Xjb de3 autolysis

All proteins were expressed in XJb(DE3) Autolysis (Zymo Research) E. coli strain. Cells were grown in LB media supplemented with 100 µg/mL ampicillin (full-length DiB proteins) or 100 µg/mL ampicillin and 50 µg/mL kanamycin (split-Zip and split proteins) at 37 °C. Expression was induced by addition 0.04% L-arabinose (full-length DiB proteins) or 0.2% L-arabinose and 10 μM IPTG (split-Zip and split proteins) at 0.8 OD. Cells were harvested after 3 hours of expression at 37 °C and were resuspended in PBS buffer, pH 7.4. Suspensions were frozen at −80 °C and thawed at room temperature three times. DNA was destroyed by short sonication and the lysates were centrifuged to obtain cell-free extracts. The proteins were first purified using gravity flow columns with TALON metal affinity resin (Clontech) and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 75 pg or Superdex 200 pg 10/300 GL column (GE Healthcare) pre-equilibrated with 50 mM sodium phosphate buffer, pH 6.0.

All proteins were expressed in XJb(DE3) Autolysis (Zymo Research, Irvine, CA, USA) E. coli strain. Cells were grown in LB or M9 media supplemented with 100 μg·mL⁻¹ ampicillin (full-length wtBlc protein) or 100 μg·mL⁻¹ ampicillin and 50 μg·mL⁻¹ kanamycin (wtBlc-split-Zip and wtBlc-split proteins) at 37 °C. Expression was induced by addition of 0.04% L-arabinose (full-length wtBlc protein) or 0.2% L-arabinose and 10 μM IPTG (wtBlc-split-Zip and wtBlc-split proteins) at 0.8 OD. Cells were harvested after 3 h of expression at 37 °C if grown in LB or after overnight expression if grown in M9 and were resuspended in PBS buffer, pH 7.4. Suspensions were frozen at −80 °C and thawed at room temperature three times. DNA was destroyed by short sonication, and the lysates were centrifuged to obtain cell-free extracts. The proteins were first purified using gravity flow columns with TALON metal affinity resin (Clontech, Mountain View, CA, USA) and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 75 pg or Superdex 200 pg 10/300 GL column (GE Healthcare, Marlborough, MA, USA) pre-equilibrated with 50 mM sodium phosphate buffer, pH 6.0.