Protein Quality Standard PQS
There is increasing awareness in the scientific community about the lack of reproducibility and reliability of results published with purified proteins. While protein production is highly regulated and controlled in the pharmaceutical industry by the authorities, there are up to date no guidelines or standards in place in the academic research to guarantee the quality of proteins included in scientific experiments. There is an urgent need to define guidelines for the scientists but also for editors and reviewers of scientific journals and funding agencies for their review processes:
A team of experts in the field of Biophysics (Association of Resources for Biophysical Research in Europe (ARBRE-MOBIEU) and Recombinant Protein Production (Production and Purification Partnership in Europe (P4EU) have developed the following Minimal Protein Quality Standard:
Members of the Protein Quality Initiative:
Bertrand Raynal – Pasteur institute, Paris, France (ARBRE)
Stefan Knauer – Bayreuth University, Germany (ARBRE)
Stephan Uebel – Max-Plank Institute for Biochemistry, Munich (ARBRE)
Rob Meijers – EMBL Hamburg, Germany (ARBRE)
Nick Berrow – Institute for research in Medicine, Barcelona, Spain (P4EU)
Kim Remans – EMBL Heidelberg, Germany (P4EU)
Ario de Marco – University of Nova Gorica, Slovenia (P4EU)
Mario Lebendiker (coordinator) – Hebrew University of Jerusalem, Israel (P4EU)
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 280nm, if applicable).
Storage conditions, i.e. final buffer composition (pH, buffers, salts and additives), storage temperature and, where applicable, freezing or lyophilization conditions.
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.
Depending on the intended use and in addition to the methods listed above:
General quality test by UV spectrum between 200nm and 340nm 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
The proposed guidelines are intended to lay the groundwork for the standardization and reproducibility of data. The goals of this document are to disseminate operative guidelines in our laboratories through the existing networks, to raise awareness amongst colleagues and collaborators and to encourage the whole scientific community to implement these guidelines in publications, e.g. as part of the supplementary information. The implementation of protein production and QC data will allow greater transparency to readers and enable efficient reproducibility in other laboratories.
In order to provide a statistical basis for that initiative and strengthen its implementation, a data collection is currently ongoing where we need your active support. To provide a representative statistic we aim to collect data on ~ 1,000 samples from 100 labs and strongly encourage you to participate. Data from 10 samples per lab would be ideal.
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