Lb broth

Genes coding for recombinant proteins were chemically synthesized with codon optimization for Escherichia coli host expression (GenScript, Piscataway, NJ, USA). Synthetic genes were cloned into pET-51b(+) vector using SalI and HindIII restriction sites yielding N-terminal Strep-Tag II Fusion and C-terminal 10xHis-Tag fusion. Obtained plasmids were transformed into competent Escherichia coli BL21(DE3) (#C2527 New England Biolabs, Hitchin, UK). All recombinant proteins were induced in 1000 mL LB broth (#2020, A&A Biotechnology) using 0.1 mM Isopropyl β-D-thiogalactoside at OD₆₀₀ = 0.5; 37 °C for 3 h with 180 rpm shaking. Cell cultures were centrifuged and pellets were lysed in 50 mL buffer containing: 50 mM NaH₂PO₄, 300 mM NaCl, 10% Triton X-100, 70,000 U/mL lysozyme (#62971 Merck, Darmstadt, Germany), Dnase I (#10104159001, Merck Germany) 15 µg/mL Ph = 8.0. Recombinant proteins were isolated from the lysate using IMAC chromatography with His-Select Nickel Affinity Gel (#P6611 Merck, Germany). Column gravity approach with 1 mL of resin has been performed (Table 2).

All chemicals and growth media were purchased from commercial sources. LB broth and LB agar medium were bought from A&A Biotechnology (Gdynia; Poland). Mannitol salt phenol−red agar, Spirit blue agar, tributyrin agar, skimmed milk, starch, carboxymethylcellulose, guaiacol, X−gal (5−bromo−4−chloro−3−indolyl−β−D−galactopyranoside), Tween 80, cottonseed oil, Lugol’s solution, Congo red dye, and PBS tablets (pH 7.4) were purchased from Merck (Darmstadt, Germany). Ultrapure H₂O (18.0 MΩ) was produced with the Milli−Q Advantage A10 system (Millipore, Billerica, MA, USA).