Site-Directed Mutagenesis of BglHI Enzyme

The single mutation H307Y was generated by PCR based site-directed mutagenesis [41 (link)], where the PCR reaction contained 10 ng of plasmid pET28_bglhi as template, 50 pmol of the primers 5’-GGCATGAACTACTACACGGCCAACTACATCAAGCAC-3’ (forward) and 5’- CGTGTAGTAGTTCATGCCGTAGAAGTCGTTGGAGCC-3’ (reverse), 200 μM dNTPs and 2.5 U of Pfu DNA polymerase (Thermo Scientific, Waltham, MA, USA) and its respective buffer. The PCR was performed with initial denaturation of the template DNA at 95°C for 3 min, followed by 30 cycles of 95°C for 1 min, 50°C for 1 min, 72ºC for 15 min and a final extension step at 72°C for 10 min. After PCR, the product was incubated with 2 U of DpnI (Thermo Scientific, Waltham, MA, USA) at 37°C for 3 h to digest the methylated template DNA. One microliter of the DpnI digested sample was used to transform E. coli DH5α. After nucleotide sequencing confirm the mutation, the plasmid (denominated pET28_307) was subsequently used to transform E. coli BL21 (DE3) for the expression of the recombinant enzyme (See the “Protein expression and purification” section).