Amicon column

The GST-DEPTOR (pGEX 5X3), His-KDM4A(aa 1-562) and His-KDM4A(aa 563-1064) (pDest17) were expressed in BL21 bacteria grown in LB. After induction with IPTG (0.2 mM) for 16 h at 20 °C, bacteria were harvested by centrifugation and lysed by sonication. The GST-fusion protein was purified on glutathione agarose beads (Sigma) and the His-fusion protein were purified with Ni-NTA agarose (Qiagen). The His-fusion proteins were eluted off the beads using 200 mM imidazole buffer and dialysed overnight against PBS, followed by concentration on Amicon columns (Millipore).

Sequences of the Fab portion of the clinical stage mAbs REGN10987, REGN10933³¹ . and CB6/Junshi^{11 (link)}. were reformatted into three different tetravalent types (sequences in Supplementary Fig. 6). Construction of the Fab-TD formats involved sub-cloning the p53 tetramerization domain (TD) linked onto the C-terminus of the immunoglobulin heavy chain CH1 domain devoid of the CH2-CH3 domains. For the Ig-TD format, the TD domain was linked directly to the C-terminus of the CH3 domain. The design of the monomeric Ig-TD (mIg-TD) format was identical to the Ig-TD format except the heavy chain core hinge region was removed. The corresponding light chains (LC) of the parental mAbs were kept intact. Expression constructs containing the modified heavy chains (HC) together with the corresponding LC constructs were expressed in Expi293F cells using Expifectamine293 Reagent according to the manufacturer’s recommendations (Thermo Fisher Scientific). Multimerized mAbs were purified directly from the culture supernatant by affinity purification. Fab-TD mAbs formats were purified using Ni–NTA and the Fc containing Ig-TD and mIg-TD mAbs formats were purified by Protein A affinity chromatography. All proteins were buffer exchanged and concentrated into PBS buffer using Amicon columns (Millipore) and aliquots were stored at 4 °C or at -80 °C for long-term storage.

The PpAs were loaded onto cation-exchange chromatography resin Hi-S cartridges (Bio-Rad), previously equilibrated with buffer with 20 mM tricine (Sigma-Aldrich), pH 8.5. To promote ionic interactions of proteins, the columns were left in oscillation at 4 °C overnight. Elution was performed using a discontinuous gradient of tricine containing 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl (1 mL/min). The eluates were passed through Amicon columns with a cutoff of 100 kDa (Millipore) to remove NaCl from the samples and concentrate the protein. The protein concentration was quantified by the Bradford method [56 (link)]. To determine the fraction in which the 110 kDa protease was present, zymograms copolymerized with bovine gelatin were made, and the proteolytic activity of each eluate was evaluated. In addition, a comparison of the protein profile by SDS-PAGE of the 60% PpAs with the eluate that showed the purified activity of 110 kDa was carried out [36 (link)]. All assays were performed in three independent experiments.

Expi293F cells were grown to a density of 1 × 10⁶ cells/mL at 37 °C with 8% CO₂ with regular agitation (150 rpm). Cells were transfected with a plasmid encoding SARS-CoV-2 RBD [56 (link)] using ExpiFectamine 293 transfection reagent, following the manufacturer’s instructions (Thermo Fisher Scientific). At day 4 post-transfection, medium from cell cultures were clarified by low-speed centrifugation. Recombinant RBD was purified by Ni-NTA agarose resin (Qiagen, Hilden, Germany), desalted and concentrated using Amicon columns (Millipore, Burlington, MA, USA) Purified SARS-CoV-2 S RBD was snap-frozen at liquid nitrogen and stored at −80 °C until further use. Protein content was confirmed as one protein of the expected size after SDS-PAGE analysis. Proteins were quantified by Pierce protein BCA assay kit (Thermo Fisher Scientific).

SF21 cells in suspension culture were transduced with baculovirus (MOI of 0.1) for 72 h, collected by centrifugation and stored at -80°C. Cell pellets were resuspended in wash buffer (10 mM Tris, 150 mM NaCl, 30 mM imidazole, pH 7.4) supplemented with EDTA-free protease inhibitor cocktail (Roche) and dispersed by passage through 18 and 25 G needles. His-tagged ORP4L was purified from lysates using metal affinity chromatography and concentrated by centrifugation in a Millipore Amicon column (30 K cut-off), diluted in storage buffer (10 mM Tris, 150 mM NaCl) and stored at -80°C [10 (link)].

Four cell lines, T1G (Tsc1 deleted [53 (link)]), T2J (Tsc2 deleted [53 (link)]), PKD1 (Pkd1 deleted [76 (link)]), and PKD2 (Pkd2 deleted [76 (link)]) were grown in complete media in a T-75 flask. Once cells reached the desired confluence of 75–80%, flasks were washed three times with sterile PBS to remove the traces of serum and then replenished with fresh media without serum for 16 h. Then, the culture media were harvested and centrifuged at 2000× g for 10 min to remove any cell debris. The supernatant was transferred on the top of an Amicon column (Millipore, Burlington, MA, USA, Cat #UFC901008) and concentrated up to 500 µL by refrigerated centrifugation as per the manufacturer’s protocol.