Anti-Glu-Glu tag antibody [FITC]

A dot immunoblotting technique has been developed to estimate the relative expression levels of tagged recombinant human proteins in mammalian cell culture media. Variations in sample denaturation, blocking agents and membrane composition and treatment were used to optimize the signal-to-noise ratio of the defined procedure. The method is rapid, with sensitivity extending to the low nanomolar range for a number of recombinant proteins. This technique should have general utility for antibody-based measurements of other tagged and non-tagged proteins in cell culture media or in biological fluids.

The MNK (Menkes disease protein; ATP7A) is a major copper-transporting P-type ATPase involved in the delivery of copper to cuproenzymes in the secretory pathway and the efflux of excess copper from extrahepatic tissues. Mutations in the MNK (ATP7A) gene result in Menkes disease, a fatal neurodegenerative copper deficiency disorder. Currently, detailed biochemical and biophysical analyses of MNK to better understand its mechanisms of copper transport are not possible due to the lack of purified MNK in an active form. To address this issue, we expressed human MNK with an N-terminal Glu-Glu tag in Sf9 [Spodoptera frugiperda (fall armyworm) 9] insect cells and purified it by antibody affinity chromatography followed by size-exclusion chromatography in the presence of the non-ionic detergent DDM (n-dodecyl beta-D-maltopyranoside). Formation of the classical vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50 = 0.7 mu M; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K-0.5 = 0.6 mu M; h = 5.0). The purified MNK demonstrated active ATP-dependent vectorial Cu-64 transport when reconstituted into soya-bean asolectin liposomes. Together, these data demonstrated that Cu(I) interacts with MNK in a co-operative manner and with high affinity in the sub-micromolar range. The present study provides the first biochemical characterization of a purified full-length mammalian copper-transponing P-type ATPase associated with a human disease.