Table 1 QC requirements and strategies for proteins that are used in specific biological applications or have particular intrinsic biochemical features
QC requirements | Strategies | |
|---|---|---|
Biological applications | ||
Protein–protein interactions studies (pull-down assays, IP, crosslinking protein interaction analysis, ITC, MST, DLS, and others) | Proteins must be properly folded and contain the correct oligomeric conformation to avoid “non-specific” sticking to soluble aggregates | |
DNA/RNA interaction assays (EMSA, ITC, MST, …) | Check that nucleases and nucleic acids were removed | Nucleic acid removal step in the purification process: anion exchange, polyethyleneimine, or streptomycin sulfate precipitation Avoid the use of external DNases or RNases |
Structural analysis (X-ray crystallography, cryo-EM, NMR) | Folded, stable protein as suggested in the QC Guidelines [1, 2] Suitable storage buffers | Conditions must be determined in which the protein remains correctly folded and in the appropriate oligomeric state at the high protein concentrations required for most structural biology applications |
Antibody fragment production | Activity evaluation | Affinity measurement, IP, ELISA |
Studies with sensitive cell lines, in vivo experiments, and production of growth factors for cell culture | QC Guidelines [1, 2] Endotoxin-free Confirmed activity | Endotoxin removal via ion exchange, SEC, and/or endotoxin removal beads. Endotoxin removal by chromatographic washing steps with non-ionic detergents such as Triton X114 |
Protein production for in vivo assays | QC Guidelines [1, 2] with emphasis on purity Non-toxic buffer for animal use Endotoxin-free | Check buffer compatibility with host animals. Final buffer free of components toxic for the animals (endotoxins) |
Antigen production for animal immunization | QC Guidelines [1, 2] with emphasis on purity Non-toxic buffer for animal use Endotoxin-free | Check buffer compatibility with host animals. Final buffer free of components toxic for the animals (endotoxins) |
Antigen production for in vitro panning | QC Guidelines [1, 2] with emphasis on purity and monodispersity Native folding | Check aggregation and presence of hydrophobic patches. If feasible, check functionality |
Compound screening, Inhibition/activity assay development | Folded, stable protein | Buffer compatibility Absence of contaminating inhibitors, interferents, etc |
Affinity/specificity measurements | Folded, stable protein | Buffer compatibility Absence of contaminating inhibitors, interferents, etc |
Intrinsic biochemical features | ||
Protein complexes | QC Guidelines [1, 2] with emphasis on a correct protein stoichiometry by SDS-PAGE and correct mass by SEC-MALS or similar methods | Careful strategy design. Optimization of expression conditions Use mild conditions that avoid complex dissociation during all the purification procedures. Recovery of the complete and stable protein complex in the correct oligomerization state and correct protein stoichiometry |
Prone-to-aggregation proteins | A repeat of the QC checks after protein storage (before usage) is highly recommended | Optimization of expression conditions Optimization of the buffer and/or storage conditions with the help of techniques such as thermofluor, nano-DSF, DLS Rapid strategy for purifying and storing the target protein as fast as possible Work at low temperature and avoid reaching critical protein concentrations that induce aggregation |
Proteins binding to divalent cations or other co-factors | QC Guidelines [1, 2] Optimally, verification of divalent cation/co-factor incorporation via spectroscopy | Addition of the divalent cation or co-factor to the growth medium and/or the purification buffers. Recovery of properly folded protein with the divalent cation (or another co-factor) correctly incorporated. Avoid the use of chelating agents in the buffers |
Proteins with inter- or intramolecular disulfide bonds | SDS-PAGE with sample buffer with or without reducing agents Assess correct disulfide bond formation by DTNB (Ellman’s reagent) or MS | Optimization of expression conditions Avoid the addition of reducing agents for the recovery of stable protein with native disulfide bonds |
Proteins with free cysteines | Assess undesirable disulfide bond formation by MS | Avoid undesirable disulfide bond formation by using reducing agents during purification and storage |