Applying Fusion Protein Technology to E. Coli - - BioPharm International

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Applying Fusion Protein Technology to E. Coli


BioPharm International


Recombinant proteins often are considered unwanted by cells and are subjected to proteolytic degradation.45 Several strategies have been developed to protect recombinant proteins from degradation, including the use of protease inhibitors,46 secretion into the periplasm or culture medium,47,48 and generating protective fusions.12,28,49-53 The compartmentalization hypothesis describes the mechanism by which gene fusions protect against proteolytic degradation.54 Fusions can promote the translocation of their partner proteins to different cellular compartments, thereby decreasing the concentration of the recombinant protein in the protease-rich cytosol. For example, SUMO can translocate from the cytosol to the nucleus and maltose binding protein (MBP) can translocate to the membrane compartment of the cell.55,56

Size of the Fusion Tag


Table 4. The Size of the Fusion Tag Influences the Yield of the Purified Protein
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 constrast, 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.

Specificity, Efficiency, and Site of Cleavage


Table 5. Abbreviations
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 and quantity.

John Hall is vice president of business development at LifeSensors, Inc., 271 Great Valley Parkway, Malvern PA 19355; 610.644.8845 x 305; Fax: 610.644.8616;

References

1. Panlou AK, Reichert JM. Recombinant protein therapeutics-success rates, market trends and values to 2010. Nature Biotechnol. 2004; 12:1513-9.

2. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990; 185:60-89.


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