“…After amplification with Pfu polymerase (Fermentas, Burlington, Ont., Canada), the remaining product was digested with Not I and Sfi I restriction enzymes (NEB), using conditions recommended by the supplier. The resulting fragments carrying the mutated scFv-encoding genes were subsequently ligated into a modified version of the pFAB5c.His vector [31] , using T4 DNA ligase (NEB). The product was used to transform electrocompetent Escherichia coli TOP10F' (Invitrogen, Carlsbad, Calif., USA), using electroporation with an ECM600 (BTX Instrument Division, Harvard Apparatus, South Natick, Mass., USA) according to the instructions from the supplier (50 F, 186 ⍀ and 1.5 kV).…”