BACKGROUND: Proteins are used as reagents in a broad range of scientific fields. The reliability and reproducibility of experimental data will largely depend on the quality of the (recombinant) proteins and, consequently, these should undergo thorough structural and functional controls. Depending on the downstream application and the biochemical characteristics of the protein, different sets of specific features will need to be checked. RESULTS: A number of examples, representative of recurrent issues and previously published strategies, has been reported that illustrate real cases of recombinant protein production in which careful strategy design at the start of the project combined with quality controls throughout the production process was imperative to obtain high-quality samples compatible with the planned downstream applications. Some proteins possess intrinsic properties (e.g., prone to aggregation, rich in cysteines, or a high affinity for nucleic acids) that require certain precautions during the expression and purification process. For other proteins, the downstream application might demand specific conditions, such as for proteins intended for animal use that need to be endotoxin-free. CONCLUSIONS: This review has been designed to act as a practical reference list for researchers who wish to produce and evaluate recombinant proteins with certain specific requirements or that need particular care for their preparation and storage.

AiCuris Anti Infective Cures AG Friedrich Ebert Str 475 42117 Wuppertal Germany

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 12840 Prague Czech Republic

European Molecular Biology Laboratory Meyerhofstrasse 1 69117 Heidelberg Germany

Evvivax Biotech Via di Castel Romano 100 00128 Rome Italy

Lab of Environmental and Life Sciences University of Nova Gorica Vipavska Cesta 13 5000 Rožna Dolina Nova Gorica Slovenia

Protein Purification Facility The Wolfson Centre for Applied Structural Biology The Hebrew University of Jerusalem 91904 Jerusalem Israel

Takis Biotech Via di Castel Romano 100 00128 Rome Italy

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