Characterization of Protein Purification

Wet cell weight was determined as described previously (Crowell et al., 2018 (link)). Sample concentrations were determined by measuring the absorbance at 280 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) was carried out under reducing conditions using Novex 12% Tris‐glycine Gels or Novex 16% Tricine Gels (Thermo Fisher Scientific) according to the manufacturer's recommended protocol and stained using Instant Blue Protein Stain (Thermo Fisher Scientific). Samples were analyzed for host cell‐protein content using the Pichia pastoris 1st generation HCP ELISA kit from Cygnus Technologies according to the manufacturer's recommended protocol. Samples were analyzed for residual host‐cell DNA using the Quant‐iT dsDNA High‐Sensitivity Assay Kit (Invitrogen) according to the manufacturer's protocol except the standard curve was reduced to 0–20 ng. Unpurified samples were not analyzed for DNA content due to interference of media components with the Quant‐iT dsDNA High‐Sensitivity Assay Kit. Instead, typical DNA content of unpurified material produced in Komagataella phaffii is used for comparison (Timmick et al., 2018 (link)). Purification yields were calculated using concentration measurements at 280 nm (purified samples) or estimated from SDS‐PAGE (unpurified samples).