Superdex 200 size exclusion chromatography

VRC01 Fab was produced and purified as previously described [40 (link)]. eOD-N276Kif, a minimal glycan, alanine-resurfaced construct possessing only three glycosylation sites (N18, N65 and N79, eOD numbering as in PDB IDs 4JPJ and 4JPK) was designed and subsequently transfected in HEK 293F (Invitrogen) suspension cells in the presence of the mannosidase I inhibitor kifunensine, to yield homogeneous Man₉GlcNAc₂ carbohydrates. The secreted protein was purified via its C-terminus His₆-tag by affinity chromatography using HisTrap nickel columns (GE Healthcare), and subsequently purified to size homogeneity by Superdex 200 gel filtration chromatography (GE Healthcare). After incubation of VRC01 Fab in molar excess of eOD-N276Kif, the complex was purified by Superdex 200 size exclusion chromatography (GE Healthcare) and concentrated to ~5 mg/ml for crystallization trials using the automated JCSG/IAVI/TSRI CrystalMation robotic system (Rigaku) at the Joint Center for Structural Genomics (www.jcsg.org) at TSRI. Crystals used for data collection were obtained with 0.16 M ammonium sulfate, 20% (w/v) PEG4000, 20% (v/v) glycerol, 0.08 M sodium acetate, pH 4.6, as the mother liquor. Prior to data collection, crystals were cryoprotected in the mother liquor supplemented with 40% glycerol and subsequently fast-plunged into liquid nitrogen.

pET14b-6xHis bacterial plasmids expressing SAMHD1 proteins were expressed in E. coli BL21 Rosetta cells, induced by 0.3 mM isopropyl-1-thio-D-galactopyranoside (IPTG) at 16 °C, and further grown overnight. After cells lysis by sonication on ice, proteins were purified by His-Trap affinity chromatography (GE Healthcare). Protein was further purified by Superdex 200 size exclusion chromatography (SEC) (GE Healthcare) in a buffer containing 20 mM Tris pH 7.8, 50 mM NaCl, 1 mM DTT, and 5% glycerol. For SEC, SAMHD1 proteins (5 μM) were preincubated with dGTP (3 mM) or without dGTP, and the mixtures (200 μL) were applied to an analytical Superdex 200 column (10 × 300 mm) at a flow rate of 0.5 mL/min. The column was calibrated with gel filtration standard (BioRad) and equilibrated with a buffer containing 20 mM Tris-HCl, pH 7.8, 50 mM NaCl, 10 μM dGTP, 5 mM MgCl2, and 5% glycerol. The elution profiles with UV traces of excitation at 280 nm were recorded.

The pET15b‐based plasmids for producing the H2A.Z.1 mutants, H2A.Z^{H2A(61–129)} and H2A.Z ^H2A(1–60), were prepared by the polymerase chain reaction method with the DNAs of the H2A and H2A.Z.1 expression vectors as templates (Kujirai et al., 2018). The human H2A, H2A.Z.1, H3.1, H4 and the H2A.Z.1 mutants were purified as described previously (Kujirai et al., 2018). Briefly, the histone proteins were produced as His₆‐tagged proteins in Escherichia coli and were purified by affinity chromatography. After removing the His₆‐tag peptide by thrombin protease treatment, the histone proteins were purified by Mono‐S cation‐exchange chromatography. The proteins were dialyzed against 2 mM 2‐mercaptoethanol and lyophilized. The H2A‐H2B dimer, H2A.Z.1‐H2B dimer, H2A.Z^{H2A(61–129)}‐H2B dimer, H2A.Z^H2A(1–60)‐H2B dimer and H3.1‐H4 tetramer were reconstituted and purified, as described previously (Fujita et al., 2020). Lyophilized H2A and H2B, or H3.1 and H4, were mixed at a 1:1 molar ratio in denaturing solution, containing 7 M guanidine hydrochloride, 20 mM Tris‐HCl (pH 7.5) and 20 mM 2‐mercaptoethanol. The H2A‐H2B dimer and H3.1‐H4 tetramer were reconstituted by refolding, in a solution containing 10 mM Tris‐HCl (pH 7.5), 2 M NaCl, 1 mM EDTA and 5 mM 2‐mercaptoethanol. The histone complexes were purified by Superdex 200 size‐exclusion chromatography (GE Healthcare).

Human TRPML1 was cloned into pEG BacMam with an N-terminal Flag tag. The protein was expressed using baculovirus-mediated transduction of mammalian HEK293S GnTI⁻ cells (ATCC). These cells tested negative for mycoplasma contamination. At 48 h post infection at 37 °C, cells were disrupted by sonication in buffer A (20 mM HEPES, pH 7.0, 150 mM NaCl) with 1 mM PMSF and 5 μg ml⁻¹ each of leupeptin and aprotinin. After low-speed centrifugation at 3470 g, the resulting supernatant was incubated in buffer A with 1% (w/v) lauryl maltose neopentyl glycol (MNG, Anatrace) for 1 h at 4 °C. The lysate was centrifuged at 34,572 g and the supernatant was loaded onto an anti-Flag M2 affinity column (Sigma). After washing twice, the protein was eluted in 20 mM HEPES, pH 7.0, 150 mM NaCl, 0.1 mg ml⁻¹ Flag peptide and 0.01% MNG, and then concentrated. The protein was purified by Superdex-200 size-exclusion chromatography (GE Healthcare) in a buffer containing 20 mM sodium acetate, pH 5.0, 150 mM NaCl and 0.06% (w/v) digitonin (Sigma). The peak fractions were collected and concentrated to 5–7 mg/ml for grid preparation. The mutated DNA constructs were generated using QuikChange Mutagenesis Kit (Agilent) (Supplementary Table 2).

Full-length gp120 proteins were produced in FreeStyleTM 293F (Invitrogen) suspension cultures or lab-adapted 293S (GnTI^-/-) suspension cultures by transient transfection using 293Fectin (Invitrogen) of a pHLSec plasmid containing gp120 with a C-terminal His_6x affinity tag. Protein was harvested from the supernatant after 96 h and purified by affinity chromatography with a HisTrap column (GE) followed by Superdex 200 size exclusion chromatography (GE Healthcare) using an AKTA Express system (GE Healthcare).

A. gossypii Scc2^378–1,479 and S. cerevisiae Scc2–Scc4 were amplified by PCR from genomic DNA (LGC Standards) and cloned into a modified version of the MultiBac vector pFBDM42 (link)43 (link). A double Strep-tag II (ds) together with a tobacco etch virus cleavage site were introduced into the C terminus of Scc2. The resultant protein expression cassettes were recombined with the DH10MultiBac cells to create a bacmid. Both constructs were expressed using the baculovirus and insect cell (High 5 cells) systems, and purified by a combination of Strep-Tactin (Qiagen), anion exchange chromatography on Poros HQ50 and Superdex 200 size-exclusion chromatography (GE Healthcare) in a final buffer of 10 mM imidazole pH 7, 300 mM NaCl and 0.5 mM tris (2-carboxyethyl) phosphine (TCEP). Mutants of A. gossypii Scc2 and S. cerevisiae Scc2–Scc4 were generated using a USER cloning method43 (link).