Impact kit

DNA sequence encoding the human BDNF pro-peptide (Val⁶⁶) and human NGF pro-peptide up to furin cleavage site was cloned into pTXB1 vector (New England Biolab, Ipswich, MA, USA) between NdeI and SpeI sites with an extra histidine residue at the C-terminus to enhance efficient cleavage of the intein affinity tag from BDNF pro-peptide and NGF pro-peptide. Recombinant BDNF pro-peptide and NGF pro-peptide were expressed in BL21 E. coli and purified to homogeneity using the IMPACT kit according to the manufacturer's protocol (New England Biolab, catalog no. E6901S). The endotoxin levels of the purified recombinant proteins for neuronal treatment are <0.5 EU/μg.

An antibody to S. lacustris was prepared by injecting rabbits with a fusion protein composed of a intein-chitin tag and residues 1–183 of the S. lacustris β-catenin protein, which showed 88.4% sequence identity (97.4% similarity) with the E. muelleri β-catenin protein. The sequence encoding the fusion protein was constructed using the IMPACT kit (New England Biolabs). After expression in Escherichia coli the fusion protein was purified on a chitin column according to the manufacturer’s instructions and dialyzed against PBS containing 1× cOmplete EDTA-free proteinase inhibitor cocktail (Sigma). Peptides were analyzed by mass spectroscopy to confirm sequence identity and injected into rabbits without further processing. The first injection was done in Freunds Complete Adjuvant, and a subsequent three injections in Freunds Incomplete Adjuvant (Sigma).
Serum samples were analyzed by a combination of ELISA and Western blotting. Sponges were fixed and labelled with antibody as described previously [53 (link)].

Cloning and expression of 6xHis tagged protein fragments of mouse cdh23 and pcdh15 were previously described in [39 (link),41 (link)]. Briefly, DNA sequences coding for both protein fragments were cloned using the NdeI and XhoI sites of the pET21a vector that includes a C-terminal His tag. All mutants used in this work were generated using the QuikChange Lightning mutagenesis kit (Stratagene) and were verified by DNA sequencing. Numbering of residues corresponds to mouse CDH23 and PCDH15 without their signal sequences.
Fragments used in SPR experiments were derived from a template provided by Dr. Yoshie Narui. Briefly, a DNA construct containing the N-terminal amino acid residues 1–205 (EC1+EC2) of mouse CDH23 was obtained by PCR amplification and subcloned using the Impact kit (NEB #E6901S) and the NdeI and SapI restriction sites of the pTXB1 vector (NEB #N6707). The pTXB1 attached the Mycobacterium xenopi GyrA intein tag at the C terminus of EC2. The tag contains an N-terminal cysteine residue that allows thiol-induced cleavage. Each intein tag contains a chitin-binding domain (CBD) for affinity purification of the fusion protein on a chitin resin. Induction of on-column cleavage, using thiol reagents such as dithiothreitol (DTT), releases the "tagless" cdh23 from the intein tag. An Ala residue was included between CDH23 EC2 and the intein tag to improve cleavage efficiency.

E. coli strain ER2566 containing the recombinant expression plasmid pXTB1-HNS (Wang H. et al., 2012 (link)) was cultured to OD₆₀₀ of 0.5 with shaking at 37°C and then protein expression was induced with 0.4 mM IPTG for 4 h at 28°C. The cells were collected by centrifugation, resuspended in ice-cold Column buffer (20 mM Tris–HCl, pH 8.0, 0.5 M NaCl, and 1 mM EDTA), and lysed by sonication. The cell debris was removed by centrifugation, and H-NS-intein fusion protein with chitin binding domain (CBD) was purified using IMPACT™ Kit (New England Biolabs, United Kingdom) according to the manufacturer’s instructions. The clarified lysate was slowly loaded onto the equilibrated chitin column, and then the chitin column was washed with 20 bed volumes of Column Buffer. Subsequently, the column was quickly washed with 5 bed volumes of the Cleavage Buffer (Column Buffer containing 80 mM DTT), and then incubated at 4°C overnight for full cleavage reaction on-column. Finally, the H-NS was eluted with Column Buffer. H-NS-containing fractions were combined and dialyzed against Column Buffer at 4°C to remove DTT. The purity of the recombinant H-NS was analyzed by SDS-PAGE (Figure 1A), and the protein concentration was determined by a Pierce BCA protein assay kit (Thermo Fisher Scientific, United States). The protein was stored in 20% glycerol at - 80°C.

The Hfq and Xoc_3982 proteins were expressed and purified using the intein-based Impact Kit (New England Biolabs, USA) as described [9 (link)]. Binding reactions were conducted in 10 μl volumes with the LightShift Chemiluminescent RNA EMSA Kit (ThermoFisher, USA); reactions were incubated at 37°C for 20 min, and 5 μl of loading buffer (50% glycerol) was then added. The interaction of Hfq and Xonc3711 sRNA was conducted in 1× binding buffer with 3’-biotinylated Xonc3711 sRNA. The interaction of sRNA Xonc3711-with Xoc_3982 mRNA was investigated using EMSA as described previously [9 (link)]. Samples were separated in 5% nondenaturing polyacrylamide gels in 0.5× TBE at 4°C and visualized by phosphoimaging on a ChemiScope 3000 mini (CLiNX, Shanghai, China).

For the tag-free purification of the putative SAH enzyme RelH_Cg, the IMPACT kit (New England Biolabs) was used analogously to the purification of the already available enzymes Rel_Cg and RelS_Cg, which were also used in the course of this study. The corresponding gene relH_Cg was introduced into the vector pTXB1 and the plasmid obtained was transformed into the expression strain E. coli ER2566. The further procedure was consistent with the purification of Rel_Cg and RelS_Cg already described in the previous study (Ruwe et al., 2017 (link)).

cDNA constructs (see Figure 
2) encoding the human Y-P30-Dermcidin (DCD) precursor (aa 1–109), Y-P30-Dermcidin precursor lacking the N-terminal signal peptide (aa 21–109), Y-P30 with (aa 1–49) and without (aa 21–49) signal peptide were subcloned into the pEGFP-N1 vector (Clontech). pEGFP-N1 was used as a negative control. For bacterial expression, Y-P30 containing the signal peptide (aa 1–49) was subcloned into a pTYB21 vector. Expression and purification of Y-P30 via the Intein system was performed according to manufacturer’s instructions (Impact kit, NEB) and previously published protocols
[25 (link)].