Journal Article

Transmembrane-Domain Trapping: A Novel Method for Isolation of cDNAs Encoding Putative Membrane Proteins

Sumio Sugano, Kiyomi Yoshitomo-Nakagawa, Yong-Shen Yu, Junko Mizushima-Sugano and Kenichi Yoshida

in DNA Research

Published on behalf of Kazusa DNA Research Institute

Volume 5, issue 3, pages 187-193
Published in print January 1998 | ISSN: 1340-2838
Published online January 1998 | e-ISSN: 1756-1663 | DOI: http://dx.doi.org/10.1093/dnares/5.3.187

Show Summary Details

Preview

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 α chain. We constructed a p18Mac vector by putting part of the IL-2 receptor α chain cDNA that encoded its signal sequence and extracellular domain, a cDNA cloning site and a poly(A) additional signal after a strong promoter SRα. If a cloned cDNA provides a transmembrane domain in-frame, the extracellular domain of the IL-2 receptor α 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 α 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.

Keywords: transmembrane domain; IL-2 receptor; membrane protein; epitope tag

Journal Article.  0 words. 

Subjects: Genetics and Genomics

Users without a subscription are not able to see the full content. Please, subscribe or login to access all content.