Expression vector construction
In silico gene design and codon optimisation for expression of TeNT in E. coli B strains was performed using Genome Compiler (http://www.genomecompiler.com/, Twist Bioscience). The TeNT gene was synthesised with 3’ Xho I and 5’ Hind III endonuclease sites by IDT (Sydney, Australia). The gene was digested from the pUCIDT delivery vector by combining 1 μg of plasmid DNA with 20 units ofXho I (NEB) and 20 units of Hind III (NEB) in 1 × cutsmart buffer (NEB) and incubating for 1 h at 37°C. One microgram of target vector (pRSET-A; ThermoScientific) was digested in the same manner. Digested DNA was separated on a gel red (Biotium) stained 1% agarose gel for 1.5 h at 70 V. DNA was visualised on a transilluminator blue light box and the bands corresponding to the TeNT gene and pRSET-A vector were excised and DNA extracted from the gel using Bioline’s gel extraction kit (Bioline). TeNT gene insert and pRSET-A vector at a molar ratio of 3:1, respectively, were combined in 1 × T4 ligase buffer (NEB). One unit of T4 ligase was added and ligation was allowed for 16 h at 16°C. One microlitre of ligation reaction was used to transform electrocompetent E. coli D10β cells (NEB). Plasmid DNA from colonies on the selection plates was extracted by miniprep (Bioline) and digested with Xho I and Hind III as previously outlined. Digested products were analysed by separation on a 1.5% agarose gel for 1.5 h at 70 V. The gene construct was designated as pRSET-TeNT.
The tetanus toxin light-chain/translocation domain (LCHn) gene was codon optimized for E. coli expression and synthesised with added 5’Xho I and 3’ Hind III endonuclease sites to facilitate sub-cloning into the pRSET-A expression vector (Thermofisher). The LCHn gene was sub-cloned into pRSET-A by digestion with Xho I andHind III followed by ligation (performed by Genscript, U.S.A.). The vector was designated pRSET-LCHn.
The x-ray crystallography-derived, three-dimensional structure of the TTc sub-domain of tetanus toxin (PDB 1AF9 (Umland et al. , 1997)) was visualised using discovery studio (Biovia, v1.7). To prevent or reduce the receptor binding capability of TTc, amino acid residue mutations R1225E and W1288A situated in the receptor binding pockets of TTc were selected and introduced to the sequence. The TTc R1225E W1288A gene was then codon optimized, synthesised with 5’ Xho I and 3’Hind III endonuclease sites and sub-cloned into pRSET-A as already outlined. The gene construct was designated pRSET-TTc R1225E W1288A.