Ni nta superflow resin

Recombinant TRIP-1 protein was expressed in bacteria using pQE-30 plasmid (Qiagen Inc, Valencia, CA) system. Briefly, 973-bp fragment corresponding to the coding region of TRIP-1 was cloned into Sph I/Sal I restriction sites in pQE-30 vector. This plasmid was transformed into E. coli bacteria. Briefly, a single bacterial colony was inoculated in 500 mL LB broth with 100 μg/mL ampicillin and 30 μg/mL kanamycin and incubated overnight in a 37 °C shaker The culture was then transferred into larger 2 L LB broth culture with 100 μg/mL ampicillin and 30 μg/mL kanamycin and incubated in a 37 °C shaker until OD 0.6–0.8 was reached. The protein was expressed by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Thermo Fisher scientific, Waltham, MA) to the culture and grown for 5 hours. The cells were then pelleted and stored at −80 °C. Protein purification was carried out using Ni-NTA Superflow resin (Qiagen) under native conditions following manufacturer’s protocol. The elution buffer with TRIP-1 recombinant protein was dialyzed, lyophilized and stored at −20 °C until use. The purified protein was then run on SDS-PAGE gel and stained with Coomassie blue to determine protein quality.

Recombinant PcTx1 was produced as described previously via expression in the periplasm of
Escherichia coli (
Saez
et al., 2011;
Saez
et al., 2015). Briefly, recombinant His
₆-MBP-PcTx1 fusion protein was isolated from cell lysates by passage over Ni-NTA Superflow resin (QIAGEN) and the His
₆-MBP tag was then removed by cleavage of the fusion protein with tobacco etch virus (TEV) protease. The released recombinant PcTx1 containing a non-native N-terminal serine to facilitate TEV cleavage (
Saez
et al., 2011) was isolated to >95% purity using reverse-phase HPLC. The isolated recombinant PcTx1 peptide is equipotent with native PcTx1 (
Saez
et al., 2011).

Lyophilized Dpo4-5m was dissolved in denaturation buffer containing 6 m Gn·HCl, and dialyzed against a series of renaturation buffers which contained 4, 2, 1, 0.5, 0.25 and 0 m Gn·HCl, respectively. Each step of the dialysis was carried out at 4 °C for 10 h with gentle stirring. The denaturation and renaturation buffers also contained 50 mM Tris-acetate (pH 7.5), 50 mM NaAc, 1 mM dithiothreitol, 0.5 mM EDTA and 16% glycerol. After renaturation, the enzyme was dialyzed against a buffer containing 10 mM potassium phosphate buffer (pH 7.0), 50 mM NaCl, 10 mM MgAc₂, 10% glycerol and 0.1% 2-mercaptoethanol. The folded polymerase was heated to 78 °C for 12 min to precipitate the thermolabile peptides, which were subsequently removed by ultracentrifugation at 19 000 r.p.m. for 40 min at 4 °C. The supernatant was incubated in Ni-NTA Superflow resin (Qiagen, Venlo, Netherlands) overnight at 4 °C, and purified according to previously described methods but without the use of Mono S column [21 (link)]. The concentration of the purified Dpo4-5m was measured spectrophotometrically at 280 nm using an extinction coefficient of 24 058 m⁻¹cm⁻¹ and calculated MW of 40.8 kDa. Approximately 100 μg of the purified synthetic polymerase was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis along with the purified recombinant protein.

Pre-equilibrated Ni-NTA Superflow resin (QIAGEN) was provided in a tube. Ump1-6His-containing or control protein extracts (EV) were added to the respective tubes and binding was allowed for 1 h at 4 °C, rotating. Afterwards, unspecific proteins were removed 4× using lysis buffer with 20 mM imidazole. Protein amounts of different mNeonGreen (NG) fusion protein [24 (link)] extracts were determined and adjusted by measuring the fluorescence using the Tecan Infinite F200 pro (filter 485 nm, 535 nm) prior to addition to the binding reaction. Binding was again allowed for 1 h at 4 °C with rotation. Proper washing was performed as described above. Samples were analyzed by fluorescence microscopy, which was carried out with the fluorescence microscope Zeiss Axioplan 2 and with the software AxioVision Rel 4.7 (Zeiss, Jena, Germany). For microscopy, a magnification of 10× was used, provided by the objective Zeiss Plan-Neofluar 10x/0.30 Ph1 (DIC I). For GFP, the following filter was used: F41-054 HQ-Cy2 HQ480/40 (excitation) Q505LP HQ527/30 (emission). Exposure times for image taking were 12 ms for brightfield and 1.5 s for GFP. For quantitative evaluation, images were analyzed using Fiji (ImageJ v1.53f, https://ij.imjoy.io).