Development and application of fully functional epitope-tagged forms of transforming growth factor-beta.
Administration of transforming growth factor-beta (TGF-beta) has been found to be of therapeutic benefit in various mouse disease models and has potential clinical usefulness. However, the ability to track the distribution of exogenously administered, recombinant forms of these proteins has been restricted by cross-reactivity with endogenous TGF-beta and related TGF-beta isoforms. We describe novel FLAG- and hemagglutinin (HA)-tagged versions of mature TGF-beta1 that retain full biological activity as demonstrated by their ability to inhibit the growth of Mv1Lu epithelial cells, and to induce phosphorylation of the TGF-beta signaling intermediate, smad 2. Intracellular FLAG- and HA-TGF-beta1 can be detected in transfected cells by confocal immunofluorescence microscopy. We also describe sandwich ELISAs designed to specifically detect epitope-tagged TGF-beta and demonstrate the utility of these tagged ligands as probes for TGF-beta receptor expression by flow cytometry. The design of these fully functional epitope-tagged TGF-beta proteins should facilitate studies such as the evaluation of in vivo peptide pharmacodynamics and trafficking of TGF-beta ligand-receptor complexes.
Laboratory of Cell Regulation and Carcinogenesis, The National Cancer Institute, National Institutes of Health, Room C629, Building 41, 41 Library Drive, MSC 5055, Bethesda, MD 20892-5055, USA.
Lawrence A
LA
Wolfraim
Gonnie M
GM
Alkemade
Biju
B
Alex
Shellyann
S
Sharpe
W Tony
WT
Parks
John J
JJ
Letterio
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods; D006388:Hemagglutinins / Q000235:genetics / Q000276:immunology; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000276:immunology; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis; D018125:Receptors, Transforming Growth Factor beta / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D053773:Transforming Growth Factor beta1
D000939:Epitopes; D006388:Hemagglutinins; C465540:N-FLAG-TGF-beta1; D009842:Oligopeptides; D010455:Peptides; D018160:Receptors, Cytoplasmic and Nuclear; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C085706:FLAG peptide