Nox protein expression, purification and structure analysis
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Flavocytochrome b558 (Cytb) is a heterodimeric integral membrane protein that serves as the electron transferase of the NADPH oxidase. Six homologues of gp91-phox, the large subunit of Cytb, have been identified (Nox family). Understanding of the structure and function of the Nox proteins is limited. To distinguish solvent-accessible and membrane or conformation sequestered regions on native structure of gp91-phox, a number of proteolytic enzyme cleavage products on the lipid reconstituted protein were identified using mass spectrometry, in this study. Affinity-purified rabbit anti-peptide antibodies binding to intact neutrophils suggested extracellular localization of gp91-phox regions, however, results using control CGD-cells suggested that these antibodies may cross-react with an unusual non-gp91-phox species in the normal and CGD-derived plasma membranes. Further, a monoclonal antibody CL5 epitope was mapped to the region 135-DPYSVALSELGDR on the gp91-phox, the prototype for the Nox family proteins. Epitopes of previously described mAb 54.1 and CL5 in gp91-phox align with Nox family proteins with high degree of identity and the use of these two monoclonal antibodies as immunoprobes for Nox family proteins was evaluated. Ab 54.1 was found to be specifically reactive with homologous Nox protein fragments expressed in E. coli. Nox3 protein expressed in HEK293H cells was also detected by 54.1, but not by CL5. Nox1 expression in stably transfected NIH 3T3 was examined using the antibodies, but no detectable binding to Nox1 was observed in immunoblotting assays and by flow-cytometry analysis. The antibodies were also used to probe for presence of potential truncated forms of gp91-phox expressed in chronic granulomatous disease (CGD) affected neutrophils with premature termination of gp91-phox synthesis. Analysis did not detect any smaller size protein fragments by immunoblotting. In addition, two other proteins were found to be crossreactive with 54.1 and CL5, they were identified as GRP58 and gelsolin, respectively, two universally expressed cytosolic proteins with regulated association with the plasma membrane. Finally, to help in ongoing structural biology efforts, a recombinant human Cytb expressing PLB-985 cell line was used to develop process of large-scale production of the protein for application in structural biology experiments.