Protein expression strategies for identification of novel target proteins

Identification of new target proteins is a novel paradigm in drug discovery . A major bottleneck of this strategy is the rapid and simultaneous express ion of proteins from differential gene expression to identify eligible cand idates. By searching for a generic system enabling high throughput expressi on analysis and purification of unknown cD-NAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eu karyotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dep endent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpr ession in Saccharomyces cerevisiae using fusions with a peptide that change s its conformation in the presence of Ca2+ ions, the FLAG(R) tag (Eastman K odak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible re ading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The a lpha-leader sequence, encoding a yeast mating pheromone, upstream of the ge ne fusion site facilitates secretion into the culture supernatant. ELF-5A c ould 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 corr ect frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of tra nsmembrane domains or patches of hydrophobic amino acids may preclude secre tion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomp lished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of isolate d proteins was in the range of 90%. In the case of unknown cDNAs, the expre ssion 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 yea st. The expression system enables high throughput production and purificati on of proteins under physiological conditions, and allows miniaturization i nto microtiter formats.