Poros hs column

We used previously published methods for tcTERT expression and purification, but implemented several modifications (Gillis et al., 2008 (link)). Briefly, we grew tcTERT in BL-21(DE3)pLysS cells using an Epiphyte3 LEX bioreactor at 37°C until they reached an OD600 of 0.6–0.8, after which the temperature was dropped to 30°C for 4–5 hr of protein production. Cells were harvested via centrifugation at 4000 x g until lysis. For TERT purification, we used buffers containing 0.75 M KCl and 10% glycerol for the capture step on Ni-NTA columns (GE Healthcare), and then further purified our sample with cation exchange on a POROS HS column (Thermo Fisher), using a salt gradient of 0.5 M KCl to 1.5 M KCl. Then, we cleaved the hexahistadine tag with Tobacco etch virus protease before purifying the cut tag from the protein with another run on our Ni-NTA columns. Finally, we used a slightly different buffer for the our size exclusion chromatography (Sephacryl S-200 16/60, GE Helathcare), containing 50 mM Tris-HCl, pH 7.5, 10% glycerol, 0.8 M KCl and 1 mM Tris(2-carboxyethyl)phosphine (TCEP). Resultant tcTERT was concentrated down to 18 mg mL⁻¹ prior to crystallography, and stored at 4°C (Gillis et al., 2008 (link)).

tcTERT was expressed and purified as described with some modifications^{42 (link),43 (link)}. An Epiphyte3 LEX bioreactor was used to grow tcTERT in BL-21(DE3) pLysS cells at 37 °C until they reached an OD₆₀₀ of 0.6–0.8, after which protein expression was induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and the temperature was decreased to 30 °C for 4-5 hours of protein induction. Cells were harvested via centrifugation at 4000 × g until lysis. For tcTERT purification, buffers containing 0.75 M KCl and 10% glycerol was used for the initial purification step on Ni-NTA columns (GE Healthcare). Samples were further purified via cation exchange on a POROS HS column (Thermo Fisher), using a salt gradient of 0.5 M KCl to 1.5 M KCl. Next, the hexahistidine tag was cleaved with Tobacco etch virus (TEV) protease overnight at 4 °C. The cut tag and TEV protease were separated from the protein with an additional Ni-NTA column chromatography step. The final chromatography step was a Sephacryl S-200 16/60, GE Healthcare column using a buffer containing 50 mM Tris–HCl, pH 7.5, 10% glycerol, 0.8 M KCl, and 1 mM Tris(2-carboxyethyl)phosphine (TCEP). Resultant tcTERT was concentrated down to 18 mg ml⁻¹ and stored at 4 °C^{42 (link)}.

Large-winged pearl shells (Pteria penguin, 6 years old) were provided by Amami South Sea & Mabe Pearl Co. Ltd. (former Amami-branch, Tasaki & Co. Ltd.), Kagoshima, Japan. Mantle and its secretory fluid were collected and stored at −80 °C or −30 °C until use, respectively. A POROS^® HS column was purchased from Applied Biosystems Thermo Fisher Scientific (Waltham, MA, USA). Resource S and HiTrap NHS-activated columns were purchased from GE Healthcare UK Ltd. (Amersham Place, Buckinghamshire, UK). TSKgel ODS-120T, COSMOSIL Protein-R, and CAPCELL PAK columns were purchased from Tosoh (Tokyo, Japan), SHISEIDO (Tokyo, Japan) and Nacalai tesque (Kyoto, Japan), respectively. Achromobacter protease I and Staphylococcus aureus V8 protease were purchased from Wako Pure Chemical Ind. (Osaka, Japan). Trehalose was purchased from Hayashibara (Okayama, Japan). PA-oligosaccharides were purchased from TaKaRa Bio Co. (Kyoto, Japan). All other reagents were of the purest grade commercially available.