Signal sequence trap to clone cDNAs encoding secreted or membrane-associated plant proteins.
A method was developed for rapid cloning of plant cDNAs encoding proteins with membrane-spanning domains. A novel expression vector was constructed for expression of plant cDNA libraries in COS cells. Fusion proteins were expressed containing at their N-terminus an endoplasmic reticulum (ER) signal peptide. After entry into the ER these proteins could traffic via the default pathway to the plasma membrane. Trapping at the cell surface occurred when the protein contained one or more membrane-spanning domains. A simple color-based immunoscreening procedure allowed the isolation of cDNAs after only two rounds of COS cell transfection and screening. Several cDNA clones encoding proteins with putative membrane-spanning domains were isolated. Among them were cytochrome b5 and full-length cDNA clones encoding putative secretory proteins targeted to the ER membrane by their N-terminal signal peptide.
Kristoffersen
P
P
Max-Delbrück-Laboratorium, Max-Planck-Gesellschaft, Köln, Germany.
Teichmann
T
T
Stracke
R
R
Palme
K
K
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D018744:DNA, Plant / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA; D015398:Signal Transduction
D018076:DNA, Complementary; D018744:DNA, Plant; D008565:Membrane Proteins; D010940:Plant Proteins