Pgex 4t vector

The full‐length CDS of AceGBF3 was cloned into pGEX‐4T vector (GE Healthcare, Chicago, IL, USA) in which it is fused to glutathione‐S‐transferase (GST) sequence. The recombinant vectors were introduced into BL21 cells to produce AceGBF3‐GST fusion protein, and expressed AceGBF3‐GST and AceMYBS1‐6xHis fusion protein was purified as described previously (Xu, 2020 ). Aliquots (20 μl) of AceGBF3‐GST and AceMYBS1‐His proteins were mixed with 1 ml binding buffer (50 mM Tris–HCl (pH = 7.5), 100 mM NaCl, 0.25% Triton‐X100 (v/v), 35 mM β‐Mercaptoethanol), then 50 μl Proteinlso GST Resin or 50 μl Proteinlso Ni‐NAT Resin (TransGen) was added and the mixture was rotated at 4°C for 3–4 h. The samples were washed four times with binding buffer, with expressed GST or 6xHis used as negative controls. 6xSDS protein loading buffer was added to samples (to 1× final) and samples were denatured by boiling for 10 min before electrophoresis. After electrophoresis the gels analyzed by Western blot using anti‐HiS (1 : 10 000 (v/v), Proteintech, 66005‐1‐lg) and anti‐GST (1 : 10 000 (v/v), Proteintech, 66002‐2‐lg; both sourced from Wuhan Sanying, Wuhan, China) antibodies.

C2AB modules from Syt-1 and Syt-7 chimeras (amino acids 96–421) were subcloned into a pGEX4T vector (GE), expressed as GST-­fusion proteins, purified from Escherichia coli lysates via GST-sepharose affinity chromatography, and thrombin cleaved in 100 mM KCl, 25 mM HEPES, pH 7.4, 5% glycerol. Syt7 C2AB (amino acids 134–403) was expressed with a His₆ tag (pTrcHis vector, Invitrogen), purified from E. coli lysates via Ni-NTA-sepharose affinity chromatography, eluted with 500 mM imidazole, and dialyzed overnight against 100 mM KCl, 25 mM HEPES, pH 7.4, 5% glycerol. Protein concentrations were determined by SDS–PAGE using BSA standards.