2.6. Design, Production and Purification of CBM-Recombinant Protein Fusions

The fusion protein with an N-terminal green fluorescent protein (GFP) domain and CBM3 (GFP-CBM3) was purchased from NZYTech-Genes & Enzymes, Lisbon (5 mg/mL). The recombinant proteins ZZ-CBM3 and CBM3-Cys (CBM3C) were cloned in Escherichia coli (E. coli) by NZYTech. The CBM3-derived recombinant constructs used throughout this study are described in Table 2.

Recombinant bi-functional CBM3 fusion proteins used in this work. Data for the native CBM3 protein are provided for comparison.

The fusion protein GFP-CBM3 was provided in 35 mM NaHepes buffer, pH 7.5, 750 mM NaCl, 200 mM imidazol, 3.5 mM CaCl₂, and 3.2 M ammonium sulfate. Hence, to recover maximal GFP-CBM3 activity, a required volume of the precipitated protein suspension was centrifuged (13,000× g for 5 min) to remove the ammonium sulfate supernatant. The resulting pellet was resuspended in the same volume of 20 mM Tris-HCl, pH 7.5, 20 mM NaCl, and 5 mM CaCl₂.

ZZ-CBM3 is a fusion of CBM3 with an N-terminal double Z-domain of protein A from Staphylococcus aureus. In this case, an insert totaling 861 base pairs was cloned into the NdeI-XhoI sites of a pET21a expression vector. The second recombinant protein, CBM3C, comprising a CBM3, a C-terminal cysteine, and an N-terminal histidine tag (6 amino acids), was constructed by inserting the coding sequences (552 base pairs) between the XhoI and NdeI sites of a pET28a expression vector (NZYTech). The peptide SSGPQQGLRANT was used to space CBM3C from the histidine tag. The full sequences of the CBM-based fusions are shown in Figure S1.

E. coli BL21 (DE3) cells were transformed with the resulting plasmids and grown in Luria–Bertani (LB) broth with the necessary selection marker (100 µg/mL ampicillin for pET-ZZCBM3-21a and 30 µg/mL kanamycin for pET-CBM3C-28a) at 37 °C and 250 rpm. Expression of the fusions was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) as previously described [37]. Cells were harvested by centrifugation 3 h after induction and resuspended in a minimal volume of TST buffer or in a buffer containing 10 mM imidazole, 50 mM NaHEPES, 1 M NaCl, and 5 mM CaCl₂, pH 7.5, for ZZ-CBM3-containing cells and CBM3C-containing cells, respectively.

Cells were sonicated (Bandelin Sonoplus) and the protein-rich supernatant was separated from debris by centrifugation. Then, ZZ-CBM3 was purified by affinity chromatography using a 1 mL column packed with IgG Sepharose 6 Fast Flow (GE Healthcare) connected to an ÄKTA 10 Purifier LC System (GE Healthcare). Unbound proteins were washed with TST buffer and bound ZZ-CBM3 was eluted with 0.5 M acetic acid, pH 2.8. After fraction collection, the pH is immediately neutralized with 3.2 M Tris buffer, pH 11 [38]. CBM3C was purified by Immobilized Metal Affinity Chromatography (IMAC) using a 1 mL nickel-containing HisTrap FF column (GE Healthcare) connected to an ÄKTA 10 Purifier LC System and an imidazole gradient [25]. The purified fusion proteins were stored at −20 °C before use.

The purity of ZZ-CBM3 and CBM3C was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using a NZYTech Low Molecular Weight (LMW) Protein Marker. Staining of the gel was performed with Coomassie Brilliant Blue, and images were obtained with a GS-800 Calibrated Densitometer (Figure S2). CBM3C protein samples were buffer exchanged to TST to remove excess imidazole using an Amicon^® Ultra-15 3k Centrifugal Filter Units, before determining the protein concentration. The concentration of the recombinant ZZ-CBM3 and CBM3C was estimated from the absorbance at 280 nm measured on a NanoDrop One Microvolume UV–Vis spectrophotometer (ThermoFisher, Waltham, MA, USA) using their molar extinction coefficient and molecular weight. The purified proteins were stored at −20 °C until required.