The use and misuse of protein tags to express and purify proteins of interest

Christian Salesse, Département d’ophtalmologie, Université Laval; Québec, Canada

Host: Cecília Roque, UCIBIO NOVA

ZOOM link: https://ucibio.pt/l/GuestSeminars

Abstract:

Studying proteins is a rather difficult task because getting a large enough amount of recombinant pure protein is very challenging, even more for difficult-to-express proteins. Many approaches are available to express proteins by use of different hosts (E. coli, P. Pastoris, mammalian cells, ) but prokaryotic hosts are still the most popular expression systems. Different strategies can be used to purify proteins such as using either their intrinsic properties or immobilized specific antibodies or ligands to a solid support. However, only a few proteins can be expressed and purified with these approaches. Hence, a large share of the currently purified proteins is expressed in fusion with a tag. Many different tags are available, such as small affinity tags as well as solubility-enhancing tags. The most commonly used small purification affinity tag is the PolyHistidines tag (see opposite Figure). Usually, a hexahistidine (His6) or even a decahistidine (His10) tag is fused to the N- or C-terminus of the protein of interest to allow its purification by immobilized metal affinity chromatography (IMAC). In contrast, solubility-enhancing tags are typically large proteins or protein domains that are highly expressed and very soluble. They can thus enhance the solubility of the protein of interest and even its expression when fused at the N-terminus (see opposite Figure). The most commonly used solubility-enhancing tags are the N-utilization substance A (NusA) and small ubiquitin-related modifier (SUMO), which however require an affinity tag for protein purification. Some solubility-enhancing tags may also be used as purification tags. Maltose-binding protein and glutathione S-transferase are the most largely used tags in this category. We have expressed and purified a significant number of proteins during the last 15 years where these approaches were extensively used. This presentation will thus provide examples where proteins were expressed and purified in fusion with a tag as well as other examples where difficulties were encountered mainly with the expression of a soluble form of proteins of interest in fusion with a tag.