Escherichia coli bl21 de3 cells

The BodoCSP4 cDNA was PCR amplified with specific primers (Table 1). The PCR products were ligated into the expression vector pBM30 according to the manufacturer’s instructions (Biomed, Beijing, China). The ligation products containing the pBM30/BodoCSP4 sequence were used to transform Escherichia coli BL21 (DE3) cells (TransGen Biotech) for protein expression and sequencing. Positive colonies were used for expression and purification of the recombinant BodoCSP4 protein. The results showed that, after sonication and centrifugation, the BodoCSP4 proteins were mainly expressed in insoluble bodies. Protein refolding was performed according to redox methods (Prestwich, 1993 (link)). In brief, 50 mM Tris buffer (pH 6.8) containing 0.2% Triton X-100 was used to wash the insoluble inclusion body, which was then dissolved in 6 M guanidine hydrochloride. The refolded protein was then collected and purified by Ni²⁺ ion affinity chromatography (GE-Healthcare, United States). The His-tag was removed by recombinant enterokinase (rEK) (Novagen, Beijing, China). The size and the concentration of BodoCSP4 were determined using SDS-PAGE and the BCA protein assay kit (CoWinbiotech, Beijing, China), respectively.

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States) unless otherwise stated. DPD was purchased from the SHANGHAI ZZBIO Co., LTD. (Shanghai, China). The Kinase-Glo Max Luminescent Kinase Assay Kit was purchased from Promega (Madison, WI, United States). NTA sensor chips were purchased from GE Healthcare (Chicago, IL, United States). All compounds used as potential inhibitors of LsrK were purchased from Topscience Biotechnology Co. Ltd. (Shanghai, China). The QS reporter strain WHQ02 (E. coli BL21 ΔTolC pWHQ01) was donated by Huiqi Wen (Institute of Microbiology and Epidemiology, Academy of Military Sciences, Beijing, China). The plasmid pWHQ01 was constructed by cloning the lsr promoter and the luxCDABE luminescent gene. BamHI and XhoI were purchased from New England Biolabs (Ipswich, MA, United States). pET-28a was purchased from Novagen (Madison, WI, United States). Escherichia coli BL21 (DE3) cells were purchased from TransGen Biotech Co., LTD. (Beijing, China). A Ni-NTA column was purchased from Sangon Biotech (Shanghai, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Bradford Protein Assay Kit were purchased from Thermo Fisher Scientific (Waltham, MA, United States).

The full-length of MdSOS2L1 was amplified by PCR adding restriction enzyme sites and inserted into the EcoRI–SalI sites of the pET-32a vector to produce a His-tagged recombinant protein. The full-length of MdALMT14 or MdALMT14^S358A coding region was cloned into the EcoRI–SalI sites of the pGEX-4T-1 vector to produce sequence encoding a glutathione S-transferase (GST) fusion protein. For recombinant protein expression, the plasmids were transformed into Escherichia coli BL21 (DE3) cells (Transgene, Beijing, China), which were then cultured and induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) in Luria–Bertani (LB) broth for 6 h at 16 °C. For pull-down analysis with the GST- and His-tagged proteins, MdALMT14–GST or MdALMT14^S358A–GST proteins were eluted from glutathione–agarose beads prior to incubation with MdSOS2L1–His attached to the tetradentate-chelated nickel resin. The protein samples were incubated for at least 4 h at 4 °C while being shaken, before centrifugation (1200 g, 2 min). The precipitates were washed at least three times to remove non-specific binding, followed by boiling (10 min, 100 °C).

The standard bacterial strains used in this study included N. farcinica IFM10152, N. asteroids DSM43757T, N. cyriaciegeorgoca DSM44484T, N. brasiliensis DSM43758T, N. otitidiscaviarum DSM43242T, N. transvalensis DSM43405T, N. veterana DSM44445T, and N. nova DSM44481T. All the Nocardia strains were procured from the German Resource Centre for Biological Materials and cultured in BHI medium (Oxoid Ltd., Hants, UK) at a 37 °C incubator. Escherichia coli BL21(DE3) cells were procured from TransGen Biotech and cultured in an LB medium containing 50 µg/mL kanamycin. The pET30a plasmid was constructed in our laboratory and used to express N. farcinica NFA49590 in E. coli. The mouse cell line RAW264.7 (National Infrastructure of Cell Line Resource, Beijing, China) was cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS).

The protein-coding region of the sortilin gene (gene number: TGGT1_290160) was obtained from DNA isolated from T. gondii RH tachyzoites by PCR using gene-specific primers (Additional file 1: Table. S1). The amplicon was cloned into pET-28a and pGEX-4T-1 vectors (Invitrogen, Carlsbad, CA, USA). The two recombinant plasmids were expressed in Escherichia coli BL21 (DE3) cells (TransGen Biotech, Beijing, China). His- and GST-tagged recombinant proteins were obtained by affinity purification, as described previously [33 (link)]. Purified recombinant proteins were detected by Western blotting using specific antibodies against HIS and GST tags (Additional file 1: Fig. S2).
Four subdomain coding regions, named sortilin-N (1–166 aa), Vps10 (160–667 aa), sortilin-C (665–848 aa), and sortilin-M (880–1033 aa), were obtained using specific primers (Additional file 1: Table. S1) as described above. The four amplified gene fragments were cloned into the pGEX-4T-1 vector and expressed in E. coli BL21 cells (DE3). Finally, recombinant GST proteins were obtained by affinity purification using Glutathione Sepharose 4 B (Cytiva, Logan, UT, USA) (Additional file 1: Fig. S3A, B), as previously described [33 (link)].

The standard strain of N. cyriacigeorgica GUH-2 was purchased from the German Resource Centre for Biological Materials and was grown at 37°C in brain heart infusion (BHI) medium (Oxoid, China). Escherichia coli BL21 (DE3) cells (TransGen Biotech, Beijing, China) were grown in LB medium at 37°C. Human monocytic THP-1 cells and murine macrophage RAW 264.7 cells were maintained in our laboratory. THP-1 cells were grown in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA), and RAW 264.7 cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 IU/ml of penicillin G (Gibco), and 100 μg/ml of streptomycin (Gibco) at 37°C in a humidified incubator with 5% CO₂. The THP-1 cells were differentiated into macrophages by incubation with 100 ng/ml PMA (Sigma, Germany) for 24 h. Wild-type C57BL/6 mice were purchased from SPF Biotechnology (Beijing, China).