , which combines features of three different transcriptional activators, the DNA binding domain of Gal4, the hormone response domain of the human estrogen receptor (ER), and the transcription activation domain of herpes-virus VP16. This chimerical protein activates transcription from GAL1-10 promoters in the presence of estradiol at micromolar concentrations, even in the presence of glucose. However, constitutive expression of this transactivator originates a high basal activity of the GAL promoters in the absence of the hormone, therefore diminishing its efficiency as a transcriptional regulator.
We have tested two novel gene/protein expression systems derived from the combination of different eukaryotic transcription elements. One of the systems activated expression of genes under the control of GAL promoters in S. cerevisiae, uncoupling it from the galactose/glucose signaling and keeping the low basal activity found in GAL promoters. The other system consisted of a cascade of estrogen-dependent activators able to stimulate transcription from an ERE-containing promoter. This new CASCADE system could constitute a simple and cost-efficient way to control heterologous protein expression in yeast, especially in projects requiring tightly controlled protein production, including functional genomics and industrial recombinant protein production.
This work was supported by PROFIT grants from the Spanish Ministerio de Educación y Ciencia (FIT-01000-2003-110 and CIT-01000-2005-32) and by the Andalusian Government (CVI-271).
- Schneider JC, Guarente L: Vectors for expression of cloned genes in yeast: regulation, overproduction, and underproduction. Methods Enzymol. 1991, 194: 373-388.View ArticleGoogle Scholar
- Johnston M, Carlson M: Regulation of carbon and phosphate utilization. The Molecular Biology of the Yeast Saccharomyces. Edited by: Jones EW, Pringle JR, Broach JR. 1992, 2: 193-281. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
- Louvion JF, Havaux Copf-B, Picard D: Fusion of GAL4-VP16 to a steroid-binding domain provides a tool for gratuitous induction of galactose-responsive genes in yeast. Gene. 1993, 131: 129-134. 10.1016/0378-1119(93)90681-R.View ArticleGoogle Scholar
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