Figure 1: Recombinant production of Dc1a.

(a) Primary structure of rDc1a. The non-native N-terminal Ser residue that is a vestige of the TEV protease cleavage site used for recombinant toxin production is highlighted in grey. Disulphide bridge connectivity is shown above the sequence. (b) Schematic representation of the pLIC-NSB3 vector used for periplasmic expression of rDc1a. The coding region includes a MalE signal sequence (MalE_SS) for periplasmic export, a His₆ affinity tag, an MBP fusion tag and a codon-optimized gene encoding rDc1a, with a TEV protease recognition site inserted between the MBP and toxin-coding regions. The locations of key elements of the vector are shown, including the ribosome-binding site (RBS). (c) SDS-polyacrylamide gel electrophoresis gels illustrating different steps in the purification of rDc1a. Lanes contain: M, molecular weight markers; lane 1, E. coli cell extract before IPTG induction; lane 2, E. coli cell extract after IPTG induction; lane 3, soluble periplasmic extract (the His₆-MBP-rDc1a fusion protein is evident at ~50 kDa); lane 4, Ni-NTA beads after loading the cell lysate; lane 5, eluate-1 from washing Ni-NTA resin with 600 mM imidazole; lane 6, elute-2 from washing Ni-NTA resin with 600 mM imidazole; lane 7, eluate-3 from washing Ni-NTA resin with 600 mM imidazole; lane 8, Ni-NTA beads after elution-3; lane 9, fusion protein sample before TEV protease cleavage; lane 10, fusion protein sample after TEV protease cleavage showing almost complete cleavage of fusion protein to His₆-MBP. (d) RP-HPLC chromatogram showing the final step in the purification of rDc1a. The arrow head denotes the peak corresponding to correctly folded recombinant rDc1a. Inset is a MALDI-TOF MS spectrum showing the [M+H]⁺ ion for the purified recombinant toxin (obs.=6,484 Da; calc.=6,485.39 Da).