Investigation of Psychrophilic SUMO Proteases: Towards a More Efficient SUMO Protease at Lower Temperature

Abstract

Covalent attachment of the fusion protein, Small Ubiquitin-like modifier (SUMO), to recalcitrant target proteins (especially transmembrane proteins) make purification of the target protein much easier by enhancing its recombinant overexpression, protecting from degradation and improving protein folding, solubility and stability. After purification of the SUMO-tagged protein, SUMO protease (Ulp1), which specifically cleaves the SUMO-tag, is required to regenerate the native target protein. However, the known SUMO protease enzyme from Saccharomyces cerevisiae i.e., S.ce. Ulp1 enzyme is not very efficient, and the cleavage reaction requires many hours of incubation with the SUMO-tagged protein at room temperature or above for partial cleavage of the SUMO-tag. Under such conditions, many target proteins, especially the integral membrane proteins, get denatured and inactivated. Herein, we aim to provide a solution to this problem by discovering and producing psychrophilic (cold-active) SUMO proteases, which will deSUMOylate the SUMO-tagged protein efficiently at low temperature thus keeping the target protein stable and active. To reach this goal, we have successfully overexpressed and purified two novel psychrophilic SUMO proteases- C.ps. Ulp1 enzyme and M.an. Ulp1 enzyme from psychrophilic yeasts, identified through bioinformatics studies. Comparative activity studies performed with different SUMO-tagged proteins have suggested that M.an. Ulp1 has better activity than the S.ce. Ulp1. Further, bioinformatics tools have been utilized to explore the probable reasons for better activity observed for M.an. Ulp1 enzyme in certain cases compared to S.ce. Ulp1 enzyme.