Protein Purification
Numerous protein purification techniques are widely utilized in both biological and biomedical research. The workflows involved in recombinant protein expression and purification rely on many factors and variables, including, but not limited to, the physical properties, biological function of the protein, and the decision of whether to use a bacterial or eukaryotic cell line for expressing the protein of interest. Significant advancements have been made in the field of recombinant protein expression and purification methodologies, alongside the availability of a wide range of commercially available systems and kits. However, proteins are complex macromolecules, and as such, optimal strategies for protein expression and purification must often be determined empirically, based on the specific characteristics of the target protein and the desired outcome,
Overview
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 purified protein.