Cysteine-terminated B-domain of Staphylococcus aureus protein A as a scaffold for targeting GABAA receptors

This study reports the preparation and characterization of cysteine-terminated B-domain (Bd-cys) of Staphylococcus aureus protein A, in combination with immunoglobulin G (IgG) antibodies directed against the ρ1 and α1 subunits of GABAA receptors, for localizing reagents of interest to the target rec...

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Bibliographic Details
Published in:Analytical biochemistry 2013-01, Vol.432 (1), p.49-57
Main Authors: Qtaishat, Nasser M., Gussin, Hélène A., Pepperberg, David R.
Format: Article
Language:English
Subjects:
antibodies
B-domain scaffold
cysteine
enzyme-linked immunosorbent assay
fluorescence
GABA receptor
guinea pigs
immunoglobulin G
oocytes
plasma membrane
polyethylene glycol
Protein A
rabbits
receptors
Staphylococcus aureus
surface plasmon resonance
surface proteins
Xenopus laevis
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Summary:This study reports the preparation and characterization of cysteine-terminated B-domain (Bd-cys) of Staphylococcus aureus protein A, in combination with immunoglobulin G (IgG) antibodies directed against the ρ1 and α1 subunits of GABAA receptors, for localizing reagents of interest to the target receptor. A cysteine residue was inserted at the C terminus of the cysteine-lacking B-domain (Bd) and used for conjugating maleimide-containing compounds. As determined by enzyme-linked immunosorbent assay (ELISA), binding of a Bd-cys-S-fluorescein conjugate to polyclonal guinea pig anti-GABAA-ρ1 and rabbit anti-GABAA-α1 IgG was similar to that exhibited by full-length protein A. Surface plasmon resonance analysis of the interaction of Bd-cys-S-PEG3400-biotin conjugate (where PEG is polyethylene glycol) with anti-GABAA-ρ1 and anti-GABAA-α1 yielded KD values of 6.4±1.9 and 0.4±0.1nM, respectively. Fluorescence anisotropy analysis of the binding of Bd-cys-S-fluorescein to the two antibodies yielded EC50 values of 65 and 18nM, respectively. As determined with biotin-reactive fluorescent reagents, Bd-cys-S-PEG3400-biotin specifically bound to the plasma membrane of Xenopus laevis oocytes that expressed α1β2γ2 or homomeric ρ1 GABAA receptors and were pretreated with the corresponding anti-GABAA IgG. The IgG-binding specificity and high affinity of Bd-cys conjugates illustrate the potential of these conjugates, in combination with a selected IgG, to localize compounds of interest at specific cell surface proteins.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2012.08.031