Protein purification strategies must consider downstream applications and individual biological characteristics

• Published in: Microbial cell factories Vol. 21; no. 1; p. 52
• Main Authors: Remans, Kim, Lebendiker, Mario, Abreu, Celeste, Maffei, Mariano, Sellathurai, Shaila, May, Marina M, Vaněk, Ondřej, de Marco, Ario
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 07-04-2022; BioMed Central; BMC
• Subjects: Acids; Analysis; Animals; Antigens; Biochemical assays; Biochemical characteristics; Biochemistry; Chemical tests and reagents; Chromatography; Chromatography, Affinity; Composition; E coli; Endotoxins; Kinases; Methods; Nucleic acids; Properties; Protein functionality; Protein purification; Protein quality control; Proteins; Purification; Purification strategies; Reagents; Recombinant proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Reproducibility of Results; Structure-function relationships; Slovenia; Protein quality control; Protein functionality; Recombinant proteins; Purification strategies
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-022-01778-5

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.
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.
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.