Hiload 16 600 superdex 200 pg column

A 30 mg cellulase preparation from MP Biomedicals (catalog number 150583) was purified by size exclusion chromatography with a Tris-HCl running buffer (pH 7.4) and a HiLoad 16/600 Superdex 200 pg column (Cytiva, Marlborough, UK) attached to an NGC medium-pressure liquid chromatography system (BioRad, Hercules, CA, USA). The eluted fractions were analyzed by SDS-PAGE, excised, digested with trypsin, desalted, and then run on the Dionex LC and Orbitrap Fusion 2 for LC-MS/MS with a 30 min run time. The raw data files were analyzed using PD 2.2 and Scaffold 5. The Aspergillus niger reference database was combined with a list of common contaminants for the searches. Protein identification was provided by the UT Austin Center for Biomedical Research Support Biological Mass Spectrometry Facility.

Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) was used to determine the oligomeric state of GapN in solution. The purified protein was separated at room temperature on a HiLoad 16/600 Superdex 200 pg column (Cytiva, Marlborough, MA, United States) in 1 × PBS (pH 7.4) buffer at a flow rate of 0.5 ml/min. The molecular weight of the eluting proteins was determined using a Dawn Heleos 8 light scattering detector and an Optilab T-rEX refractive index detector (Wyatt Technologies, Santa Barbara, CA, United States). Data were analyzed using the ASTRA software (version 6.1.5.22, Wyatt Technologies, Santa Barbara, CA, United States).

The composition of the purification buffers of the p62 PH domain are the following (IMAC: immobilized metal affinity chromatography, SEC: size exclusion chromatography).
Lysis buffer: 50 mM CHES pH 9.0, 0.3 M NaCl, 5 mM Imidazole.
Elution buffer (IMAC): 50 mM CHES pH 9.0, 0.3 M NaCl, 0.25 M Imidazole.
NaCl-buffer (SEC): 20 mM CHES pH 9.0, 0.25 M NaCl.
CsCl-buffer (SEC): 20 mM CHES pH 9.0, 0.25 M CsCl.
The DNA sequence encoding the p62 PH domain from Chaetomium thermophilum was cloned into a pBADM-11 vector (EMBL) with an N-terminal 6 × His-tag and a TEV cleavage site. P62 PH was expressed in Arctic Express (DE3) RIL cells (Agilent). After cell harvest, the pellet was resuspended and lysed in Lysis buffer, and purified in two steps. First, IMAC was performed using Ni-TED beads (Macherey–Nagel) and bound protein was eluted with Elution buffer. Second, SEC was performed using a HiLoad 16/600 Superdex 200 pg column (Cytiva) with either NaCl-buffer or CsCl-buffer. Peak fractions were pooled and concentrated with centrifugal filter units (Merck Millipore) to 11–13 mg/ml.
HEWL was purchased as dry powder (Carl Roth) and dissolved to reach a concentration of 50 mg/ml in deionized water with 0.1 M sodium acetate pH 4.5, or 0.25 M CsCl. No further purification steps were applied.

The pET28a-RccR-his plasmid was transformed into E. coli BL21(DE3), and the cells were grown in LB with 50 μg/ml kanamycin at 37°C until the optical density at 600 nm (OD₆₀₀) reached 0.6. The isopropyl-β-d-thiogalactopyranoside was supplemented at a final concentration of 0.25 mM to induce the protein expression overnight at 16°C. Cells were harvested by centrifugation and the pellet was resuspended in buffer A (10 mM Tris–HCl, pH 7.5, 1 M NaCl and 1 mM DTT), and then the suspension was disrupted by sonication and clarified by centrifugation. The supernatant was filtered and then loaded onto a 5-ml HisTrap Ni-NTA column (Cytiva). The column was washed with buffer A containing 62.5 mM imidazole for unbound proteins, and the His-tagged RccR protein was eluted with buffer A containing 500 mM imidazole. The eluted protein was concentrated to 2 ml and loaded onto a HiLoad 16/600 Superdex 200pg column (Cytiva) for further elution with buffer A. The purified protein was concentrated for subsequent experiments.

ACE2‐Fc was produced by agrobacterium‐mediated transient expression in leaves of N. benthamiana ΔXT/FT. Four days after infiltration, a total soluble protein extract was prepared and clarified as described previously.^[7, ^8] ACE2‐Fc was purified by affinity chromatography using a 5 mL HiTrap Protein A column (Cytiva) and 0.1 M glycine‐HCl (pH 3.5) for elution. Eluate fractions were immediately neutralized using 2 M Tris (pH 12.0), pooled, concentrated (Amicon Ultra‐0.5 Centrifugal Filter Units, Millipore) and dialyzed against PBS (pH 7.4) at 4°C overnight using SnakeSkin Dialysis Tubing (Thermo Fisher Scientific). Affinity‐purified ACE2‐Fc was subjected to size‐exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 200 pg column (Cytiva) equilibrated with PBS (pH 7.4) for polishing. SEC‐multi‐angle light scattering (SEC‐MALS) analysis was carried out as described recently.^[9] Production of ACE2‐Fc in HEK293 cells was performed as described previously.^[5] ACE2‐Fc separated by SDS‐PAGE was detected by Coomassie Blue staining or immunoblotting with anti‐human IgG (H+L)‐horseradish peroxidase antibody (Promega). For deglycosylation, samples were incubated with Endoglycosidase H (Endo H) or peptide‐N‐glycosidase F (PNGase F) (New England Biolabs) according to the manufacturer's instructions.