Quality control of recombinant protein
Best practice recommendations
The Association of Resources for Biophysical Research in Europe (ARBRE-MOBIEU) and the Production and Purification Partnership in Europe (P4EU) have formed a joint initiative on recombinant protein quality. We aim to develop a best practice/minimal standard for the quality control of recombinant proteins to ensure that the input material used in biophysical and biochemical research is of high quality, which, in turn, will result in optimized data quality. The prescribed tests must be both doable by all protein production labs, and, at the same time, acceptable for biophysical or structural biology labs as admission criteria.
Below you find some recommendations for what we propose as minimal and extended quality control parameters/standards as well as a summary of the guidelines. The minimal standard requires some detailed information about the recombinant protein being accessed, i.e. primary structure, accession numbers, cloning strategy, protein concentration and storage conditions. Then, a series of experiments is suggested, to establish protein purity, homogeneity and aggregation, nucleic acid content, as well as identity via a mass-spec. In addition, a series of ‘best-practice’ methods is suggested for further characterization.
Minimal information to provide in publications
- Protein name and full primary structure, by providing a NCBI (or UniProt) accession number and cloning pathway, i.e. the source of the DNA (species), expression vector and host strain, including the tags and cleavage sites used, accompanied by the full amino acid sequence of the final protein, or sufficient details to derive the full amino acid sequence of the final protein.
- Protein concentration (specifying the method used for quantification and the molar extinction coefficient at 280 nm, if applicable).
- Storage conditions, i.e. final buffer composition (pH, buffers, salts and additives), storage temperature and, where applicable, freezing or lyophilization conditions.
Minimal quality control parameters that should be tested on protein sample
- Purity: checked by SDS-PAGE, Capillary Electrophoresis (CE) or Reversed-Phase-HPLC (RP-HPLC).
- Homogeneity (aggregation state): checked preferably by Size Exclusion Chromatography (SEC) and/or Dynamic Light Scattering (DLS) or by Size Exclusion Chromatography in combination with Multi Angle Light Scattering (SEC-MALS), Field Flow Fractionation (FFF) or Field Flow Fractionation in combination with Multi Angle Light Scattering (FFF-MALS) or Analytical Ultracentrifugation (AUC).
- Identity: checked preferably by intact protein mass or by peptide mass fingerprint or Edman sequencing.
Extended quality control parameters
Depending on the intended use and in addition to the methods listed above:
- General quality test by UV spectrum between 200 nm and 340 nm to check nucleic acid content and general protein fitness/quality Mandatory if protein binds nucleic acid.
- Conformational stability/folding state: Circular Dichroism (CD), Differential Scanning Calorimetry (DSC), NMR, Fourier Transform InfraRed (FTIR).
- Homogeneity: analytical Ion Exchange Chromatography (IEX), analytical Hydrophobic Interaction Chromatography (HIC) or Isoelectric Focusing (IEF).
- Protein competent fraction, i.e. the relative amount of functionally active protein, measured as specific activity, by active site titration or other suitable methods.
- Optimization of storage conditions: long term stability, activity assay, thermal shift assay.
- Batch-to-batch consistency: use some of the methods listed above. Mandatory if more than one batch is used.