Ni nta column

A recombinant form of the T. canis mucin 1 protein (rTc-MUC-1) fused to a 6× His tag was produced in a prokaryotic expression system. In brief, the Tc-muc-1 protein-coding sequence was amplified with a primer set containing Kpn I and EcoR I restriction sites (5’-GGGGTACCATGCACGTCCTTA-3’; 5’-GGAATTCTTAACAGAAGCCGCACGT-3’), and inserted into the pCold TF plasmid (Takara Bio, Dalian) employing the Kpn I and EcoR I restriction sites. Recombinant plasmids were purified, verified by specific PCR-amplification and competent BL21 (DE3) E. coli (TransGen Biotech, Beijing) transformed. Following induction with 0.6 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), rTc-MUC-1 was expressed in a culture volume of 250 ml at 37°C for 24 h. Proteins were released from cells by sonication (1 s–on, 3 s–off for 15 min on ice) at 400 W using a Scientz-IID ultrasonic homogenizer (Scientz, China). The sonicates (25 ml each) were centrifuged at 14,000 × g and 4°C for 20 min, and supernatants filtered (0.22 μm aperture) and run through a Ni-NTA column (Sangon Biotech, Shanghai); rTc-MUC-1 was eluted from the column with 250 mM imidazole and subjected to SDS-PAGE analysis using an established protocol [32 (link)]. E. coli transformed with the pCold TF empty plasmid was used as a negative control throughout experimentation.

The expression of the recombinant enzyme was performed by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to the bacterial suspension at the initial concentration of OD600 = 0.7. After 18 h at 16°C, the induced cells were harvested by centrifugation at 4°C for 5 min at 10,000 g and washed twice with PBS buffer (pH 7.4). After sonication and centrifugation at 12,000 g for 10 min, the supernatants was obtained as the crude enzyme solution. To purify the recombinant TtBgl3, the crude enzyme solution was applied onto a Ni-NTA column (Sangon Biotech, China). The TtBgl3 protein was eluted from the column with midazolam buffer by gradient elution. Enzyme fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a 5% (w/v) stacking gel and a 12% (w/v) separating gel. After electrophoresis, SDS-PAGE was stained with Coomassie blue R-250. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Tiangen Biotech, China) (Zhang et al., 2021 (link)).

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).