Bca colorimetric assay

E. coli strain Shuffle Express was transformed with pQE30-based plasmids expressing recombinant EhPDI enzyme variants (see Table 1). Stable transformants were cultured in LB medium, supplemented with ampicillin, and protein expression was induced with 0.1 mM IPTG. Bacterial cells (from 100 mL) were harvested and lysed under native conditions, using the CelLytic B Plus Kit (Sigma-Aldrich). From the soluble fraction, recombinant proteins were purified by Ni-affinity chromatography (The QIAexpressionist, Qiagen). Eluate fractions were analyzed by SDS-PAGE and those containing more than 95% of pure protein were pooled and concentrated/desalted by ultrafiltration, using a Microsep UF Spin Filter (Pall Co.). Protein concentration was determined by performing the BCA colorimetric assay (Sigma-Aldrich), using BSA as standard.

Ten milligrams of freeze-dried REPS were dissolved in 1 mL MilliQ water and total sugar content determined by the phenol-sulfuric acid method, using glucose as standard [82 (link)] (Figure S3). Total protein concentration was also assessed using BCA colorimetric assay (Sigma-Aldrich, Milan, Italy) and bovine albumin (BSA) as standard protein for calibration curve.
The RP-HPLC analysis of the REPS solutions was performed using LC-10AVP equipment (Shimadzu, Milan, Italy), 0.1% v/v trifluoroacetic acid as solvent A and 80% v/v acetonitrile and 0.1% v/v trifluoroacetic acid as solvent B. The analyses were performed using a C₁₈ column (CPS Analitica, 150 mm × 4.6 mm, 5 μm), a loop of 20 μL, flow of 0.8 mL/min and the following solvent B gradient: 0–5 min, 0%; 5–50 min, 60%; 50–55 min, 60%; 55–60 min, 90%; and 60–65 min, 90%. The elution was monitored at 220 nm by a UV detector (Shimadzu, Milan, Italy).
FT-IR Spectroscopy was used for chemical analysis of freeze-dried REPS material, using a Perkin Elmer Spectrum 100 (PerkinElmer Inc., Paris, France). Spectra were acquired in the range 4000–250 cm⁻¹, by averaging 32 scans at a resolution of 4 cm⁻¹, using CsI cells. Data were processed using Spectrum 6.3.5 software (PerkinElmer Inc., Paris, France).