Superdex 75 26 60 column

The bacterial strains and plasmids used in this study are listed in S1 and S2 Tables. The genes encoding Fra, HemH, FfoR, and bsNos were amplified via polymerase chain reaction from genomic DNA extracted from B. subtilis 168 using either the Invitrogen Platinum Pfx or the NEB Phusion High-Fidelity DNA Polymerase, according to the manufacturer’s protocol. The DNA oligonucleotides used for amplification are listed in S3 Table. Expression constructs for C-terminal Strep-tag II or N-terminal His₆-tag fusions were constructed by using appropriate DNA restriction enzymes and subsequent DNA ligation [33 ]. The designed Strep-tag II expression constructs of hemH and ffoR, and the His₆-tag expression constructs of fra and nos were used to transform E. coli BL21(DE3) cells, as described previously [33 ,34 (link)]. Expression and purification of the His₆-tag proteins followed the procedures described in [35 (link)]. The manufacturer’s protocols of the pASK-IBA3 vector were used in the case of Strep-tag II expression constructs. In addition, all proteins were further subjected to size-exclusion chromatography on an ÄktaPurifier system (Amersham Pharmacia Biotechnology) by using a GE Healthcare preparative Superdex 75 26/60 column. All purified proteins were finally analysed by SDS-PAGE (S1 Fig.).

IL-31_K138ASNAP, NGF_R121WSNAP and the SNAP-tag were produced as previously described^{14 (link),15 (link),41 (link)}. cDNAs for CRE recombinase or Cas9 endonuclease together with a CLIP tag were cloned into a pETM-11 vector after a SenP2 cleavage site and expressed in E. Coli as fusion protein, carrying a His6-tag-SUMO at the N-terminus. His6-SUMO-CLIP-Cre and His6-SUMO-CLIP-Cas9 constructs were recovered from cell lysate with Ni-NTA resin (Qiagen, #30210) and eluted with 250 mM imidazole. An overnight (O/N) digestion with His-tagged SenP2 protease was performed at 4 °C in order to remove the SUMO tag-protein. Excess of imidazole was removed from solution through dialysis. Digested products were incubated again with Ni-NTA resin at room temperature (RT) for 1 hr. The flow-through containing only the protein of interest was collected. Further purification was performed with Ion Exchange chromatography in a HiTrapQ HP column (GE Healthcare) and finally with size exclusion chromatography in a Superdex 75 26/60 column (GE Healthcare). The protein-containing fractions were pooled and dialysed O/N against Phosphate-buffered saline (PBS), 1 mM DTT, 5% glycerol. Dialysed proteins were then concentrated (Amicon Ultra 10 kDa, Merck-Millipore), aliquoted, snap-frozen and stored at −80 °C.

For laccase purification, the protocol described in [30 (link)] was modified. All procedures were performed at 4 °C. Culture filtrate (6400 mL) was 90% saturated with (NH₄)₂SO₄, and the precipitate was separated by filtration (Whatman No. 1 filter paper), re-suspended in dH₂O, and dialyzed against dH₂O overnight. At the next stage, the preparation was stirred with DEAE-cellulose for 20 min, and the proteins were desorbed twice with 200 mM potassium phosphate buffer (KPB) pH 6.5. The resulting preparation was dialyzed against 5 mM KPB pH 6.5 and loaded on a column packed with 25 mL of DEAE-Toyopearl 650M (Tosoh, Tokyo, Japan) and equilibrated with 5 mM KPB pH 6.5. Proteins were eluted by 150 mL of 50 mM KPB pH 6.5. For further purification, fractions with laccase activity were dialyzed against 20 mM KPB pH 6.5 and subjected to FPLC size-exclusion chromatography on a Superdex 75 (26/60) column (GE Healthcare Life Sciences, Chicago, IL, USA) equilibrated with 20 mM KPB pH 6.5. Fractions with different laccase izoenzymes were transferred to 5 mM citrate-phosphate buffer pH 5.0 by dialysis and purified by an additional stage of ion-exchange chromatography on a DEAE-Toyopearl 650M column equilibrated with 5 mM citrate-phosphate buffer pH 5.0. Proteins were eluted by a linear gradient of 5–20 mM of the same buffer.