Overexpression and Purification of Scaffoldin and Cellulase Proteins

Escherichia coli BL21 (DE3) cells overproducing pET28a-scaffoldin genes or cellulases were grown at 37°C in Luria-Bertani broth supplemented with 50 μg/ml kanamycin (Sigma-Aldrich Chemical Co, St. Louis, MO, USA) to A₆₀₀ = 0.8 to 1.0. The cultures were cooled to 16°C, and protein expression was induced by the addition of 0 to 1 mM isopropyl-1-thio-β-D-galactoside - IPTG (Fermentas UAB Vilnius, Lithuania), based on the results of predetermined optimization experiments. The cultures were incubated at 16°C for an additional 16 h, the cells were harvested by centrifugation (3500 g, 15 minutes), resuspendend in Tris-buffered saline (TBS, 137 mM NaCl, 2.7 mM KCl, 25 mM Tris–HCl, pH 7.4) supplemented with 5 mM imidazole (Merck KGaA, Darmstadt, Germany) and disrupted by sonication. The sonicate was heated for 20 minutes to 60°C and centrifuged (20,000 g, 30 minutes). The supernatant fluids were mixed with 4 ml of Ni-NTA beads for 1 h on a 20-ml Econo-pack column for batch purification at 4°C. The column was washed by gravity flow with 100 ml wash buffer (TBS, 50 mM imidazole) and elution was performed with 14 ml of elution buffer (TBS, 250 mM imidazole). For purification of the scaffoldins an additional affinity-purification step was applied: the eluted fractions were incubated in a 50-ml tube with 10 ml PASC (0.75 mg/ml) for 1 h at 4°C to allow binding of the CBM. The matrix was washed three times with TBS, containing 1 M NaCl, and three times with TBS without added salt. The scaffoldin was eluted with 1% triethylamine and neutralized with 1 M 2-(N-Morpholino) ethanesulfonic acid (MES) buffer pH 5. For both scaffoldin and cellulases the buffer was exchanged by dialysis against TBS, and the scaffoldin sample was concentrated using Amicon Ultra 15 ml 50,000 MWCO concentrators (Millipore, Bedford, MA, USA). Protein concentrations were estimated by the absorbance at 280 nm. The extinction coefficient was determined based on the known amino-acid composition of each protein using the ProtParam tool on the EXPASY server (http://www.expasy.org/tools/protparam.html) [72 ].