Protein standard mix 15 600 kda

ITN-Kat (5 mg/mL) or BTN-Kat (5 mg/mL) was applied to a Superose 6 column (GE Healthcare, Amersham, UK) equilibrated in gel-filtration buffer (20 mM NaPi, pH 7.4, 150 mM NaCl). The flow rate was kept at 0.5 mL/min. Experimental and standard proteins were solubilized in gel-filtration buffer. The Superose 6 column was calibrated with standard proteins (Protein Standard Mix 15–600 kDa, Sigma-Aldrich, St. Louis, MO, USA). Elution volumes of ITN-Kat were found to be 12 mL, 4 mL, and 11 mL, and those of BTN-Kat were 11 mL, 3 mL, and 10.9 mL. To estimate their molecular masses, we used the plot of log₁₀ molecular mass against elution volume. The same data were used to calculate the predicted molecular mass of the complex using the plot of molecular mass against elution volumes.

The protein samples were analysed using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel was stained with Coomassie Brilliant Blue G-250 and destained with 10% (v/v) acetic acid^{70 (link)}. The molecular mass of the native enzyme was estimated by size exclusion chromatography (SEC) using a Hiload16/600 Superdex 200 column (GE Healthcare Bio-Sciences, Uppsala, Sweden) previously equilibrated with purification buffer (100 mM Tris-HCl pH 8.0; 200 mM NaCl; 5% (w/v) glycerol). The calibration curve was obtained using the Protein Standard Mix 15–600 kDa (Sigma-Aldrich, St. Louis, MO.), which contains standard proteins [thyroglobulin bovine (670 kDa); γ-globulins (150 kDa); Albumin (44.3 kDa); Ribonuclease A (13.7 kDa); and P-aminobenzoic acid (pABA) (0.13 kDa)], and then plotting the log of the molecular weight of the protein against the elution volume (ml). From this calibration curve, the molecular weight of MesoAmp was determined. The chromatograms were obtained by measuring the absorbance at 280 nm using an ÄKTA pure system (GE Healthcare) at a flow rate of 0.5 mL min⁻¹. All collected fractions were analysed by SDS-PAGE, and the fraction containing pure MesoAmp was used for further assays.

For the preparation of d-DctD₂, pQE30-dctD_D57Q (7 (link)) was expressed in E. coli JM109 in the presence of 1 mM isopropyl β-d-thiogalactopyranoside. To prepare d-IIA^Glc, pQE30-crr was expressed in the presence of glucose, as previously described (36 (link)). Each recombinant protein was purified using an Ni⁺-nitrilotriacetic acid affinity column (Bio-Rad). Next, 500 µL of the recombinant proteins of DctD_D57Q, d-IIA^Glc and the d-IIA^Glc/DctD_D57Q complex dissolved in a buffer (50 mM Tris-HCl [pH 8.0], 20 mM KCl, 50 mM MgCl₂, and 100 mM NaCl) were applied to the AKTA-FPLC system (Amersham Biosciences) equipped with a Superdex 200 Increase 10/300 Gl column (GE Healthcare) (37 (link)). Each sample was fractionated using a running buffer (50 mM Tris-HCl [pH 8.0], 20 mM KCl, 50 mM MgCl₂, and 300 mM NaCl) at a flow rate of 0.4 mL/min. The void volume of the column used in this assay was 7.2 mL, which is consistent with the previously reported value (38 (link)). The apparent molecular weights of the oligomeric proteins in the collected fractions were determined using an elution profile derived from the standard proteins (Protein Standard Mix 15–600 kDa, Sigma-Aldrich) as described in Fig. S1.