Precision plus protein unstained standard

The molecular weight distribution of all samples was determined according to Laemmli (1970) using SDS‐PAGE under reducing conditions. The samples were suspended with 1× Tris‐HCl treatment buffer (0.125 mol L⁻¹ Tris‐HCl, 4% SDS, 20% v/v Glycerol, 0.2 mol L⁻¹ DTT, 0.02% bromophenol blue, pH 6.8), boiled for 3 min to cleave noncovalent bonds and centrifuged at 12,100 g for 4 min (Mini Spin, Eppendorf AG, Hamburg, Germany). The electrophoresis was performed on 4–20% midi Criterion™ TGX Stain‐Free™ precast gels and the proteins were separated using the Midi Criterion™ Cell from Bio‐Rad (Ismaning, Germany). A molecular weight marker (10–250 kDa, Precision Plus Protein™ Unstained Standard, Bio‐Rad Laboratories Inc., Hercules, CA, USA) was additionally loaded onto the gel. Electrophoresis conditions were 200 V, 60 mA, 100 W at room temperature and protein visualization was performed by Criterion Stain‐Free Gel Doc™ EZ Imager (Bio‐Rad).

SDS-PAGE was used to confirm the production of both belt protein (at 25 kDa) and EGFR (at 160 kDa) (Supplementary Fig. 2b). Samples were mixed with 2 × Laemmli sample buffer (Bio-Rad Laboratories), 2.5% 2-mercaptoethanol (Sigma-Aldrich), and boiled for 5 min at 100 °C before running on precast stain-free gels from Bio-Rad Laboratories. Precision Plus Protein unstained standard (Bio-Rad Laboratories) marker was used for the stain-free imaging and Prestained NIR protein ladder (ThermoFisher) for fluorescence imaging. Gels were run at 170 V for 45 min. Stain-free imaging was performed on a Gel Doc imager (Bio-Rad Laboratories) and fluorescent images were acquired form Typhoon Gel FLA 9500 imager (GE Healthcare Life Sciences). The other proteins appearing in the stain-free gel are proteins not expressed completely during the cell-free reaction or the transcription and translation machinery of the cell-free reaction mixture. The specificity of snap surface 594 fluorophore binding to EGFR was confirmed through a fluorescence gel (Supplementary Fig. 2b).

The crude inulosucrase (cleared cell extract from an induced culture), the cleared cell extract of an uninduced culture, as well as the purified protein, were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate the protein production. Cell extract volumes corresponding to 10 µg total protein and 1 µg of purified InuGB-V3 were mixed with 4 × Roti^®-Load (Carl Roth) to a 1 × concentration and incubated at 95 °C for 5 min. The samples, as well as 10 µL of the Precision Plus Protein Unstained Standard (Bio-Rad Laboratories, Inc.), were then loaded onto a precast 12% Mini-PROTEAN^® TGX Stain-Free^™ Protein Gel (10 well, 30 µl) (Bio-Rad Laboratories, Inc.). These gels contain trihalo compounds that react with tryptophane residues of proteins in a UV-induced reaction to produce fluorescence. To separate the proteins, the gel was run at 250 V for 30 min in 1 × TGS buffer (Bio-Rad Laboratories, Inc.). Subsequently, the proteins were visualized by fluorescent detection with the stain-free enabled imager ChemiDoc^™ Imaging System (Bio-Rad Laboratories, Inc.) using an activation time of 45 s.

SDS-PAGE was carried out with Any kD Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) for 35 min at 200 V. The gel was activated for 5 min and imaged using the ChemiDoc MP imaging system (Bio-Rad). The molecular weights of protein bands were estimated using Image Lab software (Bio-Rad). The molecular mass marker comprised 5 or 10 μL of Precision Plus Protein Unstained Standard (Bio-Rad).

The molecular weight distribution of the lupin protein hydrolysates was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as described by Laemmli (1970). Lupin protein isolate, hydrolysate, and control samples were resuspended in 1 ml loading buffer (0.125 mol/L Tris–HCl, 4% SDS (w/v), 20% glycerol (v/v), 0.2 mol/L DDT, 0.02% bromophenol blue, pH 6.8), dissolved for 15 min at 30°C in an ultrasonic bath, and boiled for 5 min at 95°C to cleave noncovalent bonds. Following centrifugation at 13,000 g for 10 min (Mini Spin, Eppendorf AG), an aliquot of the supernatant was transferred to a fresh tube and supplemented in a ratio of 1:10 with loading buffer (see above). We then transferred 10 µl aliquots (5 mg/ml protein) into the wells of precast 4%–20% polyacrylamide gels (Bio‐Rad Laboratories). The samples were separated for 40 min at 200 V (60 mA, 100 W) (Amersham Biosciences Europe GmbH) at room temperature in a vertical electrophoresis cell (Bio‐Rad Laboratories). Precision Plus Protein Unstained Standard with molecular weight of 10–250 kDa (Bio‐Rad Laboratories) run alongside as size markers, and the protein subunits were visualized using a Gel Doc™ EZ Imager system (Bio‐Rad Laboratories). The molecular weight distribution was determined using Image Lab software (Bio‐Rad Laboratories).

SDS-PAGE was carried out using Any kD Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) for 35 min at 200 V. The gel was activated for 5 min and imaged using the ChemiDoc MP imaging system (Bio-Rad). Precision Plus Protein Unstained Standard (5 μL; Bio-Rad) was used as a molecular mass marker. Unless otherwise noted, 2.5 μg of protein was loaded in each well. The gels were cropped in the 15–250 kDa range because no clear bands were detected under 15 kDa (the original gel of Figs. 1b, 2b, and 3b were presented in Fig. S4, S5, and S6 see Additional file). The molecular weight of the protein bands was estimated using Image Lab software (BiFio-Rad), and the protein bands were annotated using the positions corresponding to previously reported cellulases and xylanases^{53 (link)}. Protein identification using nano LC–MS/MS systems was performed in Japan Proteomics Co. LTD.