Nickel nta beads

Substrates were amplified via PCR using the following primers:
Each substrate was cloned into the pET16b vector containing a 10x-His tag. The constructs were sequenced and transformed into BL21 (DE3) pLys E. coli. Colonies were grown overnight in LB liquid medium containing 0.1 mg/mL of ampicillin at 37°C to saturation. Cultures were then diluted into LB liquid medium containing 0.1 mg/mL ampicillin and grown at 37°C until an OD600 reading of 0.6. Cultures were then induced with 1 mM IPTG and moved to 18°C overnight. Soluble proteins were purified under native conditions. Pellets were resuspended in ~6–7 mL of lysis buffer containing: 50 mM NaH₂PO₄, 300 mM NaCl, and 10 mM imidazole pH 8. Cells were sonicated, pre-cleared, and incubated with nickel-NTA beads (Qiagen) for 3 h at 4°C. Beads were collected and washed in wash buffer containing: 50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole pH 8. The beads were eluted with 100 μL of elution buffer containing: 50 mM NaH₂PO₄, 300 mM NaCl, 500 mM imidazole pH 8. Purified protein was dialyzed overnight in dialysis buffer containing: 10 mM HEPES-KOH pH 7.7, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 10% glycerol.

The DNA sequence of lyophyllin was synthesized by Integrated DNA technologies, IDT. The sequence was cloned to pET-28a vector with 6X His tag. Constructed plasmid was transformed to OverExpress C43(DE3) cell (Cat. No. CMC0019, Sigma, St Louis, MO, USA) and grown until OD600 0.8, then induced by 0.1 mM IPTG at 25 °C overnight. The yield was about 50 μg lyophyllin/L cells. Corresponding variants were made by site-direct mutagenesis (Table S1).
To express the recombinant protein, E. coli cells were harvested by centrifugation at 11,655× g (Rotor JA-14, Beckman Coulter, Indianapolis, IN, USA) for 4 min, and then lysed by flow cell disrupter JN-Mini (JNBIO, Guangdong, China) at 1200 bar and 4 °C in buffer A (20 mM Tris pH 7.5, 100 mM NaCl, 50 mM Imidazole, 5% glycerol). The nickel NTA beads (QIAGEN, Venlo, The Netherlands) were equilibrated in the same buffer before loaded with the cell lysate. Beads were washed by buffer A for 10X column volume (CV) and then buffer B (20 mM Tris pH 7.5, 100 mM NaCl, 100 mM Imidazole, 5% glycerol) for another 10 CV. Finally, protein was eluted by buffer C (20 mM Tris pH 7.5, 100 mM NaCl, 300 mM Imidazole, 5% glycerol). It was then concentrated to suitable volume and injected to AKTA Prime (Cytiva, Marlborough, MA, USA) with Superdex 75 10/300 GL gel filtration column (Cytiva, Marlborough, MA, USA).

Plasmids obtained from the recombinant DNA library were transformed into competent E. coli BL21* (New England Biolabs, Ipswich, MA) for expression as described in [56 (link)]. Briefly, transformants were cultured in a 1 L hyper-broth media (Sigma-Aldrich, Natick, MA, USA) containing a yeast extract medium 25 g/L and a 15% glucose nutrient mix as well as ampicillin (100 µg/mL) at 37 °C under aeration conditions until cultures reached the mid logarithmic growth phase (OD600 = 0.5–0.8); protein expression was induced by the addition of 1 mM Isopropyl-β-d-thiogalactopyranoside (IPTG) (Sigma-Aldrich). After 6 h, the culture was centrifuged at 7500× g for 20 min, and the resulting pellet was collected and stored at −20 °C. The pellet was lysed in 8 M urea pH = 8.0 overnight after which Nickel-NTA beads (Qiagen, Hilden, Germany) were added. Protein column purification was performed using urea buffered at decreasing pH intervals = 8.0, 6.3, 5.9, 4.5 with the targeted protein being eluted at pH 4.5. The resulting elutants were run on an SDS-PAGE. The aliquots containing the target proteins were prepared for dialysis in 50 mM phosphate buffer at pH 5.4, and finally, water to remove the urea and induce proper folding of the proteins. The resulting solution was then lyophilized to obtain the pure protein in solid form.