His spin protein miniprep kit

To obtain the
Cas9 proteins, pETDuet plasmids containing SeHCas9 or its variants
were transformed into E. coli BL21
Star (DE3). Single transformants were inoculated in 3 mL of LB medium
and incubated at 37 °C. One percent of the overnight culture
was transferred to 50 mL of LB medium in a 250 mL shake flask and
grown at 37 °C. When the OD600 reached 0.6–0.8, protein
expression was induced by adding 0.5 mM IPTG and the flask was incubated
at 30 °C, 270 rpm for another 9–12 h. The harvested cells
were subjected to disruption by a Mini Bead Beater (Biospec). Proteins
were purified using a His-Spin Protein Miniprep Kit (Zymo Research)
as per the manufacturer’s instructions. The obtained proteins
were validated by Tricine-SDS-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE)
on a 12% protein gel, and the protein concentrations were quantified
using a Pierce BCA Protein Assay Kit (Thermo Scientific) following
the manufacturer’s instructions.

The polysheaths formed by the recombinant gp053 were precipitated from the supernatant by the addition of ammonium sulphate to a final concentration of 10%. After incubation for 10 min on ice and centrifugation at 9000× g for 15 min at 4 °C, the supernatant was removed, and the pellet was suspended in TE buffer (1/10 of the initial volume) and stored at 4 °C. The shorter, less ordered polysheaths were purified by the addition of ammonium sulphate to a final concentration of 15–20% and/or by repeated centrifugation at 9000× g for 15 min at 4 °C. Alternatively, the recombinant His-tagged protein purification using the metal-chelating sorbent was performed by using the His-Spin Protein Miniprep kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s recommendations. The concentration of the recombinant protein was determined by using a method described by Lowry et al. [45 (link)].

The transformants were cultured at 37 °C in Luria–Bertani medium containing ampicillin (50 µg/L) (Fujifilm Wako Pure Chemical) until the OD₆₀₀ was approximately 0.5. Next, the temperature was decreased to 15 °C to induce cold-shock promoters, and incubation was continued for 24 h. The cells were harvested via centrifugation (6000×g, 10 min, 4 °C) and suspended in 1 mL of protein extraction reagent (Integral Co., Tokushima, Japan). One microliter of 1000 U/mL recombinant DNase I (Takara Bio Inc. Shiga, Japan) and 0.2 mg/mL lysozyme (Fujifilm Wako Pure Chemical) were added to each sample and incubated for 15 min at room temperature. Thereafter, the samples were centrifuged (13,000×g, 20 min, 4 °C) and recombinant proteins were isolated using the His-Spin protein miniprep kit (Zymo Research Co., Irvine, CA, USA), following the manufacturer’s instructions.

The TTV-ORF-2 gene was amplified from cloned TTSuV1 genome and shuttled into the pET-28a bacterial expression vector (EMD, Millipore, Billerica, MA) in conjunction with a 6X HIS tag at both the N and C termini. Primers 5′-GTCAAGCTTTGCCGGAACACTGGGAGGAAG-3′ and 5′-ACGT CTCGAGCCAGCCATCGTCGCCGATAGTC-3′ with HindIII and XhoI restriction sites respectively, were used for amplification. The plasmid was transformed into a bacterial expression vector (BL21 DE3, Life Technologies, Grand Island, NY) and induced over-night by the addition of 1 mM IPTG at 37 °C. The over-expressed ORF2 protein was purified by affinity chromatography (His-Spin Protein Miniprep kit, Zymo Research, Irvine, CA), following the manufacturer’s instructions.

IMAC chromatography was carried out using the His-Spin Protein Miniprep Kit according to the manufacturer’s instructions (Zymo Research, Irvine, CA, USA). After preparation of the column, the reaction was resuspended in the gel matrix and incubated for 4 min at room temperature before centrifugation at 13,000× g for 10 s. The flow-through was collected as fraction E1. We then added 50 µL of distilled water to the column, followed by centrifugation as above, and the flow-through was collected as fraction E2. Finally, we added 150 µL of the washing buffer supplied in the kit, followed by centrifugation as above, and the flow-through was collected as fraction E3. Purity was assessed by SDS-PAGE. We added 1 µL (E1), 2 µL (E2), or 6 µL (E3) of the elution fractions to an equal volume of Tricine sample buffer and followed the SDS-PAGE and gel staining process described above. Bands corresponding to USCTX were excised and analyzed by mass spectrometry. For subsequent bioassays, elution fractions were lyophilized and redissolved to a volume of 10–13.3% (v/v) in medium.