Dp101 01

E. coli Transetta (DE3) cells were transformed with pGEX-4T-1-Drp1, pGEX-4T-1-Ube1, pET-28a-Ubch10, pET-28a-Ubch5, pET-28a-Ubc4, pET-28a-Ube2s, pET-28a-Ube2k, pET-28a-Ubc7, pET-28a-Apc2 and pET-28a-Apc11, respectively. The protein expression was induced by 0.5 mM IPTG for 16 h at 16 °C. Cell lysates were prepared by repeated freezing-thawing method with liquid nitrogen and water bath of 28 °C. Lysates were incubated with GST Resin (TransGen Biotech DP201-01) or Ni-NTA Resin (TransGen Biotech DP101-01) overnight at 4 °C. GST-DRP1 and GST-UBE1 were eluted with 20 mM GSH. His-UbcH10, His-UbcH5, His-Ubc4, His-UBE2S, His-UBE2K, His-Ubc7, His-APC2 and His-APC11 were eluted with 300 mM imidazole. Eluted proteins were concentrated to 3 mg/ ml with Amicon Ultra-0.5 centrifugal filter units (Millipore). Protein aliquots were frozen in liquid nitrogen immediately and then stored at −80 °C.

For analysis of pl-CSA, a recombinant VAR2CSA labeled with a 6×His tag (rVAR2), specifically interaction with pl-CSA, was expressed by inserting this fragment into a pET28a (+) vector that was transformed into Escherichia coli BL21, and was purified using a Ni²⁺ affinity column (cat. no. DP101-01; Transgen Biotech Co., Ltd., Beijing, China) from the supernatant of bacterial culture. The purified rVAR2 was used in the following experiments.

In order to express the PR1 protein with the His tag protein, the coding region without the signal peptide sequence was amplified using primer (PR1-no signal peptide) and cloned into the pEASY-Blunt1 (CE111-01, TransGen Biotech, China) vector, which was then introduced into BL21.After incubating transformed BL21 bacteria in Luria–Bertani (LB) broth at 37°C to an optical density of OD₆₀₀ = 0.6, a final dosage of 0.6 mM of isopropyl d-1-thiogalactopyranoside (IPTG) was added to promote PR1 production. At 12 hours after induction at 19°C, bacterial cells were extracted by centrifugation and suspended in a balanced buffer (300 mM NaCl, 30 mM NaH₂PO₄, and 10 mM imidazole, pH 8) and lysed with an ultrasonic cell crusher. The manufacturer’s instructions were followed to purify His6-tagged proteins from bacterial extracts using Ni-NTA resin (DP101-01, TransGen Biotech, China). Using BSA as a standard, the Lowry method was used to measure protein concentration. The used primers are provided in Supplementary Data Table S1.