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.