Akta pure system

The full-length human TPH2 gene was purchased from Sino Biological Co., Ltd, and reconstructed to pCAG with an N-terminal Twin-Strep-tag and a 3×Flag-tag. The construct was then transfected into Expi293F (Thermo Scientific) with PEI reagent and cultured for 72 h at 37°C under 8% CO₂. After that, cells were harvested, resuspended, and lysed by sonication in buffer A (50 mM HEPES pH 7.5, 150 mM NaCl, 0.1 mM FeSO4, 0.1 mM tryptophan, 0.1 mM EDTA, 10% v/v glycerol, 2% Tween-20, and 1 mM PMSF). Insoluble material was removed by centrifugation at 15,000 g and the supernatant was loaded on a 2 ml Strep-Tactin®^XT column equilibrated with lysis buffer. The column was washed successively with 2 mM ATP in buffer A to remove the endogenously expressed HSP70 protein before TPH2 was eluted in steps with three times of buffer A containing 5, 25, and 50 mM biotin. Fractions containing pure TPH2 protein were identified using SDS-PAGE and further purified using size exclusion chromatography. TPH2 was loaded on a Superose™ 6 Increase 10/300 GL column attached to an AKTA pure system (Cytiva) equilibrated in buffer B (50 mM HEPES pH 7.5, 150 mM NaCl, 0.02% w/v glyco-diosgenin). Fractions were assessed using SDS-PAGE and concentrated for cryo-EM analysis. Approximately 0.25 mg of full-length TPH2 can be obtained from 500 ml of cells.

Expression and purification of the recombinant Q5 used for the first set of ELISA assays (8 M urea batch) was carried out as previously described [17 (link)], using the Q5 gene cloned into the pRSET expression (Invitrogen, Waltham, MA, USA) transformed into Escherichia coli Rosetta^TM 2 DE3 (from Novagen, Darmstadt, Germany). The recombinant Q5 construct and the one encoding the Lci13 antigen, which was also cloned into the pRESTa plasmid [16 (link)], were then used in a second purification procedure to produce the 2 M urea batches. These used bacterial cells from one-liter cultures which were harvested and resuspended in 20 mL of buffer A (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, 2 M urea, 10 mM 2-Mercaptoethanol) and lysed with five pulses of ultrasonication at 4 °C. Soluble supernatants, after centrifugation at 20,000× g for 30 min at 4 °C, were then loaded onto a 5 mL His-Trap HP column in an AKTA Pure system (Cytivia, Marlborough, MA, USA) equilibrated with buffer A. Proteins were eluted with a two-step gradient in a 10 column volume (CV) linear gradient from 0% to 10% buffer B (buffer A + 500 mM imidazole), followed by a 20 CV linear gradient from 10% to 100% buffer B. Elutions were loaded on 15% SDS-PAGE followed by Coomassie staining to confirm the protein load and quality [1 (link)].

Approximately 1 kg or approximately 200 g wild-type (Oregon R) Drosophila embryos were collected 0–12 hr after egg deposition (AED) from population cages. The embryos were dechorionated, and nuclear extracts were prepared as described (Kamakaka et al., 1991 (link)). Protein concentration was measured by Pierce BCA assay (Thermo Fisher). The extracts were fractionated by FPLC (Figure 1A, Figure 4—figure supplement 2A) on AKTA PURE system (Cytiva Life Sciences). Aliquots of chromatographic fractions were examined by quantitative shotgun proteomics or Western blot analyses as described below. Peak SUUR or Mod(Mdg4) fractions were diluted to an appropriate ionic strength (if applicable) and used as a starting material for the next chromatographic step. Details on FPLC column sizes and run parameters are shown in Figure 1—source data 1 and Figure 4—figure supplement 2—source data 1.