A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays

被引：22

作者：

Tugendreich, S ^{[1
]}

Perkins, E ^{[1
]}

Couto, J ^{[1
]}

Barthmaier, P ^{[1
]}

Sun, DX ^{[1
]}

Tang, S ^{[1
]}

Tulac, S ^{[1
]}

Nguyen, A ^{[1
]}

Yeh, E ^{[1
]}

Mays, A ^{[1
]}

Wallace, E ^{[1
]}

Lila, T ^{[1
]}

Shivak, D ^{[1
]}

Prichard, M ^{[1
]}

Andrejka, L ^{[1
]}

Kim, R ^{[1
]}

Melese, T ^{[1
]}

机构：

[1] Iconix Pharmaceut, Mountain View, CA 94043 USA

来源：

GENOME RESEARCH | 2001年 / 11卷 / 11期

关键词：

D O I：

10.1101/gr.191601

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 similar to 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.

引用

页码：1899 / 1912

页数：14

共 67 条

[11] The development and therapeutic potential of protein kinase inhibitors [J].