E coli rosetta de3 competent cells

Recombinant SjAChE was expressed in the Escherichia coli expression system. A fragment encoding the SjAChE (G71-Y537) was amplified by PCR and ligated into the pET28b vector (Invitrogen) by using forward (5′-CGGGATCCGGGAATTTACTGTGGACAACGTG-3′ with BamHI restriction site underlined) and reverse (5′-CGCTCGAGATAACCATGCATTGTACCAGTCC-3′ with Xhol restriction site underlined) primers. E. coli Rosetta™ (DE3) competent cells (Invitrogen) were transformed with the recombinant plasmids for expression and purification as described [30 (link)]. The expressed SjAChE protein was purified from the E. coli lysate using Ni-NTA affinity chromatography (GE Healthcare Life Science, Chicago, IL, USA) under denaturing conditions, using 6 M guanidine-HCl and then refolding in buffer (300 mM NaCl, 50 mM NaH₂PO₄, 8% w/v sucrose, PH 4.5). Residual endotoxin was removed from purified proteins as described [30 (link)] and assessed by using an Endotoxin (E. coli) Standards kit (Lonza, Anaheim, CA, USA). The purified rSjAChE was used in mice vaccine/challenge experiments.

The coding regions of HcBSMTs were amplified using high-fidelity DNA polymerase KOD-Plus (TOYOBO) with corresponding primers (Supplementary Table 2) and then subcloned into pET30a vector (Novagen) through EcoRI and NotI sites. The recombinant vectors verified by sequencing were transformed into E. coli Rosetta (DE3) competent cells (Invitrogen). The induction expression of recombinant protein with isopropyl-β-D-thiogalactopyranoside (IPTG) and partial purification with Ni-NTA His·Bind Resins (Novagen) were performed as described previously (Yue et al., 2014 (link)). The pET30a vector with no insert was used as negative control.

The coding region of HcTPS6 was amplified using high-fidelity DNA polymerase KOD-Plus (TOYOBO) with the forward primer 5′-GGATCCATGGCTACTCGTCAAGCAAT-3′ and the reverse primer 5′-GTCGACGGATTTGGATAGGTTCAAAT-3′, then cloned into pET30a vector (Novagen) through BamHI and SalI sites. The resulting constructs were sequenced for no errors and transformed into E. coli Rosetta (DE3) competent cells (Invitrogen). The induction, purified and concentration determination of recombinant protein were performed as described previously [46 (link)]. Enzyme assay was carried out in 5-ml sealed glass vials in a total volume of 1 ml containing 30 mM HEPES (pH 7.5), 5 mM DTT, 20 mM MgCl₂, 20 μM GPP/FPP (Sigma) and ~10 μg recombinant HcTPS6 proteins. A PDMS fiber for solid-phase microextraction was inserted into the vial to collect volatiles. The mixture was incubated at 30 °C for 1 h and then at 45 °C for 15 min. After incubation, the PDMS fiber was injected into a gas chromatography–mass spectrometry (GC-MS) system for analysis as described above. The products were identified by comparing the mass spectra and retention times with authentic standards or known profiles of H. coronarium, or by comparing mass spectra with the NIST 08 mass spectra library.