Anti-FLAG tag antibody, clone O4 [PE]

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10 % of the total recombinant CFTR (SUMO*-CFTRFLAG-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 +/- 56 mu g SUMO*-CFTRFLAG-EGFP per billion cells at 80 % purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment future CF drug discovery efforts, including biophysical and structural studies.

Altered beta-adrenergic receptor (beta-AR) density has been reported in cells, animals, and humans receiving beta-blocker treatment. In some cases, beta-AR density is upregulated, but in others, it is unaffected or even reduced. Collectively, these results would imply that changes in beta-AR density and beta-blockade are not related. However, it has still not been clarified whether the effects of beta-blockers on receptor density are related to their ability to activate different beta-AR signaling pathways. To this aim, five clinically relevant beta-blockers endowed with inverse, partial or biased agonism at the beta(2)-AR were evaluated for their effects on beta(2)-AR density in both human embryonic kidney 293 (HEK293) cells expressing exogenous FLAG-tagged human beta(2)-ARs and human lymphocytes expressing endogenous beta(2)-ARs. Cell surface beta(2)-AR density was measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Treatment with propranolol, carvedilol, pindolol, sotalol, or timolol did not induce any significant change in surface beta(2)-AR density in both HEK293 cells and human lymphocytes. On the contrary, treatment with the beta-AR agonist isoproterenol reduced the number of cell surface beta(2)-ARs in the tested cell types without affecting beta(2)-AR-mRNA levels. Isoproterenol-induced effects on receptor density were completely antagonized by beta-blocker treatment. In conclusion, the agonistic activity of beta-blockers does not exert an important effect on short-term regulation of beta(2)-AR density.