A transmembrane trap method for efficient cloning of genes encoding proteins possessing transmembrane domain.
To facilitate searching for genes encoding cell membrane proteins, we developed a method for isolating cDNAs that contain sequences for hydrophobic transmembrane runs. This cloning strategy, termed the "transmembrane (TM) trap method," utilizes a vector that directs the cell surface expression of mouse CD4 fusion protein when an insert encoding hydrophobic transmembrane sequences is cloned in-frame with correct orientation. We applied this novel method to isolation of cytokine receptor cDNAs. Our strategy enabled efficient isolation of relatively rare species encoding receptors such as IL-2Rgamma, IL-3Rbeta, IL-4Ralpha, IL-5Ralpha, and IL-6Ralpha. This method also could be used to isolate cDNAs for intracellular molecules with a transmembrane region, e.g., bcl-2. These results indicate that the TM trap method provides an efficient cloning strategy for identification of various families of genes encoding proteins with one or more transmembrane regions.
Miyoshi
S
S
Department of Immunology, University of Tsukuba, Tsukuba, Japan.
Motohashi
T
T
Nakamura
Y
Y
Osawa
M
M
Hiroyama
T
T
Kim
D K
DK
Tokumoto
Y
Y
Nakauchi
H
H
D000818:Animals; D001483:Base Sequence; D015704:CD4 Antigens / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics*; D051379:Mice; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
D015704:CD4 Antigens; D018076:DNA, Complementary; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins