E coli strain bl21 de3 competent cells

Site-directed mutagenesis was performed with TaKaRa MutanBest kit (TaKaRa, Beijing, China). Briefly, the ilvC sequence was amplified by PCR from the cDNA template and PCR products were inserted into pOT2 plasmid. pOT2-Ilvc plasmid was amplified with designed mutant primers by PCR, and blunted using Blunting Kination Enzyme Mix, then ligated using ligation solution I in the kit. Plasmids were transformed into Escherichia coli Migula (Enterobacterales: Enterobacteriaceas) DH5α (TransGen, Beijing, China) and verified by DNA sequencing. Five amino acid residues contacting both NADP(H) and Mg²⁺ are conserved among bacteria, fungi, and plants; thus, their 5 active-site residue mutageneses were performed as mentioned above, respectively [15 (link)].
The recombinant ILVC protein was expressed in E. coli [16 (link)]. Briefly, ilvC with a 6× His-tag sequence at the C-terminus was amplified by PCR from the cDNA template or pOT2-Ilvc, and PCR products were inserted into pET-28b (+) vector (Novagen, Beijing, China), then transformed into E. coli strain BL21 (DE3)-competent cells (TransGen, Beijing, China). ILVC protein expression was induced by the addition of isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM and purified with Ni-NTA agarose (Qiagen, Chatsworth, CA, USA) using a nickel-ion affinity column (Qiagen). Protein purity was monitored by SDS-PAGE.

A 444 bp DNA fragment coding the TcCTL15-CRD domain was amplified using the primers listed in Table S1. After that, the amplicons were digested with EcoR I and Xho I and then ligated to the pET-28a expression vector (Novagen, Darmstadt, Germany). The pET-28a-TcCTL15 construct was transformed into E. coli strain BL21 (DE3) competent cells (TransGen, Beijing, China) via induction with a final concentration of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37 °C for 4 h. The recombinant TcCTL15 (rTcCTL15) was expressed as inclusion bodies and subjected to denaturation, refolding, and purification according to a method described earlier [7 (link)]. The purified rTcCTL15 fused with a His-tag was checked using 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with anti-His mouse polyclonal antibodies (TransGen, Beijing, China). The successfully purified rTcCTL15 was quantified using the total protein quantitative assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with bovine serum albumin (BSA) as a standard and was then ready for the binding and agglutination assays.