Protein purification

The expression and purification of recombinant proteins facilitates production and detailed characterization of virtually any protein. Although a wide variety of heterologous expression systems have been developed and are currently used to produce recombinant proteins, the purification of the proteins obtained can still be problematic.

Classical purification procedures can be employed, but in most cases recombinant DNA techniques permit the construction of fusion proteins in which specific affinity tags are added to the protein sequence of interest; the use of these affinity tags simplifies the purification of the recombinant fusion proteins by employing affinity chromatography methods. Ideally a tag should be small, and have a minimal effect on the structure, activity, and properties of the recombinant protein. Different affinity tags have different sizes and properties. The 6xHis tag has a size of just 0.84 kDa, compared to 26 kDa for the glutathione S-transferase tag, 30 kDa for protein A, and 40 kDa for maltose-binding protein.

The FLAG tag consists of just 8 amino acids, but is highly immunogenic, which means that the FLAG tag must be removed before a recombinant protein can be used to produce antibodies. In contrast, the His tag has extremely low immunogenicity and rarely interferes with protein structure or function, making His-tagged proteins suitable for all kinds of downstream applications without cleaving the tag.