Specific detection by flow cytometry of histidine-tagged ligands bound to their receptors using a tag-specific monoclonal antibody.
Engineering proteins to contain a histidine (His)-tag has proved to be very useful for the purification and analyses of these molecules. In the present study, we demonstrate that the binding of His-tagged ligands to their receptors may be visualised by flow cytometry making use of a selected monoclonal antibody (mAb) against the His-tag. Employing this method, a recombinant C3a (rC3a) anaphylatoxin with a His-tag at its N-terminus could be shown to bind to C3a receptor (C3aR)-expressing RBL-2H3 transfectants with a half-maximal effective concentration (EC50) of about 3 nM which is well within the range of published affinity constants. Binding of a recombinant interleukin-8 (rIL-8) molecule with a C-terminal His-tag to RBL-2H3 cells which stably express the IL-8 receptors CXCR1 or CXCR2 could also be demonstrated using the tag-specific mAb. Furthermore, aminoterminally tagged C5a molecules of rat or human origin could be shown to bind to the human C5a receptor (C5aR). However, the fluorescence signal of the binding of rat rC5a to the human C5aR was distinctly higher over a wide range of ligand concentrations than the signal of human rC5a binding although both ligands were equally potent in the induction of chemotaxis in C5aR-expressing cells. Thus, the tag-specific mAb was able to interfere with the binding of human but not rat rC5a to the human C5aR. This observation is in agreement with the hypothesis of a two binding site model for the interaction of human C5a with its receptor whereas a different binding mode may apply for rat C5a. Our data demonstrate that the selected His-tag specific mAb may be a valuable tool for the visualisation of the binding of recombinant ligands to their receptors and may also provide useful information on the specific binding properties of the ligands.
Wilken
H C
HC
Department of Immunology, University of Göttingen, Germany.
Rogge
S
S
Götze
O
O
Werfel
T
T
Zwirner
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000378:metabolism; D015926:Complement C3a / Q000378:metabolism; D015936:Complement C5a / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006639:Histidine / Q000276:immunology*; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins; D051381:Rats; D044087:Receptor, Anaphylatoxin C5a; D019707:Receptors, Chemokine / Q000378:metabolism; D011951:Receptors, Complement / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism*; D018123:Receptors, Interleukin / Q000378:metabolism; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D016209:Interleukin-8; D008024:Ligands; D008565:Membrane Proteins; D044087:Receptor, Anaphylatoxin C5a; D019707:Receptors, Chemokine; D011951:Receptors, Complement; D011971:Receptors, Immunologic; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D011993:Recombinant Fusion Proteins; C085567:complement C3a receptor; D006639:Histidine; D015926:Complement C3a; D015936:Complement C5a