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How to remove the tag in the reconstructed protein?

Reconstructed protein are commonly fusion protein with tag(s) which helps purification or to enhance solubility. Generally speaking these tags will be barely used after the purified protein is harvested. Furth.er more, these fusion tags may result in changes in protein conformation, loss or alternation of biological activity and introduce in toxicity. For these reasons, it may be advisable to remove the tag(s) from the protein after purification.
When designing a fusion protein, how the tag(s) shall be removed must be carefully considered. The proteases are divided into 2 groups, exoprotease and endoprotease, depending on the location of cutting. As a consequence exprotease cut protein one aa by one aa while endoprotease cut the protein into 2 (or more) fragments. Another specification is the cutting terminal, some enzyme performs cutting activity only on the N-terminal, while others may cut from C-terminal or on both side. Here we have a list shows some of the commonly used cleavage site and cutting enzymes.

Protease

Cleavage site

Location and residue

pH

Inhibitor

Salt sensitivity

Chaotrope sensitivity

Enzyme-to-protein ratio

Endoprotease

Enteropeptidase (EK)

DDDDK↓

N-, none

C-, DDDDK

5.5 – 8.5

Reducing agents

 

 

0.1% (wt/wt)

Thrombin

LVPR↓GS

N-, GS

C-, LVPR

6 - 9

Reducing agents

≤ 0.15M

≤ 0.1M urea

1 – 10% (wt/wt)

Factor Xa

LVPR↓GS

N-, none

C-, IEGR

6 - 9

Reducing agents, chelating agents, phosphate ions.

≤ 0.15M

≤ 0.1M urea

1 – 10% (wt/wt)

TEV Protease

ENLYFQ↓(G/S)

N-, G/S

C-, ENLYFQ

5.5 – 8.5

Thiol alkylating agents

≤ 0.1M

2M urea

1 – 3% (wt/wt)

Rhinovirus 3C Protease          

LEVLFQ↓GP

N-, GP

C-, LEVLFQ

5.5 – 8.5

Thiol alkylating agents

 

 

 

SUMO Protease

SUMO↓X

N- only, no residue

6 - 9

 

≤0.5M

≤ 0.1M GnCl

≤ 2M urea

0.1% (wt/wt)

Immoblized subtilisin (BPN)

Propeptide↓X

N- only, no residue

7.2

 

 

≤ 4M urea

1:1 (mol/mol)

Exoprotease

Carboxypeptidase A

 

C-terminal amino acids except Pro, Lys and Arg.

 

Reducing agents,

chelating agents

 

 

 

Carboxypeptidase B

 

C-terminal Lys and Arg.

 

Reducing agents,

chelating agents

 

 

 

DAPase           

 

N-terminal dipeptides

 

Reducing agents,

thiol alkylating agents.

 

 

 

 

These 2 groups of protease are often used in tag removal but the difference is significant. Exoprotease is not used as frequent as endoprotease. The reason is due to the advantages of adding tag on the N-terminal of the protein like enteropeptidase and TEV protease can remove N-terminal affinity tags without leaving behind any nonnative residues. In some cases there are compelling reasons to add a tag to the C-terminus of a recombinant protein: C-terminal tag is less likely to interfere with the removal of signal peptides and the secretion of proteins. For example, TEV tag is added to the C-terminal and cut by TEVease, result in 6 aa residues remaining. Carboxypeptidase can be used to remove the residues, which lead to a total cleavage of the tag.
Endoprotease is very well developed and much more widely used for our bench works. 2 concerns arise when choosing tag removal strategy: (1) how to remove the protease after digestion, (2) non-specific cutting on the reconstructed protein. Since the tag removal method has been well developed, more and more commercial products have been launched to solve these concerns.
The first concern is straightforward to solve, either by adding additional chromatography step or by immobilize the enzyme to a fixed support. SUMO tag combined with his tag has been well recognized by the researchers, it allows easy removal of the enzyme by metal chelate chromatography or beads. 
The second concern requires more experience and test thus more difficult to solve. A significant amount of factors are influencing the accuracy of cleavage: the enzyme-to-substrate ratio (lower is better), temperature, pH, salt concentration, length of exposure, etc. TEV protease, thrombin and EK all have clearly defined cutting site, but they have all been reported to have unexpected cutting of the target protein in some instances. Some solutions have been already taken to avoid randomized cutting. TEV protease has been re-engineered to increase its specificity (and stability), resulting in AcTEV (Invitrogen) and ProTEV (Promega). FXa is recommended to use at pH 6.5 to minimize non-specific cleavage.
One last concern shall be mentioned as well. Normally the protein shall be dissolved in neutral buffer, but different condition have to be tested in case of unexpected situation occurs. These factors will be taken into consideration: pH, salt level, chaotropes or detergents. The most robust of the enzymes cited appear to be the SUMO proteases, the Profinity enzyme, and the TEV protease. Thrombin, fXa, and EK are much more sensitive to high salt concentrations or to the presence of chaotropes or reducing agents.
Each protein is unique, any protein expression project shall in the beginning be considered as test. We design and we test the design, in case any of the difficulties mentioned above happens, we will examine and provide more solutions. 

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  • Antibody
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