Novex 16 tricine gels

Whole protein samples were prepared from culture samples taken at specific time points after induction by addition to ice-cold 10% (vol/vol) trichloroacetic acid. Pelleted protein samples were then washed with cold acetone before resuspension in nonreducing Laemmli buffer. Electrophoresis on Novex 16% Tricine gels (Thermo Fisher Scientific, Waltham, MA) was performed according to manufacturer recommendations. Proteins were transferred to a 0.2-μm polyvinylidene difluoride membrane by the iBlot2 system with a pre-programmed protocol (20 V for 1 min, 23 V for 4 min, 25 V for 1 to 2 min). After blocking with 4% (wt/vol) milk–Tris-buffered saline; His-tagged and c-myc tagged proteins were detected separately. Antibodies and substrate were acquired from Thermo Fisher Scientific (Waltham, MA). The mouse α-His primary antibody was used at a 1:2,000 dilution, and the secondary goat-anti-mouse Alexa Fluor 680 was used at a 1:20,000 dilution. α-c-myc (9E10) mouse monoclonal was used at a dilution of 1:1,000 overnight, followed by secondary antibody (goat-anti-mouse horseradish peroxidase) at 1:1,000, with SuperSignal West Femto Maximum Sensitivity Chemiluminescent Substrate (Thermo Fisher) for detection. Blots were scanned on Amersham Imager 600 RGB (GE Healthcare, Chicago, LA) and Bio-Rad-ChemiDoc machines, respectively, and analyzed using LI-COR ImageStudioLite version 4.0.21 software.

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).

IL-7 samples were separated in Novex 16% tricine gels (Invitrogen, cat. no. EC6695BOX) as recommended by the supplier. Proteins in gels were stained with the SilverQuest Silver Staining Kit (Invitrogen cat. no. LC6070) or transferred onto PVDF membranes using the Trans-Blot Turbo Transfer System with associated materials and protocols (Biorad, cat. no. 1704150). For Edman sequencing, proteins on the PVDF membrane were stained with Coomassie Brilliant Blue (Coomassie R-250, Thermo Scientific, cat. no. 20278) and analyzed with the Procise 491cLC protein sequencer (Applied Biosystems) or the PPSQ-51A protein sequencer (Shimadzu). For the analysis of signaling molecules, proteins in cell lysates were separated in 4-12% Tris-glycine gels under reducing conditions and transferred to PVDF membranes. Membranes were blocked for 1 h in 5% BSA with TBST buffer (150 mM NaCl, 0.1% Tween 20, 50 mM Tris, pH 7.5) and incubated overnight with anti-pSTAT3 (Tyr⁷⁰⁵) (Cell Signaling, cat. no. 9138) or anti-β-actin (Proteintech, cat. no. 20536-1-AP) antibodies. After washing, the blot was incubated with peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG for 1 h at room temperature. Finally, Western blot images were developed using the Vilber Lourmat Fusion system (Labtech International) and Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).