Multiplex expression cloning of blood-brain barrier membrane proteins.
The blood-brain barrier (BBB) is a vascular endothelial interface that separates the brain interior from the bloodstream. Membrane proteins resident at the BBB play important functional and regulatory roles. The current study describes the development and successful implementation of a multiplex expression cloning (MEC) method to allow facile identification of BBB membrane proteins. The overriding goal of the MEC approach was to mine a BBB cDNA library and selectively isolate membrane protein-encoding cDNAs. This selection process was achieved via fluorescence-activated cell sorting (FACS) of cDNA-expressing mammalian host cells for those cells that were immunolabeled with a BBB membrane protein-specific polyclonal antiserum (BMSPA). After optimization of the host cell expression system, four selection rounds allowed the isolation of a panel of 15 unique cDNAs that encoded BBB membrane proteins. The identified proteins display significant diversity in structure, function and in vivo expression levels. The MEC approach thus proved effective for conducting moderate throughput membrane proteome analyses of the BBB while limiting any biases caused by membrane protein insolubility or low in vivo expression levels that can complicate other proteomic approaches.
Agarwal
Nitin
N
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA.
Shusta
Eric V
EV
D000818:Animals; D001812:Blood-Brain Barrier / Q000166:cytology / Q000378:metabolism*; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006801:Humans; D007106:Immune Sera / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015203:Reproducibility of Results
D007106:Immune Sera; D008565:Membrane Proteins