Pet28a

E. coli DH5α (Thermo Fisher Scientific, Shanghai, China) was used for cloning. Full-length XynSPP2 and catalytic domain (XynSPP2ΔFn3) were subcloned into the expression vector pET-28a (Novagen, San Diego, CA, United States). Overnight cultures of E. coli BL21 (DE3) cells (Thermo Fisher Scientific, Shanghai, China) harboring recombinant plasmids (pET-28a-XynSPP2 or pET-28a-XynSPP2ΔFn3) were prepared to inoculate 200 ml of ZYM 5052 autoinduction medium supplemented with 100 μg/ml kanamycin, and 34 μg/ml chloramphenicol in a 2 L shake flask (Studier, 2005 (link)). After 4-h incubation at 37°C and 250 rpm, the cultures were cooled and further incubated for 24 h at 20°C before harvested by centrifugation. The pellet was resuspended in sodium phosphate buffer (50 mM, pH 7), and the cells were broken by sonication. The cell-free extract was obtained by centrifugation at 25,000 × g for 30 min at 4°C. Protein purification was conducted by Ni-affinity chromatography as described previously (Han et al., 2018 (link)). The homogeneity of the recombinant protein was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The pure fractions were pooled and protein concentration was determined by Bradford using bovine serum albumin as the standard.

Sequences encoding phb were amplified using PCR with two specific primers (Table 1) containing a BamHI site and an EcoRI site added on the 5’ and 3’ ends, respectively. The resulting amplifications of phb were ligated to pET28a (Invitrogen Life Technologies, USA). Plasmid DNAs were transformed into DE3 (Novagen, Germany). A single colony of DE3 harboring the targeting plasmids was inoculated into 10 ml of Luria-Bertani (LB) medium supplemented with kanamycin (Kana⁺) (25 mg/l) and shaken at 37°C for 6-8 h. The pre-culture broth was added to LB medium (Kana⁺) at a ratio of 1: 100, and shaken at 200 rpm at 37°C for several h until the OD600 value reached 0.4-0.8. Isopropyl-β-d-thiogalactoside (IPTG) was added at a final concentration of 1 mM, and the culture was continually incubated at 37°C and shaken at 200 rpm for 10-12 h. After ultrasonication and centrifugation, the sediment was collected and re-suspended with PBS. Recombinant His-PHB proteins in these re-suspended liquids were purified using Ni-Agarose His according to the manufacturer's instructions (CWBIO, China) and assessed using 10% SDS-PAGE.

The genomic DNA of F. nucleatum was used as a template. The gene encoding FnYggS (UniProt: A0A117MW82, residues 1–223) was cloned into the expression vector pET-28a(+) (Invitrogen, Waltham, MA, USA) at the NcoI and XhoI restriction sites. The expression and purification of FnYggS have been previously described [32 (link)]. Site-directed mutagenesis for FnYggS-S201A was performed using two subsequent PCRs. The mutant sequence was confirmed through DNA sequencing, and the expression and purification of the recombinant protein were found to be the same as those of native FnYggS.

The plasmids used for the expression of the FnYggS mutants, which were used in the analysis of PLP binding, were constructed by two subsequent PCRs. The first round of PCR was used to amplify the upstream mutated segment using the forward primer F 5′-GGG CCA TGG GCC ACC ATC ACC ATC ACC ATA TGA GTA TAA AAG CAA ATG TTG AAG-3′ and the reverse primer 5′-TAT CTT ATA ATC ACT AGC CAT TCC CAT TGA AAG-3′. The first round of PCR was used to amplify the downstream mutated segment using the forward primer 5′-CTT TCA ATG GGA ATG GCT AGT GAT TAT AAG ATA-3′ and the reverse primer R 5′-GGG CTC GAG TTA TTT AAA AAT TTT TGT TCC AAC-3′. The second round of PCR introduced an overhang using DNA fragments generated in the first round of PCR as templates and the primers F and R, and they were cloned into the expression vector pET-28a(+) (Invitrogen, USA) at the NcoI and XhoI restriction sites. Furthermore, the plasmids were transformed into E. coli BL21 (DE3) cells that were cultured in Luria-Bertani (LB) medium. The FnYggS mutant was expressed and purified by a method similar to that used for native FnYggS.

In accordance with the two predicted heptad repeat regions of MERS-CoV S protein (AFS88936.1) [11 (link)] from the Learn coil-VMF website [22 (link)] and the solved crystal structures of MERS-6HB [10 (link),11 (link)], we used the sequences of HR1 from amino acid 984 to 1062 and HR2 from amino acid 1245 to 1289 for our construction. The gene of MERS-5HB was synthesized as a codon of HR1–SGGRGG–HR2–GGSGGSGG–HR1–SGGRGG–HR2–GGSGGSGG–HR1 and subcloned into the vector pET-28a(+) (Invitrogen, Carlsbad, CA, USA) for expression in the Escherichia coli strain BL21 (DE3) (Invitrogen). The bacteria were grown at 37 °C in 2× to an optical density (OD₆₀₀) of 0.8 and induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 22 °C for 8 h. The cells were harvested and re-suspended in phosphate buffer saline (PBS) containing 500 mM NaCl, then lysed with 1% Triton X-100 by ultrasonication on ice. The lysate was clarified by centrifugation and the supernatant was passed over a nickel-nitrilotriacetic acid (Ni-NTA) Resin (Qiagen, Hilden, Germany) column pre-equilibrated with PBS containing 500 mM NaCl. The protein was eluted with different concentrations of imidazole. Purified protein was concentrated to a proper concentration determined by the experimental requirements and stored at −80 °C for further analysis.