Fusion tag for increasing protein solubility and facilitate purification
The overexpression of recombinant proteins often results in the formation of inclusion bodies made of insoluble proteins which complicate the large scale production of proteins for therapeutic purposes.
To improve the solubility and facilitate the purification of "difficult-to-solubilize" proteins, the team of Prof Christian Salesse has developed a highly soluble protein tag enabling single-step purification of recombinant proteins. This new protein fusion tool, TagR (single recoverin moiety) or 2XTagR (tandem copies of recoverin), is available within cloning vectors into which a target protein gene can be fused. Recoverin is a protein known to undergo a conformational switch when it binds calcium. In the calcium-free state, the hydrophobic residues of the N-terminus of recoverin are sequestered within a deep hydrophobic pocket but, upon calcium binding, they become available for binding to hydrophobic structures (membranes or chromatography resins).
In Escherichia coli, yields of TagR- and 2XTagR-fused proteins can exceed 30 mg/L and these proteins can be purified very efficiently in a single elution step. Alternatively, after a few wash(es), in situ (in-column) protease treatment can enable the release and elution of the highly purified recombinant protein, in the absence or presence of detergent.
Salesse C and L Cantin (2017-2018). Recoverin as a fusion protein tag to improve expression, solubility and purification of proteins. WO2017011909A1, CA2991804A1, US20180208634A1, EP3325627A1, JP2018521690A. Assignee: Université Laval.
Calvez P, TF Schmidt, L Cantin, K Klinker, and C Salesse (2016). Phosphatidylserine allows observation of the calcium-myristoyl switch of recoverin and its preferential binding. J Am Chem Soc 138: 13533-13540.
Desmeules P, S-É Penney, and C Salesse (2006). Single-step purification of myristoylated and nonmyristoylated recoverin and substrate dependence of myristoylation level. Anal Biochem 349: 25-32.
During development, TagR was shown to be more efficient than widely used tags such as glutathione-S-transferase (GST) or maltose-binding protein (MBP).
SOVAR and Université Laval seek a partner for co-development or commercialization of this technology.
The laboratory of Prof Salesse has constructed a series of TagR and 2XTagR cloning vectors.