Clonejet pcr cloning vector

The purified PCR products were first cloned and inserted into the pre-linearized CloneJet PCR cloning vector (Thermo Fisher Scientific, USA) according to the user manual, and then the recombinant plasmids was transformed into E. coli DH5α by the heat-shock method [47 ]. After that, 500μL of SOC medium was added, and the transformation mixtures were incubated at 37°C for 1h at 250 rpm for recovery and expression of antibiotic resistance genes encoded in the recombinant plasmids. After incubation, 100μL of each transformation culture was plated onto 2XYT*1 agar media supplemented with carbencillin (50μg/mL) according to Sambrook and Russel [47 ]. Recombinant colonies were screened by colony-PCR using specific/vector primers to verify the presence and proper orientation of the insert. The positive clones were selected for culture and plasmid extraction using a plasmid DNA miniprep kit (ThermoFisher Scientific, USA) according to the user manual. Both the cloned genes and the expression vector (pQE-Trisystem) (Qiagen, Hilden, Germany) were digested using the FastDigest KpnI and NotI restriction enzymes (Thermo Fisher Scientific, USA) and then purified and ligated using T4 DNA ligase (Thermo Fisher Scientific, USA). Five microliters of the recombinant plasmids were transformed into competent E. coli BL21 (DE3) cells as aforementioned.

Thirteen chimeric GH5 genes were optimized for expression in E. coli, and the gene sequences were synthesized by GenScript, U.S.A. The PCR fragments and synthesized gene sequences were cloned into CloneJET^™ PCR cloning vector (Thermo Scientific) and subsequently subcloned into the pET21b expression vector (Novagen) using the restriction sites NdeI and XhoI. Site-directed mutagenesis and short-fragment substitutions were performed using the QuikChange Lightning Site-directed Mutagenesis Kit (Agilent Technologies) and In-Fusion^™ HD Cloning system (Clonetech). The proteins were expressed in BL21(DE3) pLyss at 37°C for 4–6 hrs under the induction of 0.5 mM IPTG (isopropyl β-D-thiogalactopyranoside). Harvested cells were resuspended in binding buffer (50 mM sodium phosphate, pH 8, 300 mM NaCl) and disrupted by sonication. Lysates were centrifuged at 10000 x g for 60 min and the supernatants were loaded onto nickel-charged 1 ml HiTrap FF crude columns (GE Healthcare) with a flow rate of 0.5 ml/min. The proteins were eluted by 150–300 mM imidazole. All chromatographic steps were performed using an AKTA FPLC system (GE Healthcare). The purified protein samples were confirmed by SDS-PAGE under denaturing conditions and their concentrations were determined by Bradford assay.