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.
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy.
Francesca
F
Cesaratto
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy; Immunobiology Division, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba.
Alejandro
A
López-Requena
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy.
Oscar R
OR
Burrone
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy. Electronic address: burrone@icgeb.org.
Gianluca
G
Petris
D000911:Antibodies, Monoclonal / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D055571:Secretory Pathway
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010455:Peptides; D010450:Endopeptidases; C099488:TEV protease
ER; ERAD; Glycosylation; Sec-TEV; Secretory pathway; TEV protease