Figure 2.

Expression and enzymatic characterization of recombinant wild-type human phospholipase Cζ (PLCζ) protein. (A) One μg of bacterially-expressed, affinity-purified NusA-hPLCζ fusion protein analyzed by 7% SDS-PAGE (left panel) or by immunoblot analysis with either anti-PLCζ polyclonal (V-37; 1:10,000 dilution; middle panel) or anti-NusA monoclonal antibody (1:20,000 dilution; right panel). (B) The PIP₂ hydrolysis enzyme activities of recombinant hPLCζ, mPLCζ, and rPLCδ1 purified by nickel affinity chromatography as NusA-fusion proteins (20 pmol) determined with the [³H]PIP₂ cleavage assay, n = 3 ± standard error of the mean (SEM), using two different preparations of recombinant protein and with each experiment performed in duplicate. In control experiments with NusA alone, no specific PIP₂ hydrolysis activity was observed (data not shown). (C) Effect of varying [Ca²⁺] on the normalized PIP₂ hydrolysis enzyme activity of purified, recombinant hPLCζ, mPLCζ, and rPLCδ1 NusA-fusion proteins. For these assays, n = 2 ± SEM using two different batches of recombinant proteins and with each experiment performed in duplicate.