A signal sequence trap based on a constitutively active cytokine receptor
Tetsuo Kojima
& Toshio Kitamura
Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan.
Correspondence should be addressed to Toshio Kitamura kitamura@ims.u-tokyo.ac.jpsignal sequence trapretrovirus cDNA library
Targeting of secreted and cell-surface proteins to the cell membrane is mediated by a short hydrophobic stretch of amino acids, termed the signal sequence. We have developed a method that detects signal sequences in cDNA fragments based on their ability to redirect a constitutively active mutant of a cytokine receptor to the cell surface, thereby permitting interleukin-3 (IL-3)-independent growth of Ba/F3 cells. Retrovirus-mediated expression of the fusions in IL-3−dependent cells was followed by selection of clones for growth in the absence of IL-3. Infection of cells with 5 106 viral particles in a pilot experiment led to the isolation of 150 known and 48 novel cDNA clones, and all the known cDNA clones were found to encode secreted and cell-surface proteins. In addition, we isolated type II membrane proteins, which have not been detected by existing signal sequence trap strategies.
106 viral particles in a pilot experiment led to the isolation of 150 known and 48 novel cDNA clones, and all the known cDNA clones were found to encode secreted and cell-surface proteins. In addition, we isolated type II membrane proteins, which have not been detected by existing signal sequence trap strategies.
