Fusion Tags for Protein Expression and Purification - Fusion tags can improve the yield and solubility of many recombinant proteins. Of course, no single tag or cleavage method will answer every need

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Fusion Tags for Protein Expression and Purification
Fusion tags can improve the yield and solubility of many recombinant proteins. Of course, no single tag or cleavage method will answer every need.


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Proteases


Table 2. Proteases commonly used for tag removal
An integral part of the choice of a fusion tag is the choice of the method for removing the tag after purification. This step almost always involves using a protease to cleave a specific peptide bond between the tag and the protein of interest. A small number of highly specific proteases are routinely used for this purpose and are listed in Table 2. These include the tobacco etch virus (TEV) protease; thrombin (factor IIa, fIIa) and factor Xa (fXa) from the blood coagulation cascade; an enzyme involved in the cleavage or activation of trypsin in the mammalian intestinal tract, enterokinase (EK); proteases involved in the maturation and deconjugation of SUMO, SUMO proteases (Ulp1, Senp2, and SUMOstar); and a relative newcomer to the field, a mutated form of the Bacillus subtilis protease, subtilisin BPN' (Bio-Rad's Profinity eXact system). Many of these enzymes have been genetically engineered to enhance their stability (e.g., AcTEV, ProTEV) or their specificity, (e.g. SUMOstar, Profinity). With the exception of the SUMO proteases, all of these enzymes have the potential to cleave within the protein of interest.12–13 The SUMO proteases recognize not only their specific cleavage site, xaa-Gly-Gly/yaa, but also the tertiary structure of SUMO itself, giving them a very high degree of specificity. Bryan, et al., have attempted to introduce the same level of specificity into the Profinity system by mutating both the subtilisin prodomain as well as the active site of subtilisin to increase the affinity of the enzyme for the prodomain and to decrease the likelihood of digestion within the protein of interest.14 One interesting consequence of this is that the affinity for the prodomain is so high that these researchers observed product inhibition of the enzyme. Essentially, the enzyme carries out one catalytic cycle and is then inhibited by the prodomain, which is retained in the active site, thus preventing further cleavage by this otherwise promiscuous enzyme. Because capture on the immobilized, mutant subtilisin matrix is an integral part of the system, the column must have a capacity (in moles of subtilisin) equimolar with the fusion protein. Although this is not problematic on the research scale, it could become prohibitively expensive at the multigram scale.

The principle concerns with using a protease for removing a tag are 1) removing the protease following digestion, and 2) non-specific digestion of the target protein by the protease. Resolving the first concern is relatively straightforward, although in most cases it involves an additional chromatography step. Recombinant forms of TEV and its variants and of the SUMO proteases are all produced with a hexahistidine (His6) tag, allowing easy removal of the enzyme by metal chelate chromatography. Alternatively, some of these enzymes have been immobilized on solid supports, allowing their removal by simple filtration or centrifugation steps. Thrombin, fXa, and EK, which generally are produced from natural sources, can be removed by affinity chromatography, for instance, on benzamidine-agarose. With the Profinity system, cleavage and separation from the enzyme are combined in a single step.

The second concern is more difficult to resolve. Non-specific cleavage is influenced by a number of parameters, such as the enzyme-to-substrate ratio (lower is better), temperature, pH, salt concentration, and length of exposure. TEV protease, thrombin, fXa, and EK all have well defined recognition sequences, but all of them have been found to cause "nicking" of the target protein in some instances. TEV protease has been re-engineered to try to increase its specificity (and stability), resulting in AcTEV (Invitrogen, Carlsbad, CA) and ProTEV (Promega, Madison, WI). Whether or not such engineering has reduced non-specific proteolysis remains to be seen. In addition, other tricks must be used with the native enzymes. For instance, one supplier recommends using fXa at pH 6.5, well below its pH optimum, to minimize non-specific cleavage. Of course, this requires the use of higher enzyme-to-substrate ratios and longer digestion times to achieve complete cleavage. Two of the enzymes listed (SUMO proteases and the Profinity enzyme) seem to be immune to this problem. SUMO proteases have evolved to recognize both the tertiary structure of SUMO as well as the cleavage sequence, xaa-Gly-Gly/yaa. The Profinity enzyme has been extensively mutated to derive a version that has very high affinity for the prodomain of the original enzyme. Thus, it also recognizes the tertiary structure of the prodomain as well as the cleavage sequence Phe-Met-Ala-Lys/yaa. On the other hand, SUMO proteases act catalytically (i.e., with a low enzyme-to-substrate ratio) whereas the Profinity enzyme requires equimolar concentrations of enzyme and substrate.

One final consideration should be mentioned. Although one would ideally have a protein that is fully soluble in phosphate buffered saline at neutral pH, the reality is that for many proteins to be soluble at useful concentrations, they require more acidic or more basic pH levels, high or low salt levels, or the presence of chaotropes or detergents. It is therefore essential that the protease of choice retain substantial activity under adverse conditions. 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.


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