Fusion protein tag to improve solubility and purification of proteins
Recoverin is a protein known to undergo a conformational switch when it binds calcium (Desmeules et al., 2006; Calvez et al., 2016). 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).
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 having research, industrial, or therapeutic applications, the team of Prof Salesse has developed a highly soluble protein tag enabling single-step purification of recombinant proteins. This new protein fusion tool is composed of a cloning vector into which a target protein gene can be fused to either TagR (single recoverin moiety) or 2XTagR (tandem copies of recoverin; patent pending).
Expressed at very high level in Escherichia coli (yields in excess of 30 mg/L of culture), TagR- and 2XTagR-fused 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). Recoverin as a fusion protein tag to improve expression, solubility and purification of proteins. WO2017011909A1. 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.
SOVAR and Université Laval seek a partner for co-development or commercialization of this technology, determined to be more efficient than widely used tags such as glutathione-S-transferase (GST) or maltose-binding protein (MBP).
The laboratory of Prof Salesse possesses a collection of TagR and 2XTagR cloning vectors.