GCN 4 - Based Expression System ( pGES ) : Translationally Regulated Yeast Expression

The expression of foreign proteins in Saccharomyces cerevisiae is a powerful tool for basic research and the biotechnological industry. In spite of the potential of S. cerevisiae, only a few useful expression vectors have been developed for this yeast. These vectors are based on an increasing transcription rate in combination with an increase in gene dosage. Most vectors are maintained as plasmids, which forces growth of cultures on poor selective media. Expression of the yeast Gcn4 protein is regulated at the translational level and increases strongly under amino acid starvation. Because under these conditions protein synthesis in general ceases, it is conceivable that regulatory elements that control Gcn4 expression could support selective expression of foreign genes. We cloned DNA fragments residing upstream from the GCN4 coding sequence (including the 5′ UTR) and ligated them to a cDNA that encodes the human serum albumin (HSA) gene. These GCN4 regulatory elements induced efficient HSA expression at the translational level under amino acid starvation. The GCN4/HSA cassette promoted efficient, inducible expression on either a multicopy or integrative plasmid. The integrated cassette induced a high level of HSA in dense cultures grown on rich media. Thus, the GCN4-based expression system (pGES) provides high protein quantities. pGES is the first expression vector to be induced at the translational level. INTRODUCTION The expression of proteins in microorganisms is a powerful tool for basic research and for various medical and industrial needs. A variety of advanced expression systems have been developed in prokaryotes, in particular in the bacterium Escherichia coli (9,20,28). In this organism, a foreign protein can be expressed efficiently, reaching a level of 10%–30% of its total protein content (7,9,20,28). However, many eukaryotic proteins expressed in bacteria are not active. For various reasons, proteins expressed in bacteria must be extensively purified and assayed in rigorous and expensive quality-control systems. Therefore, although powerful, the bacterial systems are disadvantageous in many cases (3,27). An attractive alternative is the baker’s yeast, Saccharomyces cerevisiae. Most foreign proteins expressed in this eukaryote are accurately processed posttranslationally and are biologically active (3,10,21,27). S. cerevisiae has been widely used in the food industry and is regarded as a safe organism; the risk of side effects and the odds of the presence of pathogenic agents are minimal (3,10,27). Although S. cerevisiae was used successfully for the production of several proteins for therapeutic and diagnostic uses (10), and in spite of its great potential as a protein factory, this organism is not widely used for the production of proteins. The major reason for this seems to be that the level of expression obtained in yeast is significantly lower than that obtained in bacteria (3,10,21,23,24). Only a few yeast expression vectors are available. All vectors (including bacterial and mammalian vectors) contain strong promoters (usually a promoter of a glycolitic gene; e.g., GPD1, ADH1 or PGK1) (21,23,24). In addition, they contain the 2 μm origin of replication or a multicopy integration system that promotes maintenance of the vector in 60–100 copies per cell (10,22). Some of the vectors contain an inducible promoter, the most widely used being the GAL1-10 promoter (21,23). The strategy of all yeast expression vectors is therefore based on a high transcription rate and the presence of many copies per cell. Most yeast expression vectors are present in the cell as nonintegrated plasmids. This forces the growth of expressing cultures on synthetic media under selective conditions. When grown in laboratory flasks, these cultures enter the stationary phase at a low cell density, and the quantities of proteins obtained are low. When grown in industrial fermentors, high cell concentrations could be obtained but would require special treatments and long, expensive incubation time. Here, we describe the development and use of a novel type of expression vector that is fully inducible and can be used in rich media. This vector is based on the regulatory elements of the GCN4 gene. GCN4 encodes a transcriptional activator with target genes that encode amino acids and nucleotides biosynthetic enzymes (15,16). Under optimal growth conditions on media supplemented with amino acids and pyrimidine and purine bases, Gcn4 is not expressed. When cells are starved for amino acids, Gcn4 expression is dramatically induced, and consequently, transcription of biosynthetic genes Research Report 552 BioTechniques Vol. 28, No. 3 (2000) GCN4-Based Expression System (pGES): Translationally Regulated Yeast Expression