A genetic selection for isolating cDNAs encoding secreted proteins.
We describe a simple, rapid technique for simultaneously isolating large numbers of cDNAs encoding secreted proteins. The technique makes use 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 sequence is used in conjunction with this strain. Heterologous secreted genes fused appropriately upstream of this defective invertase provide the necessary signals to restore secretion, allowing the yeast to grow on sugars such as sucrose or raffinose. This microbial growth selection facilitates scanning cDNA libraries containing millions of clones, enabling the wholesale identification of novel secreted proteins without the need for specific bioassays. The technique is similar to one previously described (Klein et al. (1996) Proc. Natl. Acad. Sci. USA 93, 7108-7113). We describe results using a cDNA library derived from activated human peripheral blood mononuclear cells (PBMC). Genes identified from this library encoded signal sequences of proteins of diverse structure, function, and cellular location such as cytokines, type 1 and type 2 transmembrane proteins, and proteins found in intracellular organelles. In addition, a number of novel secreted proteins were identified, including a chemokine and a novel G-protein-coupled receptor. Since signal sequences possess features conserved throughout evolution, the procedure can be used to isolate genes encoding secreted proteins from both eukaryotes and prokaryotes.
Jacobs
K A
KA
Genetics Institute, Inc., Cambridge, MA 02140, USA. kjacobs@genetics.com
Collins-Racie
L A
LA
Colbert
M
M
Duckett
M
M
Golden-Fleet
M
M
Kelleher
K
K
Kriz
R
R
LaVallie
E R
ER
Merberg
D
D
Spaulding
V
V
Stover
J
J
Williamson
M J
MJ
McCoy
J M
JM
D000595:Amino Acid Sequence; D018925:Chemokines / Q000235:genetics; D018076:DNA, Complementary / Q000302:isolation & purification*; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D011506:Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012641:Selection, Genetic; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D043324:beta-Fructofuranosidase
D018925:Chemokines; D018076:DNA, Complementary; D021382:Protein Sorting Signals; D011506:Proteins; D011956:Receptors, Cell Surface; D007371:Interferon-gamma; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase