A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays.
To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.
Tugendreich
S
S
Iconix Pharmaceuticals, Mountain View, California 94043, USA.
Perkins
E
E
Couto
J
J
Barthmaier
P
P
Sun
D
D
Tang
S
S
Tulac
S
S
Nguyen
A
A
Yeh
E
E
Mays
A
A
Wallace
E
E
Lila
T
T
Shivak
D
D
Prichard
M
M
Andrejka
L
L
Kim
R
R
Melese
T
T
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D012680:Sensitivity and Specificity; D048051:p38 Mitogen-Activated Protein Kinases
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases