Ni sepharose resin

The genes encoding the subunits of the human γ-secretase complex, except for Aph-1aL, were cloned into the same expression vector as described above. The gene encoding human Aph-1aL was fused N-terminally with the N11-SUMO tag and C-terminally with the TEV protease recognition site and the FLAG tag. The T4 phage lysozyme-human PS1 fusion protein (T4L-PS1)26 (link) was produced by the P-MF method, with mixed micelles (3.3 mg/ml total or polar extract of porcine brain and 2.0 mg/ml digitonin) and without PEG8000. The proteoliposome fractionation, the membrane extraction, and the metal affinity purification were performed as described in the previous section, with some modifications: the use of 50 mM Tris-HCl buffer (pH 7.0) for the glycerol density gradient, 50 mM Tris-HCl buffer (pH 7.0) containing 1% βDDM, 0.1% CHS, and 400 mM NaCl for membrane extraction, Ni-Sepharose resin (GE Healthcare Life Sciences), the wash buffer [50 mM Tris-HCl buffer (pH 7.0) containing 0.05% βDDM, 0.002% CHS and 400 mM NaCl] and the elution buffer [50 mM Tris-HCl buffer (pH 7.0) containing 0.05% βDDM, 0.002% CHS, 200 mM imidazole, and 400 mM NaCl] for metal-affinity purification. The enzymatic activity of PS1 was assayed by the method using an intramolecularly-quenched fluorogenic peptide probe, Nma-Gly-Gly-Val-Val-Ile-Ala-Thr-Val-Lys(Dnp)-D-Arg-D-Arg-D-Arg-NH₂45 (link).

ZIKV NS5 RdRp polymerase was cloned at pETTRX by the LIC method and expressed and purified according to the protocol described in [92 (link)]. Briefly, NS5 RdRp polymerase was expressed in ZYM 5052 auto-induction medium and purified in four steps: (i) a HisTrap HP 5.0 mL with a Ni Sepharose resin (GE Healthcare, Sao Carlos, Brazil); (ii) a buffer exchanged by dialysis and a concomitant TEV protease cleavage from 6His-TRX-tag; (iii) an inverse HisTrap HP 5.0 mL to separate protein from 6His-TRX-tag and (iv) a size-exclusion chromatography at a XK 16/60 Superdex 75 column (GE Healthcare, Sao Carlos, Brazil).

Production and purification of the six recombinant proteins were performed as previously described [23 (link)]. Cells were grown in Terrific Broth (TB) and when the OD₆₀₀ reached 0.7–0.8, protein production was induced by addition of arabinose (final concentration 0.02%) for all variants. Protein production was carried out overnight at 24 °C. The fusion enzymes were purified using Ni Sepharose resin (GE Healthcare, Bio-Sciences AB, Chicago, IL, United States). Purified enzymes (in 50 mM Tris–HCl, pH 7.5) were flash frozen in liquid nitrogen and stored at −80 °C.

The expression and purification procedures for the SARS-CoV-2 Omicron S protein were described previously (39 (link)). In brief, the gene encoding stabilized Omicron S ECD HexaPro was constructed and expressed using FreeStyle 293-F cells (Thermo Fisher Scientific, R79007). Protein was purified from filtered cell supernatants using Ni Sepharose resin (GE Healthcare, 17526801) before being subjected to additional purification by gel filtration chromatography using a Superose 6 10/300 increase column (GE Healthcare, 17517201) in 1× PBS (pH 7.4).
All the antibodies were described and prepared as previously described (16 (link), 39 (link)). In brief, 2 plasmids containing the light chain and heavy chain of antibodies were transiently cotransfected into Expi293 cells (Thermo Fisher Scientific, A14528) at a 1:1 ratio with ExpiFectamine 293 (Thermo Fisher Scientific, A14525). After 7 days, antibodies were purified from filtered cell supernatants using protein G Sepharose and dialyzed into 1× PBS (pH 7.4).

Transformed E. coli cells were pre-cultured in 10 mL of Luria-Bertani (LB) broth medium containing 0.04 mg/mL ampicillin and 0.034 mg/mL chloramphenicol at 37 °C for 16 h and then transferred to 400 mL of terrific broth medium containing 0.04 mg/mL ampicillin. Bacterial growth was monitored with OD₆₀₀ every 30 min prior to isopropyl β-d-thiogalactopyranoside (IPTG) induction. After the culture reached OD₆₀₀ of 1.5 (approximately 3 h), over-expression of His-tagged KaiC was induced by adding IPTG to a final concentration of 0.1 mM. The cells were further harvested for approximately 5 h until OD₆₀₀ reached ~6. The OD₆₀₀ was measured after 10-fold dilution of the culture medium. The harvested cells were collected by centrifugation and then kept at −20 °C until use.
Multiple open columns filled with Ni-Sepharose resin and PD-10 (GE Healthcare, Chicago, IL, US) were used to purify multiple KaiC mutants simultaneously. Both KaiA and KaiB were expressed as GST-tagged forms and purified after the cleavage of the GST-tag, as described previously [21 (link)].