Protocol abstract
Nature Protocols 1, - 2439 - 2447 (2006)
Published online: 29 December 2006 | doi:10.1038/nprot.2006.373
Subject Categories: Biochemistry and protein analysis | Genetic analysis | Genomics and proteomics | Isolation, purification and separation | Model organisms | Nucleic acid based molecular biology
Identification of eukaryotic secreted and cell surface proteins using the yeast secretion trap screen
Sang-Jik Lee1,2, Byung-Dong Kim2 & Jocelyn K C Rose1
Abstract
Secreted and cell surface proteins play essential roles in numerous essential biological processes in eukaryotic organisms, but are often more difficult to isolate and identify than proteins that are localized in intracellular compartments. However, several high-throughput 'gene-trap' techniques have been developed to characterize these 'secretomes', including the yeast secretion trap (YST) screen. This method involves fusing cDNA libraries from the tissue or cell type of interest to a yeast (Saccharomyces cerevisiae) invertase reporter gene, transforming the resulting fusion library into an invertase-deficient yeast strain and plating the transformants on a medium containing sucrose as the sole carbon source. A yeast cell with a transgene encoding a secreted or cell surface protein can synthesize a secreted invertase fusion protein that can rescue the mutant, and the plasmid DNA can then be sequenced to identify the gene that encodes it. We describe a recently improved version of this screen, which allows the identification of genes encoding secreted proteins in 1–2 months.
- 228 Plant Sciences Building, Department of Plant Biology, Cornell University, Ithaca, New York 14853, USA.
- Center for Plant Molecular Genetics and Breeding Research, Seoul National University, Seoul 151-921, Korea.
Correspondence to: Jocelyn K C Rose1 e-mail: jr286@cornell.edu
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