Ni nta ni2 nitrilotriacetate agarose

E. coli BL21 (λDE3) cells and pET28a were purchased from Novagen (Darmstadt, Germany) and were used to express proteins. Restriction enzymes, T4 ligase, modification enzymes, DNA polymerase, dNTPs, and all antibiotics were obtained from TaKaRa Biotech (Shiga, Japan). PCR primers were synthesized by Invitrogen (Carlsbad, USA). Ni-NTA (Ni²⁺-nitrilotriacetate) agarose was purchased from Qiagen (Hilden, Germany). 7H9 and 7H10 broths were purchased from Becton, Dickinson Company (New Jersey, USA). Antibodies were obtained from the Wuhan laboratory animal center of CAS (Wuhan, China).

The recombinant plasmid for C-terminal His-tagged R27-encoded E protein overexpression was obtained following the instructions of ALICator Ligation Independent Cloning and Expression System (Thermo Scientific) using primers RepECtermFor and RepECtermRev (Supplementary Table S1). Recombinant clones were sequenced before overexpression.
Transformed BL21(DE3) cells were grown at 37°C in LB until the cultures reached an OD_600nm of 0.5–0.6. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was subsequently added to a final concentration of 1 mM. Upon 3 h incubation at 37°C, cells were centrifuged and pellets were frozen, resuspended in lysis buffer (50 mM NaH₂PO₄ pH 8, 1 M KCl, 10 mM imidazole suplemented with 1 mg ml^-1) of lysozyme and a protease inhibitor cocktail (cOmplete ULTRA Tablets Mini, EDTA-free, EASYpack Roche) and disrupted by sonication on ice. The lysate was centrifuged at 20,000 × g for 30 min at 4°C, and the supernatant was then treated with Ni-NTA (Ni²⁺-nitrilotriacetate)–agarose (Qiagen). The resin was washed extensively with the same buffer, and the protein was eluted using 200 mM imidazole as described previously (Nieto et al., 2000 (link)).

Escherichia coli (E. coli) BL21 (λ DE3) and pET28a were purchased from Novagen (Darmstadt, Germany) and used to express proteins. Restriction enzymes, T4 ligase, modification enzymes, DNA polymerase, dNTPs were obtained from TaKaRa Biotech (Shiga, Japan). PCR primers were synthesized by Invitrogen (Carlsbad, USA). Ni-NTA (Ni²⁺-nitrilotriacetate) agarose was purchased from Qiagen (Hilden, Germany). 7H9 and 7H10 broths were purchased from Becton, Dickinson Company (New Jersey, USA). Cordycepin (3’-deoxyadenosine, from Cordyceps militaris, C2689) was purchased from Tokyo chemical industry CO., LTD. (Tokyo, Japan).