Superdex 200 increase 10 300 gl size exclusion chromatography column

Gel filtration chromatography
was performed using AKTA GO Fast
Protein Liquid Chromatography (FPLC) equipment (Cytiva). The samples
were injected into a Superdex 200 Increase 10/300 GL size exclusion
chromatography column (Cytiva) and were run at 0.5 mL min^–1 in PBS. A loop of 100 μL was used, and 50 μL was injected
in each run.

The Trypanosoma brucei threonine tRNA (TbtRNA^Thr_CGU) sequence was cloned in a pUC19 vector, between a T7 promoter and a BstNI cleavage site. The vector was linearized with BstNI and used as a template for in vitro transcription. The reaction was carried out in T7 reaction buffer (40 mM Tris pH 8; 5 mM DTT; 1 mM spermidine; 0.01% Triton X-100; 30 mM MgCl₂), with 4 mM of each NTP. For a 10 mL reaction, 0.5 mg of linear DNA and 0.4 mg of T7 RNA polymerase were used. The reaction was incubated at 37 °C for 8 h. The transcribed RNA was isopropanol precipitated, Urea-PAGE purified and desalted following standard procedures. The tRNA^Thr was refolded by heating at 95 °C for 10 min in 10 mM Tris pH 7.5 and 50 mM NaCl, slow cool down and adding MgCl₂ to 1 mM when it reached 37 °C. After refolding, TbtRNA^Thr_CGT was further purified by size-exclusion chromatography, in a Superdex 200 Increase 10/300 GL size-exclusion chromatography column (Cytiva) pre-equilibrated with refolding buffer (10 mM Tris pH 7.5 and 50 mM NaCl, 2 mM MgCl²).

SEC-SAXS experiments were performed at beamline B21 of the Diamond Light Source synchrotron facility (Oxfordshire, United Kingdom). Protein samples at concentrations >5 mg/mL were loaded onto a Superdex™ 200 Increase 10/300 GL size exclusion chromatography column (Cytiva) in 20 mM HEPES pH 7.5, 150 mM KCl at 0.5 mL/min using an Agilent 1200 HPLC system. The column outlet was fed into the experimental cell, and SAXS data were recorded at 12.4 keV, detector distance 4.014 m, in 3.0 s frames. Data were subtracted and averaged, and analysed for Guinier region Rg and cross-sectional Rg (Rc) using ScÅtter 4.0 (http://www.bioisis.net), and P(r) distributions were fitted using PRIMUS (Konarev et al., 2003 (link)). Crystal structures and models were fitted to experimental data using CRYSOL (Svergun and Koch, 1995 (link)).

To assess oligomeric state of the fusion proteins, 7.5–300 µg of purified recombinant protein was loaded onto a Superdex 200 Increase 10/300 GL size-exclusion chromatography column (Cytiva) using a 300–500 µl loop. Proteins were eluted using a mobile phase of PBS, pH 7.4 at a flow rate of 0.5 ml/min.

A gene encoding hACE2 residues 1 to 615 followed by a C-terminal HRV 3C cleavage site and a TwinStrepII-tag was cloned in the pXLG expression vector. HEK293F cells transfected with this construct were grown in a TubeSpin bioreactor in FreeStyle293 medium for 72 h at 37 °C with 8% CO₂ and agitation at 180 rpm. The secreted hACE2 protein was purified from the cell culture medium using a 1-ml StrepTactin Superflow high-capacity cartridge (IBA). After elution, the C-terminal TwinStrepII-tag was removed by cleavage with His6-tagged HRV 3C protease, followed by an immobilized metal ion affinity chromatography step to remove the HRV 3C protease from the sample. Finally, the untagged hACE2 protein was injected into a Superdex200 Increase 10/300 GL size exclusion chromatography column (Cytiva) equilibrated in 50 mM Hepes, pH 7.2, 150 mM NaCl, and 10% glycerol. The protein was concentrated to 1.9 mg/ml, flash-frozen in liquid nitrogen, and stored at −80 °C.

HOIL-1 helix-RBR (residues 233-510, C460A), UbcH7 (C86K)-Ub conjugate and M1 di-Ub were mixed at a 1:1.5:1.5 molar ratio and purified using a Superdex 200 Increase 10/300 GL size-exclusion chromatography column (Cytiva) equilibrated in buffer containing 10 mM HEPES pH7.9, 100 mM NaCl. Fractions containing all three proteins were pooled, concentrated to 10 mg/mL and used in crystallisation screens. Initial hits were obtained from the BCS PEG Smears screen (Molecular Dimensions) condition 28% PEGSB, 0.2 M NaCl, 0.1 M Na-phosphate pH 6.2. Hits were optimised by varying drop ratio and seeding. Final crystals were obtained by mixing 2.6 µl protein solution with 1 µl reservoir solution (28% PEGSB, 0.2 M NaCl, 0.1 M Na-phosphate pH 6.2). Crystals were cryo-preserved in reservoir solution supplemented with 15% v/v glycerol before cryo-cooling in liquid nitrogen.