Ni nta column

G3BP1_OHu02150C_pET-28a(+)-TEV, YTHDF3_OHu08859C_pET-28a(+)-TEV, YTHDF3-EGFP_YTHDF3_OHu08859C_pET-28a(+)-TEV, G3BP1-RFP_G3BP1_OHu02150C_pET-28a(+)-TEV were custom cloned from GenScript. The plasmids were transfected to BL21(DE3) pLysS competent cells (Promega). Positive colonies were picked up and grown at 37°C in LB media supplemented with 50 μg/ml of kanamycin until the early exponential phase (OD600 0.4–0.8). Isopropyl-β-d-1-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 0.5 mM and protein expression was induced by further incubating the culture at 37°C for 5 h. The cells were harvested by centrifugation at 7000 rpm for 10 min, resuspended in 10 ml lysis buffer (10 mM imidazole in 1× PBS) and subjected to sonication. The suspension was then cleared by centrifugation at 10 000 rpm for 30 min at 4°C and the resulting supernatant was passed through a 1 ml Ni-NTA column (Thermo Scientific). After washing the column with washing buffer (50 mM imidazole in PBS), recombinant proteins were eluted with elution buffer (250 mM imidazole in PBS) and dialyzed extensively against PBS to remove the imidazole.

N‐GFP‐NCAP‐His_pET‐28a(+)‐TEV and N‐RFP‐G3BP1‐His_pET‐28a(+)‐TEV were custom synthesized from GenScript. The plasmids were transfected to BL21(DE3) pLysS competent cells (Promega). Positive colonies were picked up and grown at 37 °C in LB media supplemented with 50 μg mL⁻¹ of kanamycin until the early exponential phase (OD600 0.4–0.8). Isopropyl‐β‐d‐1‐thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 0.5 mm, and protein expression was induced by further incubating the culture at 37 °C for 5 h. The cells were harvested by centrifugation at 4700  g for 10 min, resuspended in 10 mL lysis buffer (10 mm imidazole in 1X PBS) and subjected to sonication. The suspension was then cleared by centrifugation at 9600  g for 30 min at 4 °C temperature, and the resulting supernatant was passed through a 1 mL Ni‐NTA column (Thermo Scientific). After washing the column with washing buffer (50 mm imidazole in PBS), recombinant proteins were eluted with elution buffer (250 mm imidazole in PBS) and dialysed extensively against PBS to remove the imidazole.

CAMKK2 kinase domain (132–470) was cloned in a pNIC-Bio2 vector in fusion with N-terminal 10xHis tag followed by a TEV protease cleavage site and a C-terminal biotin ligase recognition sequence. This construct was used in the expression of CAMKK2 in E. coli BL21(DE3)-R3-BirA [44 (link)]. Protein was purified in a Ni-NTA column (Thermo Scientific, Waltham, MA, USA) followed TEV digestion overnight, dialysis to remove imidazole and re-purification in Ni-NTA to remove undigested samples and TEV protease (made in house with an N-terminal 6xHis tag). As a last step, this sample was loaded to a HiLoad Superdex 200 16/600 column (GE Healthcare, Chicago, IL, USA) for final polishing and buffer exchange.
Tracer displacement assay was measured in 15 µL volume containing 5 nM of our C-terminal biotinylated CAMKK2 kinase domain, 2 nM of Europium-labeled streptavidin in buffer 50 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% Brij-35 and 8 nM of tracer 236 (measured K_D of 8.13 ± 0.9 nM) as described [45 ].

Protein purification was carried out under denaturing conditions using a Ni- NTA column (Thermo Fisher Scientific, Germany). For this purpose, bacterial pellets were resuspended in native buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0). Ultrasonicator was used to perform the sonication. Inclusion bodies were resuspended in denaturing buffer (100 mM NaH₂PO₄, 10 mM Tris–Cl, 8 M urea, pH 8.0). In the next step, the Ni–NTA column was equilibrated with denaturing buffer before sample injection. Then the supernatant was transferred onto the column. The column was washed with 5 ml of washing buffer (100 mM NaH₂PO₄, 10 mM Tris–Cl, 8 M Urea, pH 6.3) to remove unbound proteins. Elution buffers (100 mM NaH₂PO₄, 10 mM Tris–Cl, 8 M Urea, pH 4.5) were used to elute the His-tagged protein. Finally, the purified protein was subjected to urea removal and refolded by a dialysis bag (12 kDa MWCO) against phosphate‐buffered saline (PBS) (pH 7.5). Samples were run on 12% SDS-PAGE.

The catalytic domain of PDE5A (residues 535-862; GenBank accession number BC126233.1) was subcloned into the T7 promoter-driven expression vector pET21b with a 6 × His-tag at the C-terminus (Hsieh et al., 2020 (link)). The recombinant plasmid was transformed into E. coli strain BL21 (DE3) and grown in an autoinducing medium (Studier 2005 (link)) containing 50 μg/mL ampicillin at 37 C until OD600 = 0.6–0.7, then induced protein expression at 15°C for 40 h. The PDE5A protein was purified through the Ni-NTA column (Thermo Scientific) and further purified by the HiPrep™26/60 Sephacryl™-S-200HR column (GE Healthcare). A typical batch cell yielded over 10 mg of the PDE5A protein from 1 L of autoinducing medium with a purity >95% based on SDS‒PAGE. The protein was concentrated to a certain concentration using centrifugal filters and stored in a storage buffer (50 mM NaCl, 20 mM Tris-HCl pH 7.5, 1 mM β-mercaptoethanol, 1 mM EDTA, and 5% glycerol).

Recombinant His-tag pilin fusion proteins were produced as perisplam-directed proteins as described before^{51 (link)}. Fragments of pilE, pilV, and comP genes lacking the region coding for the amino-acid residues 1 to 28 of the full-length proteins were amplified by PCR from the existing plasmids pmal-PilE, pmal-PilV, and pmal-ComP^{9 (link)} and subcloned in pET22b (Novagen) between BamHI and XhoI restriction sites. The primer sequences used are provided in Supplementary Table 2.
The pET22b plasmid carries a signal peptide to direct the recombinant protein into Escherichia coli periplasm, where disulfide bond formation can occur. These plasmids were transformed in the E. coli BL21 (DE3) (Thermo Fisher Scientific) strain and grown at 30 °C for 3 h in lysogeny broth (LB), and 16 h at 16 °C in LB + 1 mM isopropyl β-d-1-thiogalactopyranoside. The fusion proteins were extracted from the perisplasm and loaded onto a Ni-NTA column (Thermo Scientific). Bound His-pilin were eluted using elution buffer (50 mM NaH₂PO₄, pH 8, 300 mM NaCl, 250 mM imidazole) and then dialyzed using an Amicon 10k device and resuspended in PBS at a concentration of 1 mg/ml. Purity and quality of recombinant proteins were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining.