ANALYTICAL BIOCHEMISTRY 243, 127–132 (1996) ARTICLE NO. 0490 Signal Sequence Trap to Clone cDNAs Encoding Secreted or Membrane-Associated Plant Proteins Peter Kristoffersen, Thomas Teichmann, Ralf Stracke, and Klaus Palme 1 Max-Delbru ¨ ck-Laboratorium in der Max-Planck-Gesellschaft, Carl-von-Linne ´-Weg 10, D-50829 Ko ¨ln, Germany Received June 25, 1996 molecular approaches may be more efficient to identify A method was developed for rapid cloning of plant and clone genes encoding membrane proteins. Bacte- cDNAs encoding proteins with membrane-spanning rial expression systems are not suitable for such ap- domains. A novel expression vector was constructed proaches, since bacterial expression of membrane pro- for expression of plant cDNA libraries in COS cells. teins often leads to low expression levels and incorrect Fusion proteins were expressed containing at their N- folding. However, transient expression of cDNAs in terminus an endoplasmic reticulum (ER) signal pep- mammalian cells has proven to be a useful strategy to tide. After entry into the ER these proteins could traf- isolate cDNA clones encoding secreted and membrane- fic via the default pathway to the plasma membrane. bound proteins from animal cells (1, 2). Membrane pro- Trapping at the cell surface occurred when the protein teins that can be identified by expression in COS cells contained one or more membrane-spanning domains. may also be good candidates for large-scale production A simple color-based immunoscreening procedure al- needed for functional and structure analysis. The ad- lowed the isolation of cDNAs after only two rounds vantage of heterologous gene expression in COS cells of COS cell transfection and screening. Several cDNA when compared to expression in yeast cells seems to clones encoding proteins with putative membrane- be the lack of hyperglycosylation of secreted and mem- spanning domains were isolated. Among them were cy- brane proteins which often seems to occur in yeast cells. tochrome b 5 and full-length cDNA clones encoding pu- We have devised a method, using transient expres- tative secretory proteins targeted to the ER membrane sion in COS cells, to clone plant cDNAs encoding pro- by their N-terminal signal peptide.  1996 Academic Press, Inc. teins with signal sequence or membrane-spanning domains. Using a color-based screening procedure combined with plasmid DNA rescue from positively Membrane proteins are important for a broad range stained COS cells and an efficient Escherichia coli of cellular processes and include, for example, cell sur- transformation protocol positive cDNA clones can be face receptors, ion channels, and metabolite transport- isolated very rapidly. ers. At present only few plant genes encoding mem- brane proteins have been isolated. The development of MATERIALS AND METHODS methods for rapid isolation and systematic identifica- tion of such proteins is therefore of general interest. Materials Approaches to identify membrane proteins typically in- Zea mays L., var. Mutin (D/FR 7205034), was ob- volve the isolation of proteins followed by conventional tained from Kleinwanzlebener Saatzucht, Einbeck, automated sequencing techniques. However, the puri- Germany. COS-1 cell line was kindly provided by F. W. fication of membrane proteins often turns out to be very Kluxen, Institute of Physiological Chemistry, Univer- challenging, because frequently only small quantities sity of Du ¨ sseldorf, Germany. Dulbecco’s modified Ea- of membrane proteins are available and because sepa- gle’s medium (DMEM) 2 was obtained from Biochrom ration and sequence analysis by automated Edman degradation of peptides derived from these hydropho- bic proteins can be difficult. Therefore, in the long term, 2 Abbreviations used: AEC, 9-amino-3-ethylcarbazole; BSA, bovine serum albumin; ER, endoplasmic reticulum; HRP, horseradish per- oxidase; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modi- fied Eagle’s medium; FCS, fetal calf serum; LB, Luria broth; CMV, 1 To whom correspondence should be addressed. Fax: 49(0)221- 506-2613. E-mail: palme@mpiz-koeln.mpg.de. cytomegalovirus. 127 0003-2697/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.