Rapid expression cloning of receptors using epitope-tagged ligands and high-speed cell sorting.
BACKGROUND
In this study we describe a new approach for expression cloning of receptors.


METHODS
Our approach was based on highly efficient transfer of retroviral cDNA libraries into target cells and detection of receptor-ligand interaction with the use of an antibody directed against an epitope tag on recombinant ligands. Detection of the complex and isolation of receptor-transduced cells were achieved by flow cytometry and rare event high-speed cell sorting. Recovery of the cDNA coding for the receptor(s) was achieved by polymerase chain reaction.


RESULTS
As a proof-of-concept study we set out to clone the receptor for B-lymphocyte stimulator protein (BlyS), not known at the start of the project but reported while this work was in progress. First, we detected binding of epitope-tagged BlyS to IM9 cells. Second, human T-lymphoblasts (CEM cells), which do not bind BlyS, were transduced with a retroviral cDNA library generated from IM9 cells. Transduced CEM cells binding epitope-tagged BlyS protein were identified by flow cytometry. After three sequential rounds of cell sorting, transduced CEM cell populations with high binding capacity for BlyS were identified. To determine the cDNAs conferring binding to the transduced CEM cells, the integrated proviral DNAs were amplified by polymerase chain reaction and analyzed by DNA sequencing. Rescued cDNAs contained Transmembrane Activator and calcium-modulator and cyclophilin ligand (CAML) Interactor (TACI) and B-Cell Maturation factor (BCMA) sequences, representing two published receptors of BlyS.


CONCLUSIONS
Our data demonstrated that flow cytometry and high-speed cell sorting combined with transduction of retroviral cDNA libraries and binding of epitope-tagged orphan ligands as a selectable phenotype can be used efficiently for expression cloning of receptors. Of particular interest was our finding that apparently it is not necessary to purify the ligand but that conditioned medium containing the ligand can be used instead. Thus we concluded that our approach shortens the time to identify receptors for many orphan ligands and helps to exploit these receptors as drug targets.
Robeva
Anna S
AS
Novartis Institute for Biomedical Research, Functional Genomics, Novartis Pharmaceuticals Corporation, East Hanover, New Jersey 07936, USA.
Yan-Neale
Yan
Y
Burfeind
Patricia
P
Bodian
Dale L
DL
Chirn
Gung-Wei
GW
Kolbinger
Frank
F
Labow
Mark
M
Vallon
Rüdiger D W
RD
D000906:Antibodies / Q000276:immunology; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D002648:Child; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D018985:Epitopes, B-Lymphocyte / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression / Q000235:genetics / Q000276:immunology; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008024:Ligands; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000276:immunology; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000276:immunology; D012190:Retroviridae / Q000235:genetics; D012985:Software Design; D014161:Transduction, Genetic / Q000379:methods*; D016662:Virus Integration / Q000235:genetics
D000906:Antibodies; C120197:BLyS receptor; D018076:DNA, Complementary; D018985:Epitopes, B-Lymphocyte; D008024:Ligands; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor