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Protein Expression Strategies for Identification of Novel Target Proteins

Novartis Forschungsinstitut, Vienna, Austria, Institute of Applied Microbiology, University of Agriculture and Forestry, Vienna, Austria

E. Wasserbauer

Novartis Forschungsinstitut, Vienna, Austria

A. Einhauer

Institute of Applied Microbiology, University of Agriculture and Forestry, Vienna, Austria

C. Ortner

Institute of Applied Microbiology, University of Agriculture and Forestry, Vienna, Austria

A. Jungbauer

Institute of Applied Microbiology, University of Agriculture and Forestry, Vienna, Austria

F. Hammerschmid

Novartis Forschungsinstitut, Vienna, Austria

G. Werner

Novartis Forschungsinstitut, Vienna, Austria

Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cD-NAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca²⁺ ions, the FLAG(r) tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The a-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of isolated proteins was in the range of 90%. In the case of unknown cDNAs, the expression product with the highest molecular mass was assumed to represent the correct reading frame. In summary, we consider the YEpFLAG-1 system to be a very efficient tool to overexpress and isolate recombinant proteins in yeast. The expression system enables high throughput production and purification of proteins under physiological conditions, and allows miniaturization into microtiter formats.

Journal of Biomolecular Screening, Vol. 5, No. 2, 89-97 (2000)
DOI: 10.1177/108705710000500205


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