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 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 cDNAs, 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 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 alpha-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.
Schuster
M
M
Novartis Forschungsinstitut, Vienna, Austria.
Wasserbauer
E
E
Einhauer
A
A
Ortner
C
C
Jungbauer
A
A
Hammerschmid
F
F
Werner
G
G
D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D062792:MARVEL Domain-Containing Proteins; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D009842:Oligopeptides; D010448:Peptide Initiation Factors / Q000032:analysis / Q000235:genetics*; D010455:Peptides; D010957:Plasmids; D011510:Proteolipids; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae; D000097574:Eukaryotic Translation Initiation Factor 5A
D062792:MARVEL Domain-Containing Proteins; D008565:Membrane Proteins; D009842:Oligopeptides; D010448:Peptide Initiation Factors; D010455:Peptides; D011510:Proteolipids; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C094765:PLP2 protein, human; C085706:FLAG peptide