Protein Purification

Numerous protein purification methods are widely used in both biological and biomedical research. Recombinant protein expression and purification workflows depend on many variables. These variables include but are not limited to the physical properties and biological function of the protein, and whether a bacterial or eukaryotic cell line should be used to express the protein of interest. Significant advancements have been made in the area of recombinant protein expression  and purification methodology along with a plethora of commercially available systems and kits. However, proteins are complex macromolecules, and optimal protein expression and purification strategies must be determined empirically.  

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Critical Factors for Protein Purification

Protein structure and function are often critical factors to consider when selecting a protein purification strategy. Recombinant protein biochemical or biological activity is partially determined by discrete domains within the protein, of which are often reliant on the protein being folded into secondary, tertiary, and quaternary structures. 

Protein folding is collectively referred to as the higher-order structure (HOS) and is essential for the correct three-dimensional shape and function of the protein. Additionally, protein solubility is a highly desirable attribute for successful protein purification and is influenced by numerous factors including, size and N- and C-terminal elements. Recombinant proteins commonly incorporate N- and C-terminal tags, which are small sequences that are used for immunohistochemical detection and purification, or protein affinity chromatography, depending on the specific N- and C-terminal tag and intended downstream application.

Protein Purification Methods and Applications

Whether researchers aim to study protein function or seek to scale up protein purification using strategies for downstream, industrial-scale biologics and pharmaceutical production, there are numerous protein purification methods, reagents, and tools available. The selected protein purification method will partially determine the sample preparation workflow. Affinity chromatography is a suitable initial purification step for purifying solubilized recombinant proteins that contain relevant tags; however, unwanted proteins are likely to also bind to the affinity resin column and will elute in the final wash along with the desired protein of interest. If additional purification is necessary, supplementary purification strategies are employed including, size-exclusion chromatography or ion exchange chromatography. Importantly, many affinity tags can be removed as researchers may want to remove any non-native sequences from the final purified protein.

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