The remaining inclusion body was
Riton X-100 to remove membrane proteins. The remaining inclusion body was washed with 5 M and 6 M urea and solubilized in 10 ml buffer B (100 mM Tris/HCl pH 8, 8 M urea). After 10 fold dilution in buffer B the protein was refolded in 20 fold volume of refolding buffer (50 mM MES, 1-Bromo-2-fluoro-4-methoxy-5-nitrobenzene pH 6, 800 mM arginine) for 2 hours at 8 . Refolding was done in a total volume of 50 ml. The refolded protein was then purified and concentrated by hydrophobic interaction chromatography. For this purpose the refolded protein solution was first slowly further diluted 4 fold with ddH2O and then brought to 1.5 M ammonium sulfate. This solution was loaded onto a 0.3 ml EMD-Propyl (Merck) column (MoBiTec GmbH, G tingen) and step-eluted with 0.5 ml of 100 mM Tris/HCl pH 8 in 0.5 ml. The active fractions 2-Bromo-1,3-difluoro-4-nitrobenzene (tested with activity plates) were dialysed against the storage buffer (20 mM sodium phosphate, pH 7.0, 50 (v/v) glycerol, 1 mM DTT, 0.1 mM EDTA, and 150 mM NaCl) and stored at -20 . The protein concentration was determined spectrophotometrically and calculated according to Ehresmann et al. [21]. Total yield of a 2 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/11834444 litre culture of CelA-SSO1949-CelA was approximately 3 mg of purified protein.Activity plates: hydrolysis of carboxymethylcellulose (CMC)Activity plates enabled qualitative determination of cellulase activity of the chimeric proteins. 2.1 g Gelrite (Sigma) have been autoclaved in 240 ml ddH2O and 30 ml of 0.5 M sodium phosphate pH 3, 30 ml of 2 CMC solution and 3 ml of 1 M MgCl2 were added and poured into Petri dishes. After solidification 20 l of protein extract were applied and allowed to dry. The plate was incubated overnight at 75 . Then, the plate was washed PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13485127 with 100 mM sodium phosphate pH 6 and stained with Congo Red for 30 min. Next, the plate was destained with 1 M NaCl. An active cellulase yields a white spot on a red background.Fluorescent activity assayThe quantification of enzyme activity was done by FRET (fluorescence resonance energy transfer) basedKufner and Lipps BMC Biochemistry 2013, 14:11 http://www.biomedcentral.com/1471-2091/14/Page 9 ofassay. The bifunctionalized cellohexaoside sodium N-2-N[(S-(4-deoxy-4-dimethylamino-phenylazophenylthioureido-D-glucopyranosyl)-(14)–D-glucopyranosyl-(14)-D-glucopyranosyl-(14)–D-glucopyranosyl-(14)–Dglucopyranosyl-(14)–D-glucopyranosyl)-2-thioacetyl] aminoethyl-1-naphthylamine-5-sulphonate was offered as substrate [18]. The reaction was followed by monitoring fluorescence, which increases in the course of the enzymatic reaction. The measurement was performed in 0.1 M sodium phosphate buffer at various pH and temperatures and 1 M substrate on a PerkinElmer LS50B spectrofluorometer equipped with a thermostatically controlled cuvette holder (80 ). Excitation was at 340 nm and emission was observed at 490 nm. The fluorescent substrate is very stable under these conditions and has a half-live of several hours [9]. Initial rate constants were determined at different substrate concentrations in the presence of 120 ng hybrid protein. The Michaelis enten constant Km, kcat and the maximal velocity vmax were calculated by nonlinear regression to the Michealis-Menten equation.Competing interest The authors declare that they have no competing interests. Authors’ contributions KK carried out the cloning, expression, purification and characterization of the enzymes. GL did the SCHEMA analysis and conceived the study. KK and GL wrote the manuscript and approved the final manuscript. Both authors read and appro.
