What methods did researchers use to create this figure Disre

BoNT/A-LC (PBN4) 300 SNAP-25 14.2 FING. 4 The L chain of BoNT/A cleaves SNAP-25 and requires Glu 224 for activity. In the experiments shown in the left part of the figure, SNAP-25 was transiated in vitro in the presence of [35S)methionine and combined at the end of the incubation with BONT/A-L chain and TeTx- L chain that were translated in vitro in parailel incubations using un- labelled amino acids. In the experiments shown in the right three lanes, BONT/A components were used that were purified from bacteria. Note that neither the H chain nor the two mutant L chains or TeTx L chains caused cleavage of SNAP-25. METHODS. For the construction of the GIn 224 and Lys 224 mutants, two new singular restriction sites, BamHI and Nrul were generated by PCR in positions 1,002 and 1.063 The sites flank the sequence coding for a conserved HExxHxxH motif that is essential for Zn binding, thus allowing replacement of the internal sequence by appropriate synthetic oligonucleotides. In vitro translation was as given in Fig. 3 but linearized DNA was used as template for the generation of mRNA. SNAP-25 mRNA was translated in the presence of P5s)methionine. BoNT/A-LC mRNA was translated in an unlabelled form, both under control of SP6 pro- moter in pSP72 (Promega, Madison, WI. At the end of the translation period (60 min, 30 °C), cycloheximide was added to both translation assays (250 uM final concentration) before mixing of the samples. Incu- bation was for 2 h at 37 C. Electrophoresis and fluorography were as described in Fig. 3

Solution

Botulinum neurotoxins (BoNT) is produced by the anaerobic gram-positive bacteria of species Clostridium botulinum, C. baratii, and C. butyricum. It consist of seven different immunologically serotypes from A to G. BoNTs are synthesized approx.150-kDa single-chain protoxins which is processed by proteolytic cleavage to form a disulfide-linked dimer.

The techniques used are sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Gel electrophoris, PCR and Western blotting