Ni2 nta superflow column

The coding sequence of Vd2LysM was amplified from V. dahliae cDNA and cloned into the pETSUMO (Invitrogen) expression vector according to the manufacturer's instructions prior to E. coli Origami (DE3) transformation. A single transformant was selected and grown in Luria broth medium until an optical density at 600 nm (OD₆₀₀) of 0.9 was reached. Heterologous production of Vd2LysM was induced with 1 mm Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) at 26°C during ∼20 h. Cell pellets were lysed using lysozyme from chicken egg (Sigma, St Louis, MO, USA) and Vd2LysM was purified from the soluble protein fraction using an Ni²⁺‐NTA Superflow column (Qiagen). Purified protein was dialysed against 200 mm NaCl and concentrated over Amicon ultracentrifugal filter units (Molecular weight cut‐off (MWCO) = 3 kDa; Millipore, Billerica, MA, USA).

Antibodies were computationally designed as previously described [18 (link),37 (link)]. Complementary peptides designed to bind specific epitopes were grafted onto the CDR loops of domain antibodies [18 (link),37 (link)]. The expression and purification of the antibodies were carried out as previously described [18 (link)]. In brief, DesAb constructs were expressed and purified using pRSET-B vector in E. coli Rosetta-gami 2 (DE3, Merck Millipore, Burlington, MA, USA), cells were grown, harvested and lysed, and the antibodies (circa 18 kDa) were purified using a Ni²⁺-NTA Superflow column (Qiagen, Hilden, Germany) [18 (link)]. The His-tagged antibodies were eluted by increasing the concentration of imidazole, and were subsequently dialyzed thoroughly in PBS. Protein concentration was determined by absorbance at 280 nm [18 (link)]. DesAb aliquots were stored at −20 °C until use and never thawed more than once. The sequence of DesAb_18-24 is MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPKLEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIGWVRRAPGKGEEWVASIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAAGSVFVGTEAEEEAAAWGQGTLVTVSSGT and that of DesAb_34-40 is MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPKLEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIGWVRRAPGKGEEWVASIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAAGSIRETALLYEEEAAAWGQGTLVTVSSGT.