Cloning and Characterization of Thermostable Cellulases

The genomic DNA of Ca. bescii (DSM 6725) was used as a template for the cloning of endoglucanase Cel5D with and without its CBM28. GH5-g, GH5-t and GH5-v were cloned in pET28a (Novagen, Darmstadt, Germany) using the gDNA template, primers, and restriction enzymes listed in Additional file 1: Table S1. The dockerin sequences were determined like explain in Kahn et al. [54 (link)]. PCRs were performed with Phusion High Fidelity DNA polymerase F530-S (New England Biolabs, Inc, Massachusetts, United States), PCR products, and plasmids were synthetized with Fastdigest enzymes (Thermo scientific, USA). Ligation was performed with T4 DNA ligase (Fermentas UAB, Vilnius, Lithuania). PCR products were purified using a HiYield™ Gel/PCR Fragments extraction kit (RBC Real Biotech, Valencia, CA).
GH9-lk-v, GH9-v, GH48-lk-t, GH48-t were synthesized in pET21a by GenScript (USA). All enzymes were equipped with a His-Tag for purification by immobilized metal ion affinity chromatography (IMAC). The monovalent scaffoldins ScafT, ScafG, and ScafV and the trivalent scaffoldin ScafGTV were described previously [33 (link), 55 (link)–57 (link)]. Competent Escherichia coli XL1 cells were used for plasmid maintenance and production.