Solubility-Enhancing Tags

Solubility-enhancing tags are generally large peptides or proteins that increase the expression and solubility of fusion proteins. Fusion tags like GST and MBP also act as affinity tags and as a result, they are very popular for protein purification. Other fusion tags like NusA, thioredoxin (TRX), small ubiquitin-like modifier (SUMO), and ubiquitin (Ub), on the other hand, require additional affinity tags for use in protein purification.

No single fusion tag can increase the expression and solubility of all target proteins. However, some fusion tags have been more successful than others in increasing the solubility of many proteins. A comparison of some popular fusion tags showed that large proteins like NusA and MBP are more effective in solubilizing proteins than the smaller affinity tags or GST.^4–7 Novel tags like Skip and T7 protein kinase, in turn, have been shown to be successful in expressing hard-to-express proteins in E. coli. ⁸ Similarly, ubiquitin-based tags have been used to increase the solubility and expression level of proteins. SUMO tags are emerging as a viable alternative for increasing both the expression and solubility of otherwise hard-to-express proteins.⁹ The SUMO tag can be cleanly excised using SUMO protease, which recognizes the conformation of SUMO protein rather than a specific sequence within SUMO. Initially, the SUMO system was confined to E. coli, as highly conserved SUMO proteases are present in eukaryotes that cleaved the SUMO tag. However, the recently developed SUMOstar tag, a modified version of SUMO, is not recognized by the native eukaryotic protease and is specifically cleaved by the genetically engineered SUMOstar protease. Thus, the SUMOstar system can be used effectively in both prokaryotic and eukaryotic systems. The usefulness of the SUMO system was substantiated by the study of Marblestone, et al., who examined the effects of various fusion partners on total and soluble expression yield.^9–10 They evaluated the expression and solubility of three model proteins fused to the C terminus of MBP, GST, TRX, NusA, Ub, and SUMO tags. The tags were ranked in terms of increased total expression as

and increased soluble expression as

SUMO ~ NusA > Ub ~ GST ~ MBP ~ TRX.

Overall, SUMO and NusA were equally good in terms of increasing the expression and the solubility of fusion proteins. However, SUMO offers certain advantages over NusA, in that SUMO is smaller and because it can be cleaved off precisely from the target protein without leaving behind any residues.

The Rainbow tag is yet another new development in tag fusion technology. This technology allows the continuous monitoring of correctly folded proteins throughout the process of expression and purification. The Xavin mononucleotide (FMN)-binding domain of cytochrome P450 reductase (displaying a blue-green or yellow color, depending on the oxidation state of the FMN cofactor) and the red colored, heme-binding cytochrome b5 are used as tags, and the rainbow tags are visible with the naked eye.¹¹ The use of rainbow tags, however, requires an additional affinity tag for purification.