Ni sepharose 6 fast flow

In order to express the Pdc1 variants incorporating NT site-specifically, plasmid pET55-PDC1^Tyr38NT, pET55-PDC1^Tyr157NT, or pET55-PDC1^Tyr344NT, which expresses Tyr38NT-, Tyr157NT-, or Tyr344NT-Pdc1, respectively, constructed as described above was used. E. coli BL21 (DE3) strain harboring each of these plasmids and the plasmid pDule-3-nitroTyrosine (5B) (Addgene)^{60 (link)} were cultured in M9CA medium at 37 °C. When OD₆₀₀ reached 0.6, 0.1 mM isopropyl-β-D-thiogalactopyranoside and 1 mM NT were added and E. coli cells were further cultured for 16 h at 16 °C. After harvesting cells by centrifugation, the C-terminally His-tagged Pdc1 was purified by Ni Sepharose™ 6 Fast Flow (Cytiva) following the manufacturer’s protocol.

Briefly, E. coli strain, BL21 (DE3), was separately transformed with plasmids encoding the proteins. Then, the cGAS proteins were expressed and purified as previously described^{1 (link)}. The E. coli cells were grown at 37 °C, until an OD₆₀₀ of 0.6 was reached. The temperature was then reduced to 20 °C, and the cells were induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 24 h at 20˚C. The resultant cells were harvested by centrifugation at 5000 x g for 15 min and washed twice with cold phosphate-buffered saline (PBS). The collected cells were broken by ultrasonic wave and centrifugated at 24,000 x g for 30 min to remove unbroken cells and debris. The soluble fraction was incubated by Ni Sepharose 6 Fast Flow (Cytiva), washed with binding buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 30 mM imidazole), and eluted with elution buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 300 mM imidazole) as previously described^{1 (link)}. Concentration of resultant proteins and buffer exchange were performed using an Amicon Ultrafree centrifugal filter (Millipore) with a cutoff of 10 kDa. The proteins were labelled with Alexa Fluor™ 488 using the Alexa Fluor™ 488 Protein Labelling Kit (ThermoFisher, Waltham, MA, USA). The estimated degree of labeling was 2 mol of Alexa Fluor 488 per mol of protein.

Ni-Sepharose 6 Fast Flow was from Cytiva. Mutagenic primers were also ordered from Sigma-Aldrich (Merck; St. Louis, MO, USA). T4 ligase was purchased from ThermoFisher and the restriction enzyme BsaI was purchased from New England Biolabs. The E. coli NEB 10-beta and BL21-AI (NEB, Ipswich, MA, USA) strains were used as hosts for cloning and protein expression, respectively. All other reagents and chemicals were purchased from Sigma-Aldrich.

Recombinant plasmids, including pET30a-P97R1, pET30a-mhp390, pET30a-P46, and pET30a-LTB-linker-P97R1-linker-mhp390-linker-P46 were constructed for transformation into E. coli BL21 (DE3). The 6× His-tagged recombinant proteins (P97R1, mhp390, P46, and LTB-P97R1-mhp390-P46) were expressed and subsequently purified using Ni Sepharose 6 Fast Flow (Cytiva, Marlborough, MA, USA). The purified recombinant proteins were separated by 12% SDS-PAGE gels, and their concentration was quantified through the BCA Protein Assay Kit (Beyotime, Shanghai, China). The proteins were then stored at −70 °C for future use.

Reagents were obtained from the following sources: Ni Sepharose 6 Fast Flow (Cytiva, 17531802), Glutathione Sepharose 4 Fast Flow (Cytiva, 17513201), tris(2-carboxyethyl)phosphine (Thermo, 75259), EDTA-free protease inhibitor cocktail (Roche, 11873580001), PreScission Protease (Absin, abs01243), Anapoe-X-100 (Anatrace, 9002-93-1), ATP (Roche, 11140965001), MgOAc (Sigma-Aldrich, M0631), L-glutamic acid potassium (Sigma-Aldrich, G1501), biotinylated anti-His antibody (Bioss Antibodies, bs-0287R-bio), streptavidin (Sangon Biotech, A610492), LD555-MAL (Lumidyne, 04), LD655-MAL (Lumidyne, 10), GDP (Sigma-Aldrich, G7127), GTPγS (Roche, 10220647001), GMppNHp (Sigma-Aldrich, G0635), mPEG-SVA-5000 (Laysan Bio, 170-106), Biotin-PEG-SVA-5000 (Laysan Bio, 170-124), benzoic acid (Sigma-Aldrich, 242381), protocatechuate 3,4-dioxygenase (Sigma-Aldrich, P8279), and n-dodecyl-β-D-maltoside (Avanti Polar Lipids, 850520). All lipids were obtained from Avanti Polar Lipids: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, 850457); 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS, 840034); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, 810144); and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhodamine-PE, 810150).

The gene of VHHs were each inserted into the Nco I–Sac II site of the pRA vector, which included FLAG and poly-histidine tags^{27 (link)}. E. coli BL21(DE3) cells were transformed with the constructed expression vectors, grown overnight at 28 °C on LB agar, and then cultured in 2× YT broth; both media contained 100 µg/mL ampicillin. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM at OD₆₀₀ = 0.8, and the cells were shaken at 160 rpm for overnight at 28 °C. IPTG was added to the flask to a final concentration of 1 mM. The cells were shaken at 160 rpm at 20 °C overnight. The cells were harvested by centrifugation, resuspended in phosphate-buffered saline (PBS), and sonicated. Insoluble matter was removed by centrifugation. Variants were purified from the supernatants by IMAC (Ni Sepharose™ 6 Fast Flow; Cytiva, IL, USA) and SEC (HiLoad 26/600 Superdex 75 pg; Cytiva, IL, USA). The procedure of the preparation of galectin-3 was described previously^{4 (link)}.