10 kda cutoff concentrator

The codon-optimized nucleotide sequence of scFv16 was synthesized by GenScript and subcloned into an expression vector pFastBac1 with an 8x His tag at the C-terminus. The scFv16 used in this paper was the same as that used in the structures of the CB1-Gi-scFv16^{35 (link)}. In brief, scFv16 was expressed in secreted form from Trichuplusia ni Hi5 insect cells and purified by Ni-NTA chromatography. The supernatant was incubated with Ni-NTA resin (GenScript) at 4 °C for 2 h. The resin was then loaded to a gravity column and washed with 15 column volumes (CV) of wash I buffer containing 20 mM HEPES (pH7.5), 100 mM NaCl and 10 mM imidazole; followed by 15 CV of wash II buffer containing 20 mM HEPES (pH7.5), 100 mM NaCl and 30 mM imidazole. The protein was eluted with elute buffer containing 20 mM HEPES (pH7.5), 100 mM NaCl and 250 mM imidazole. The elute was collected and further purified using a Superdex 200 10/300 column (GE Healthcare). Monomeric fractions were pooled, concentrated 10 mg/mL with a 10-kDa cut-off concentrator (Millipore), and flash frozen in liquid nitrogen, then stored at −80 °C for further use.

A 684 bp synthetic gene encoding the astacin domain of T. circumcincta DPY-31, with codons optimised for E. coli, was generated by GeneArt (Life Technologies, UK) and cloned into the pET-28a(+) vector (Novagen, Germany) using NdeI and XhoI. This construct was fully sequenced, transformed into E. coli BL21 (DE3) cells, and recombinant protein expression induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16 °C, 250 rpm overnight. Cells were harvested and stored at −80 °C then resuspended in native lysis buffer, pH 8.0 (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole), 1.7 mg/ml of lysozyme and 0.1 mg/ml of DNaseI. The lysed cells were sonicated and purification of the protein from the soluble cell lysate was performed using Ni-NTA resin columns (Qiagen, UK) under native conditions, with 20 mM imidazole in the wash buffer (50 mM NaH₂PO₄, 300 mM NaCl) and 250 mM imidazole in the elution buffer (50 mM NaH₂PO₄, 300 mM NaCl). The eluates were concentrated together using a 10 kDa cut-off concentrator (Millipore, Germany) and then analysed by SDS–PAGE and Western blotting using an anti-histidine (His) G antibody (Life Technologies, UK) and an anti-mouse IgG (whole molecule) alkaline phosphatase conjugate (Sigma, UK). Protein concentration was determined using the Bradford assay.