Related Experiment Videos
Transmembrane-domain trapping: a novel method for isolation of cDNAs encoding putative membrane proteins
S Sugano1, K Yoshitomo-Nakagawa, Y S Yu
1Department of Virology, The Institute of Medical Science, The University of Tokyo, Japan. ssugano@ims.u-tokyo.ac.jp
Summary
We developed a novel method to isolate complementary DNAs (cDNAs) encoding membrane proteins. This system effectively identifies cDNAs that provide transmembrane domains for cell surface expression of chimeric proteins.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- Identifying genes encoding membrane proteins is crucial for understanding cellular functions.
- Existing methods for isolating membrane protein cDNAs can be inefficient or lack specificity.
Purpose of the Study:
- To develop and validate a novel screening system for isolating cDNAs of putative membrane proteins.
- To demonstrate the system's efficacy in identifying previously unknown membrane protein genes.
Main Methods:
- Construction of a p18Mac vector incorporating the IL-2 receptor alpha chain extracellular domain and a cloning site.
- Transfection of cells with a cDNA library generated using the p18Mac vector.
- Screening for cell surface expression of the chimeric protein to identify positive clones.
Main Results:
- Screened 2000 cDNA clones, identifying 5 positive clones expressing the IL-2 receptor alpha chain extracellular domain on the cell surface.
- Partial sequencing revealed known genes (amyloid precursor protein, mitochondrial proteins) and novel gene candidates.
- The system demonstrated effectiveness in isolating cDNAs encoding putative membrane proteins.
Conclusions:
- The developed screening system is effective for isolating cDNAs of putative membrane proteins.
- This method facilitates the discovery of novel membrane proteins and aids in functional studies.
- The system offers a valuable tool for molecular biology and drug discovery.