Pdest17 plasmid

Wild-type (WT) pmar-lig, (accession number KGF9828), as well as the point mutants pmar-lig_R120A, pmar-lig_R120D, pmar-lig_R120D/G359K and pmar-lig_C145S/C332S were ordered as codon-optimized synthetic constructs from the Thermofisher GeneArt service. Synthetic genes were supplied sub-cloned into pDONR221 and were transferred into the pDEST17 plasmid (Invitrogen) as described previously (21 (link)). Protein expression and purification of both Pmar-Lig and the ATP-dependent DNA ligase from Alteromonas macleodii (Ame-Lig) were carried out as previously described (21 (link),22 (link)). T4 DNA ligase (T4-Lig) was expressed from pET14b-T4_ligase using standard conditions (induction by addition of 0.5 mM IPTG followed by incubation at 22°C overnight). Purification followed the same scheme as for Pmar-Lig and Ame-Lig, with the exception that tag removal was omitted and single round of IMAC was performed prior to gel filtration.

Destination vector for protein expression in fusion with monomeric avidin (mAvidin), by mean of recombination cloning, was prepared as follow: DNA sequence encoding triple mutant avidin (N17I, N54A and W110K) protein (S1 Fig) was synthesized with 3’ and 5’ restriction sites NdeI and NotI respectively and subcloned into pDEST17 plasmid (Invitrogen, Life Technologies, Burlington, ON, Canada) to obtain a novel Gateway cloning plasmid, p17-Avi, compatible with the one-step and restriction enzyme-free recombination Gateway cloning methodology.