Fig. 5

flCDKL5 production with a high copy number bicistronic plasmid. a Comparison of flCDKL5 expression levels in P. haloplanktis TAC125 recombinant strains harboring either a low copy number monoistronic plasmid (p79C-107 (G)) or an optimized high copy number bicistronic plasmid (pB40-BCD2-107 (L) M10V) using Coomassie staining after SDS-PAGE (left panel) and anti-CDKL5 Western blot (right panel). The arrow indicates flCDKL5 produced with the pB40-BCD2-107 (L) M10V plasmid. NI, not induced strain. b Solubility analysis of 107 (L) M10V protein after cellular lysis in native conditions. Total cellular extracts and soluble and insoluble fractions achieved after centrifugation were resolved via SDS-PAGE and the target protein was detected with an anti-CDKL5 Western blot. c Catalytic activity of enriched 107 (L) M10V on pure EB2. After chromatographic enrichment, either 107 (L) M10V (flCDKL5), or a catalytically inactive variant (flCDKL5 KD) was incubated with EB2 and MgATP and then assayed for catalytic activity. A commercial preparation of GST-CDKL5(1–498) from insect cells (CDKL5 ΔC) was used as a positive control. The total amount of the full-length enzyme and the substrate were detected by Coomassie staining (left panel, highlighted by arrows), while phosphorylated EB2 was observed with anti-EB2 pSer222 antibody (right panel)