Aprotinin

Analytical SEC experiments of the FixK₂ protein derivatives were performed at room temperature on a Superdex 200 10/300 GL column (Cytiva, Little Chalfont, UK) using an ÄKTA PURE protein purification system (Cytiva, Little Chalfont, UK). After equilibrating the column with elution buffer (40 mM Tris-HCl, pH 7.0, 150 mM KCl 0.1 mM EDTA), 100 µL protein samples were injected and separated at a flow rate of 0.75 mL.min⁻¹. Absorbance was recorded at 280 nm. The following proteins were used as standards for calibration (Figure S2): conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa) (Cytiva, Little Chalfont, UK). Gel filtration experiments were repeated at least three times with independent preparations of each protein at a range of at least five concentrations. The UNICORN™ system control software (Cytiva, Little Chalfont, UK) was employed to program the chromatography runs and for preliminary analyses of the data by adjusting for injection times.

Hydrodynamic properties of proteins were analyzed using a TSKgel SuperSW2000 4.6 × 300 mm column (Tosoh Biosence, Tokyo, Japan). Prior to separation, the column was equilibrated in Buffer B pH 7, and 10 µM protein solutions were injected. Runs were performed at room temperature at a flow rate of 0.3 mL min⁻¹. Bovine ribonuclease A and aprotinin (Cytiva, Chicago, IL, USA) were used as calibration standards. All elution profiles were baseline-corrected and were not further normalized. In each case, the most representative elution profile of three repeated runs is shown.

Size-exclusion chromatography was performed using a Superdex 75^® 10/300 column on an FPLC instrument (AKTA, Cytiva Life Sciences, Marlborough, MA, USA). A volume of 100 µL AfLEA1 protein in 50 mM phosphate buffer at pH 7.0 was injected at a 0.1 mL/min flow rate. Low molecular weight standards (Cytiva Life Sciences, Marlborough, MA, USA), including aprotinin (6.5 kDa), ribonuclease A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (44 kDa), conalbumin (75 kDa), and a blue dextran 2000 tracking polymer, were used as molecular weight markers.

The oligomeric state of purified recombinant untagged PhaR protein was determined by analytical SEC experiments on a Superdex 75 10/300 GL column (GE Healthcare, Uppsala, Sweden). In these experiments, the pH of the mIVT buffer (protein and column) was increased to 8, as this pH improved the solubility of the PhaR protein in solution. After equilibration of the column with buffer, protein sample aliquots of 200 µL at a concentration ranging 5 to 30 µM were injected and separated at a flow rate of 0.5 mL min⁻¹ on an ÄKTA^TM purifier Fast Protein Liquid Chromatography (FPLC) purification system (Pharmacia Biotech, Uppsala, Sweden). The absorption profile of the eluent was simultaneously recorded at 220 and at 280 nm. The following proteins were used as standard for calibration (Figure S4): conalbumin (75 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa), and aprotinin (6.5 kDa) (Cytiva, Little Chalfont, UK). Gel filtration experiments were repeated at least twice with independent preparations over a range of at least three concentrations. The UNICORN™ system control software version 5.11 (GE Healthcare, Uppsala, Sweden) was employed to program the chromatography runs and for preliminary analyses.

To examine possible effects of the Cys86 mutation on the oligomeric assembly, purified BvPgbs were diluted with 50 mM NaP containing 150 mM NaCl pH 7.0 to 1 mg/mL (5.2 µM) solutions. Analytical size-exclusion chromatography (SEC) was carried out with a Superdex 7510/300 GL column (CV = 24 mL) on an ÄKTA™ Avant system (Cytiva Life Science, Uppsala, Sweden) calibrated with conalbumin (75 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa), and aprotinin (6.512 kDa) as molecular weight (M_w) standards (Cytiva Life Science). All protein solutions were run in volumes of 100 µL with a protein concentration of 1 mg/mL. The M_w of the BvPbg solutions was estimated by fitting the measured retention volume (V_r) to a semi-log plotted calibration curve (Supplementary Figure S1).