Size of the Fusion Tag
The size of the fusion tag plays a crucial role in the overall yield of the purified protein. As seen in Table 4, the yield
of the purified target protein is dictated by the yield of the fusion protein: the larger the fusion tag, the lesser the overall
yield. Split SUMO, which is 47 amino acids long, is only 19% of the fusion when Split SUMO is fused to a target protein that
is 200 amino acids in length (47/247). In contrast, NusA, which is 495 amino acids long, is 71% of the fusion with the same
target protein (495/695). Therefore, if expression yielded one gram of fusion protein for both Split SUMO–Target and NusA–Target,
the yield of the purified target after cleavage would be 0.810 g and 0.288 g, for Split SUMO and NusA, respectively.
Table 4. The size of the fusion tag influences the yield of the purified protein.
Specificity, Efficiency, and Site of Cleavage
The quality, quantity, and activity of the purified protein are influenced by the specificity, efficiency, and site of cleavage.
Cleavage of the fusion usually is necessary because the fusion interferes with the structural or functional properties of
the recombinant protein.57 Fusions can be cleaved by either chemical or enzymatic strategies.58,59 These methods include the use of engineered cleavage sites that are recognized by the proteases and are positioned between
the fusion tag and the protein target. Proteases that have been employed to cleave fusion tags include tobacco etch virus
(Tev) protease,60 factor Xa, thrombin protease,59 and the SUMO protease.12–16 Problems associated with proteolytic cleavage of fusion tags are low yield, precipitation of the protein of interest, labor-intensive
optimization of cleavage conditions, expense of proteases, failure to recover active, structurally intact protein,61 and the generation of non-native N-terminal amino acids (not the case with SUMO and ubiquitin fusions12–16 ). As a result, choosing the right chemical or enzymatic strategy is crucial for achieving active protein of high quality
Table 5. Abbreviations
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