SIGNAL SEQUENCE TRAP TO CLONE CDNAS ENCODING SECRETED OR MEMBRANE-ASSOCIATED PLANT-PROTEINS

A method was developed for rapid cloning of plan cDNAs encoding protei ns with membrane-spanning domains. A novel expression vector was const ructed for expression of plant cDNA libraries in COS cells. Fusion pro teins were expressed containing at their N-terminus an endoplasmic ret iculum (ER) signal peptide. After entry into the ER these proteins cou ld traffic via the default pathway to the plasma membrane. Trapping at the cell surface occurred when the protein contained one or more memb rane-spanning domains. A simple color-based immunoscreening procedure allowed the isolation of cDNAs after only two rounds of COS cell trans fection and screening. Several cDNA clones encoding proteins with puta tive membrane-spanning domains mere isolated. Among them were cytochro me bg and full-length cDNA clones encoding putative secretory proteins targeted to the ER membrane by their N-terminal signal peptide. (C) 1 996 Academic Press, Inc.