Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells

Tobacco etch virus protease (TEVp) is a unique endopeptidase with stringent substrate specificity. TEVp has been widely used as a purified protein for in vitro applications, but also as a biological tool directly expressing it in living cells. To adapt the protease to diverse applications, several TEVp mutants with different stability and enzymatic properties have been reported. Herein we describe the development of a novel engineered TEVp mutant designed to be active in the secretory pathway. While wild type TEVp targeted to the secretory pathway of mammalian cells is synthetized as an N-glycosylated and catalytically inactive enzyme, a TEVp mutant with selected mutations at two verified N-glycosylation sites and at an exposed cysteine was highly efficient. This mutant was very active in the endoplasmic reticulum (ER) of living cells and can be used as a biotechnological tool to cleave proteins within the secretory pathway. As an immediate practical application we report the expression of a complete functional monoclonal antibody expressed from a single polypeptide, which was cleaved by our TEVp mutant into the two antibody chains and secreted as an assembled and functional molecule. In addition, we show active TEVp mutants lacking auto-cleavage activity. (C) 2015 The Authors. Published by Elsevier B.V.