1.2.2 Constructions
Con 4
Con4：a fusion protein where a solubility and folding partner is fused N-terminal to the target protein (e.g., SUMO, sortase A). An affinity tag at the N-terminus is required for purification. SUMO has emerged as an alternative for the production, solubility and correct folding of otherwise intractable proteins. The SUMO tag can be removed using a specific protease (e.g., the yeast SUMO protease-1 Ulp1) that recognizes the conformation of the ubiquitin partner rather than a specific sequence. The use of SUMO is mostly constrained to E. coli, since highly conserved SUMO proteases are present in eukaryotes.
1 Background 2 Tandem Affinity Tag 3 Self-cleaving purification tags 4 Conclusions and Inspiration
1 Background
1.1
1.2 Affinity Tags
Traditionally, a purification tag has implied use of an affinity separation method. With these methods, the affinity tag is expressed as a fusion partner with the desired target. The tag binds strongly and selectively to an immobilized ligand on a solid support, and cell and process contaminants are washed away. Affinity chromatography typically yields purities >90% in a single column step.
1.2.2 Constructions
Con 2
Con2：aid solubility and folding like maltose-binding protein (MBP) , glutathione Stransferase (GST) or small ubiquitin modifying protein (SUMO), thioredoxin (Trx), among others. Some tags such as MBP or GST are used for both affinity purification and solubility. GST binding glutathione-Sepharose resin with the slow binding kinetics is time consuming. MBP can be purified on amylose but may result in protein degradation. His tag both native and denaturing conditions can be used during purification
1.2.1 Common Affinity Tags
6H IMAC (immobilized metal-ion affinity chromatography) Imidazole WSHPQFEK (DYKDDDDK) EDTA
(链亲和素)
SLAELLNAGLGGS and TKDPSRVG
mild condition good for complexes
1.2.2 Constructions
Fig. 1. Diffecorporate and remove affinity tags.
1.2.2 Constructions
Con 1
Con1：a linker region including a specific sequence for endoprotease cleavage increase accessibility of the affinity tag and is often required for effective endoprotease cleavage
1.2.2 Constructions
Con 3
Con3：the simplest genetic design-a fusion protein designed for exopeptidase removal of the tag (only for N-terminal tags, using TAGZyme)
1.2.2 Benefits
• • • • • (i) improve protein yield (ii) prevent proteolysis (iii) facilitate protein refolding (iv)protect the antigenicity of the fusion protein (v)increase solubility