Dpni endonuclease

Easy single-primer site-directed mutagenesis was performed as described in [33 (link)]. Oligonucleotide mutagenesis primer (5′-GAG ATG GTG GCG CGC GAG AAC CTG TAT TTC CAA TCG GTG CCT TAT GTC CG-3′) and check-primer (5′-AGA TGG TGG CGC GCG AG-3′), designed for the selection of mutant clones, were synthetized in (Evrogen, Moscow, Russia). Eighteen cycles of polymerase chain reaction (PCR) were performed on the templates of the PSP- and PSP-E125A-expressing plasmids [28 (link)] using Tersus Plus PCR kit (Evrogen, Moscow, Russia) according to the manufacturer’s recommendations. The PCR products were treated with DpnI endonuclease (Thermo Fisher Scientific, MA, USA), which digested the parental DNA template, and then transformed into E. coli Match1 competent cells. The mutant clones were selected by PCR performed directly on colonies using Taq DNA polymerase (Evrogen, Moscow, Russia) and check primer with T7 reverse universal primer. Plasmid DNA purified from mutant clones was sequenced to ensure the absence of random mutations associated with PCR. The second run of mutagenesis was performed for preparations of PSPmodE75 on the template of the PSPmod-expressing plasmid. All mutated proteins were verified by Maldi-TOF mass spectrometry.

Easy single-primer site-directed mutagenesis was performed as described38 (link) with minor modifications to make point mutations listed in Supplementary Table S2. The synthetic oligonucleotide primers designed to switch amino acids (one primer for each mutant) and those designed for the selection of mutant clones are listed in the Supplementary Table S3. Eighteen cycles of PCR were performed on the template of the HUSpm-expressing plasmid32 using the Tersus Plus PCR kit (Evrogen, Moscow, Russia) according to the manufacturer’s recommendations. The PCR products were treated with DpnI endonuclease (Thermo Fisher Scientific, Massachusetts, United States), which digested the parental DNA template, and then transformed into E. coli Match1 competent cells. The mutant clones were selected by PCR performed directly on colonies using Taq DNA polymerase (Evrogen, Moscow Russia) and check primers (Supplementary Table S3) with an appropriate T7 universal primer. Plasmid DNA purified from mutant clones was sequenced to ensure the absence of random mutations associated with PCR. The expression and purification of mutant proteins was performed in the same way as described for HUSpm32 . The purity of the mutants was estimated by SDS–PAGE with Coomassie staining, and the protein concentration was measured using the Bicinchoninic Acid Protein Assay Kit (Sigma-Aldrich, St. Louis, USA).

Easy single-primer site-directed mutagenesis was performed as described in^{61 (link)}. The synthetic oligonucleotide primers designed for the changing of amino acids (one primer for each mutant) and those designed for the selection of mutant clones are listed in the Supplementary Table S5. Eighteen cycles of PCR were performed with the template of the HUSpm-expressing plasmid using the Tersus Plus PCR kit (Evrogen, Moscow, Russia) according to the manufacturer’s recommendations. The PCR products were treated with DpnI endonuclease (Thermo Fisher Scientific, Massachusetts, United States), which digested the parental DNA template, and then transformed into E. coli Match1 competent cells. The mutant clones were selected by PCR performed directly on colonies using Taq DNA polymerase (Evrogen, Moscow, Russia) with check primers (Supplementary Table S5) and an appropriate T7 universal primer. Plasmid DNAs purified from mutant clones were sequenced to ensure the absence of random mutations associated with PCR. The expression and purification of mutant proteins were performed in the same manner as described for the wild type HUSpm. The purity of the mutants was estimated by SDS–PAGE with Coomassie staining, and the protein concentrations were measured using the Bicinchoninic Acid Protein Assay Kit (Sigma-Aldrich, St. Louis, USA).