A GENETIC SELECTION FOR ISOLATING CDNAS ENCODING SECRETED PROTEINS

We describe a simple, rapid technique for simultaneously isolating lar ge numbers of cDNAs encoding secreted proteins. The technique makes us e of a facile genetic selection performed in a strain of Saccharomyces cerevisiae deleted for its endogenous invertase gene. A cDNA cloning vector which carries a modified invertase gene lacking its leader sequ ence is used in conjunction with this strain. Heterologous secreted ge nes fused appropriately upstream of this defective invertase provide t he necessary signals to restore secretion, allowing the yeast to grow on sugars such as sucrose or raffinose. This microbial growth selectio n facilitates scanning cDNA libraries containing millions of clones, e nabling the wholesale identification of novel secreted proteins withou t the need for specific bioassays. The technique is similar to one pre viously described (Klein et al. (1996) Proc. Natl. Acad. Sci. USA 93, 7108-7113). We describe results using a cDNA library derived from acti vated human peripheral blood mononuclear cells (PBMC). Genes identifie d from this library encoded signal sequences of proteins of diverse st ructure, function, and cellular location such as cytokines, type 1 and type 2 transmembrane proteins, and proteins found in intracellular or ganelles. In addition, a number of novel secreted proteins were identi fied, including a chemokine and a novel G-protein-coupled receptor. Si nce signal sequences possess features conserved throughout evolution, the procedure can be used to isolate genes encoding secreted proteins from both eukaryotes and prokaryotes. (C) 1997 Elsevier Science B.V.