Transmembrane-domain trapping: a novel method for isolation of cDNAs encoding putative membrane proteins.
We have developed a method that enables us to isolate cDNAs of putative membrane proteins. The system is designed to isolate a cDNA which can provide the transmembrane domain to the extracellular part of the IL-2 receptor alpha chain. We constructed a p18Mac vector by putting part of the IL-2 receptor alpha chain cDNA that encoded its signal sequence and extracellular domain, a cDNA cloning site and a poly(A) additional signal after a strong promoter SRalpha. If a cloned cDNA provides a transmembrane domain in-frame, the extracellular domain of the IL-2 receptor alpha chain will be expressed on the surface of the transfected cells. Otherwise, the chimeric protein will be either secreted or retained inside the transfected cells. We made a cDNA library using p18Mac and screened for cDNA clones which allowed the expression of the extracellular domain of the IL-2 receptor alpha chain on the cell surface. Of the 2000 clones screened, 5 clones were scored as positive. Partial sequence analysis revealed that one clone encoded the amyloid precursor protein, two others encoded mitochondrial proteins and the rest were new. These results suggest the system is effective in isolating cDNAs encoding putative membrane proteins.
Sugano
S
S
Department of Virology, The Institute of Medical Science, The University of Tokyo, Japan. ssugano@ims.u-tokyo.ac.jp
Yoshitomo-Nakagawa
K
K
Yu
Y S
YS
Mizushima-Sugano
J
J
Yoshida
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification*; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D015375:Receptors, Interleukin-2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D015704:CD4 Antigens; D018076:DNA, Complementary; D000939:Epitopes; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins