Fig. 2

Development of bicistronic plasmids for the production of flCDKL5. a Stepwise optimization of the production of flCDKL5. b Schemes of Monocistronic (I) and Bicistronic (II) plasmids for the expression of flCDKL5. (I) In the monocistronic configurations we tested two inducible systems (Step 1): p79C dependent on the LacR/pLacZ couple; pMAV dependent on the GalR/pGalT couple. Furthermore, a mutagenesis approach was used to overcome a translation abortion due to a polyA sequence (AAAAAGAAAAAAAAAA, Step 2). (II) The Bicistronic Designs (BCDs) were developed by testing three different combinations of SD1, SD2 and Leader peptide encoding sequences (Step 3). Additionally, a methionine encoding sequence (GTAATG) was mutated to abolish an internal translation start (Step 4) and different permutations of N- and C-terminal tags were applied to increase the preservation and detectability of flCDKL5 extremities (Step 5). Finally, the protein yield was increased by replacing the parental plasmid replication origin with a new one named B40 (Step 6). LacR and pLacZ, the regulator and the promoter of the lacZ gene in P. haloplanktis TAE79, respectively; GalR and pGalT, the regulator and the promoter of the GalT gene in P. haloplanktis TAC125; 5’ UTR, 5’ untranslated region; SD1 and SD2, Shine Dalgarno 1 and 2, respectively; Leader, upstream sequence encoding a short peptide coupled to flCDKL5 translation through the overlap of a stop and a start codons (TAATG); Cm(R), chloramphenicol resistance marker; OriC, E. coli pMB1 replication origin; OriR, Antarctic replication origin; OriT, origin for plasmid conjugative transfer [33]. The underlined sequences were targeted for nucleotide substitution