基于亲和层析的两种蛋白质纯化策略

A general drawback of endoprotease-mediated tag cleavage is the need for high ratios of enzyme to protein and long incubation time required for complete tag removal. An important limitation is that highly specific endopeptidases are typically expensive and unavailable in quantities. In addition, many of these proteases are highly bioactive (e.g. thrombin凝 血酶 and Factor Xa) and must be separated completely from the target protein, often by an additional purification step.
1.2.2 Constructions
Fig. 1. Different
strategies to incorporate 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 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
1.3 Tag removal methods
In many scenarios, the presence of a purification tag might alter the activity of the target protein, or cause a therapeutic protein to become immunogenic. Thus, tag removal is a crucial requirement for many of these methods. The following section reviews various tag removal strategies and how improvements in these methods might have an impact on future use.
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.
without requiring a specific sequence at the C-terminus of the cleavage site
a cysteine peptidase that sequentially removes dipeptides from the N-terminus until a stop position
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
Affinity tags have also been used to increase the sensitivity of binding assays for tagged ScFv (单链抗体single-chain antibody fragment, scFv)
1.2.3 Negative effects
1st step 2nd step
3rd step
The process is designed for the initial purification of the his-tag protein using IMAC; a tag removal step where a protease (or peptidase)—that also contains a his-tag— is added to the his-tag protein and a final subtractive IMAC purification. In this last step, process impurities such as unprocessed his-tag protein, the protease (or peptidase) and any unspecific binder are retained in the column while the detagged ‘pure’ protein is eluted.
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
Two variants of Affinity Tags for Purification: Tandem Affinity Tag & Self-cleaving purification tags
汇报人: 宋健
2015.06.03
Cell review-2013-Fong B.A.-The potential role of self-cleaving purification tags in commercial-scales process
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.
1.3.1 Proteases
Proteases are enzymes that cleave proteins at specific sequences and are therefore potential candidates to catalyze tag removal. Exopeptidases(aminopeptidases and carboxypeptidases) catalyze the removal of amino acids from the end of a protein and can be used for the removal of very small tags such as His6. Several affinity tagged process endoproteases are therefore used for tag removal such as Thrombin, virus-deriwk.baidu.comed TEV protease, 3C protease , Granzyme B and Caspase-6, used for the removal of His6, GST, FLAG.
• • • • • • (i) a change in protein conformation (ii)lower protein yields (iii) inhibition of enzyme activity (iv) alteration in biological activity (v)undesired flexibility in structural studies (vi)toxicity