Ni nta affinity resin

The expression and purification of DREP1 (amino acid residues, 10–90), DREP3 (amino acid residues, 111–195), and DREP4 (amino acid residues, 39–130) were performed as described in previous studies [24 (link), 26 (link), 27 (link)]. Briefly, each plasmid containing the CIDE domain was transformed into BL21 (DE3) Escherichia coli competent cells (Sigma-Aldrich, St. Louis, Missouri, USA), and then expression was induced by treating the bacteria with 0.5 mM isopropyl β-D-thiogalactopyranoside overnight at 20 °C. The bacteria were then collected, resuspended, and lysed by sonication in 50 mL lysis buffer (20 mM Tris-hydrochloride [HCl] pH 7.9, 500 mM sodium chloride [NaCl], and 10 mM imidazole). Next, the bacterial lysate was centrifuged, the supernatant collected, and eventually applied to a gravity-flow column packed with Ni-NTA affinity resin (Qiagen, Hilden, Germany). Unbound bacterial proteins were removed from the column by washing with buffer (20 mM Tris-HCl pH 7.9, 500 mM NaCl, 60 mM imidazole, and 10% glycerol), and the target proteins were eluted using an elution buffer (20 mM Tris-HCl pH 7.9, 500 mM NaCl, and 250 mM imidazole). The protein was further purified using a Superdex 200 gel-filtration column (GE Healthcare, Chicago, Illinois, USA). All the mutant proteins were expressed and purified by the same method as used for the wild-type CIDE domain.

His-tag pull-down assay was performed as described with some modifications^{31 (link),32} . First, a PreScission protease cleavage site (LEVLFQ↓GP) was inserted into the site between C terminal of MucB (or MucA^peri) and His-tag, and the His-tag of MucB (or MucA^peri) was removed by HRV 3C protease digestion. Then, 20 µg MucA^peri with His-Tag was incubated with excessive MucB (without His-tag) and 20 µL Ni–NTA affinity resin (Qiagen) for 30 min. After centrifuging at 15,000 × g for 3 min, the Ni–NTA resin was washed three times with solution buffer containing 25 mM imidazole. Fractions were eluted with solution buffer containing 300 mM imidazole and determined by 15% SDS-PAGE gel and visualized by Coomassie Brilliant Blue stain.

FolAsp sequences lacking the N-terminal signal peptide were cloned into the pET-28a expression vector via the overhangs added by the primers during amplification (Supplementary Table 1). The His-FolAsp fusion proteins were expressed in E. coli BL21 cells as described previously (Gamir et al., 2016 (link)). Briefly, isopropyl β-D-thiogalactoside (IPTG) was added into the medium to induce target protein expression and then cultured at 25°C and 180 rpm for 16 h. The harvested cells were resuspended in lysis buffer containing 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride (PMSF) and lysed with a microfluidizer. The proteins were purified using Ni-nitrilotriacetic acid (Ni-NTA) affinity resin (Qiagen) following the manufacturer’s instructions. The eluted fraction was desalted on a Sephadex G-25 column (PD-10, GE Healthcare) equilibrated with 50 mM Tris-HCl (pH 8). The protein concentrations were determined with an Omni-Easy Instant BCA Protein Assay Kit (Epizyme). The purified protein was concentrated with a 10-kDa MWCO Amicon Ultra Centrifugal Filter (Merck Millipore) and visualized by Coomassie-stained SDS-PAGE.

The constructs were transformed into E. coli BL21(DE3)-RIL (Novagen). Overnight cultures were diluted 1:100 in LB containing 100 μg/ml ampicillin and 10 μg/ml chloramphenicol and cultured until the O.D.600 nm reached 0.6. Protein expression was induced with 0.5 mM IPTG and the cells were kept at 25 °C overnight. Bacterial cells were harvested by centrifugation and resuspended in a lysis buffer (50 mM Tris-HCl pH8.9, 250 mM NaCl, 1 mM dTT, 1 mM PMSF).
Recombinant His-tagged or GST-tagged proteins were purified by standard affinity chromatography using Ni-NTA affinity resin (Qiagen) or GST-sepharose 4B affinity resin from GE Healthcare (South Burlington, VT, USA). The tags were cleaved using tobacco-etch virus protease (TEV) on the column at 4 °C for 18 hrs. After cleavage the tag GST or His was removed from the protein samples using Ni-NTA affinity resin or GST-Sepharose 4B affinity resin. The purified GST was eluted from the resin with a 10 mM reduced glutathione elution buffer.