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Fig. 6 | Microbial Cell Factories

Fig. 6

From: Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Fig. 6

flCDKL5 and EB2 expression with a tricistronic plasmid. a Scheme of the TCD plasmid for the co-expression of flCDKL5 and EB2. The overall backbone was based on the optimized bicistronic pB40-BCD2 plasmid. Hence, it harbored a high copy number replication origin (B40); the selected Leader ORF and the SD1 are derived from LacZ; the SD2 and SD3 upstream of EB2 and flCDKL5 cistrons, respectively, harbor nucleotides perfectly complementary to 16S rRNA (underlined sequences in the inset). flCDKL5 was produced as 107 (L) M10V with the indicated tags and site-specific mutation, while EB2 was synthesized with an N-terminal cMyc and a C-terminal 6xHis tags. The translational coupling of the three cistrons was achieved through the overlap of a stop and a start codon (TAATG). b Co-expression of EB2 and either catalytically active flCDKL5 wt or inactive flCDKL5 KD. After recombinant expression, P. haloplanktis TAC125 recombinant crude lysates were analyzed via Western blot with an anti-His antibody to detect total proteins (left panel) and with an anti-EB2 pSer222 to detect specifically phosphorylated EB2 (right panel). Each assay was performed with biological triplicates (independent cultures) to have a qualitative idea of the robustness of the in cellulo assay. The two arrows serve to distinguish the flCDKL5 specific signal from the EB2 one

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