Protein Purification
Various of protein purification methods are widely used in the fields of biology and biomedical research. Many varialbes determines the expression and purification process of recombinant proteins, which includes but are not limited to the physical properties and biological functions of the proteins, and also whether bacteria or eukaryotic cell cell lines should be used to express the target proteins. Significant progress has been made in the field of recombinant protein expression and purification methods, while a large number of commercially available systems and reagent kits have emerged. However, proteins are complex macromolecules and the optimal protein expression and purification strategy must be determined based on experience.
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Key Factors in Protein Purification
Protein structure and function are the typical key factors to consider when selecting protein purification strategies. The biochemical or biological activity of recombinant proteins partially depends on the discontinuous domains within the protein, which typically depends on the secondary, tertiary, and quaternary structures of the proteins.
Protein folding, collectively referred as higher-order structures (HOS), is essential for maintaining the proper three-dimensional shape and function of proteins. In addition, protein solubility is a key attribute of successful protein purification and also influenced by many factors, including molecular size, N-terminal and C-terminal elements. Recombinant proteins typically contain N-terminal and C-terminal tags, which are short sequences used for immunohistochemical detection, purification and protein affinity chromatography, depending on the specific N-terminal and C-terminal tags and expected downstream applications.
Protein Purification Methods and Appplications
Regardless of the purpose of researchers, to study protein function or expand the scale of protein purification for downstream industrial production of biologics and pharmaceuticals by proper strategies, many protein purification methods, reagents, and tools are available. The selected protein purification method will partially determine the sample preparation process. Affinity chromatography would be a suitable initial purification step for purifying soluble recombinant proteins with related tags; However, unwanted proteins may also be binded to affinity resin columns and eluted together with the desired target proteins during the final elution process. If additional purification processes needed, supplementary purification strategies can be used, including exclusion chromatography or ion exchange chromatography. Importantly, as researchers may expect to remove any unnatural sequences from the final purified proteins, many affinity tags can be removed.
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