Cesaratto Francesca, López-Requena Alejandro, Burrone Oscar R, Petris Gianluca
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy.
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.
J Biotechnol. 2015 Oct 20;212:159-66. doi: 10.1016/j.jbiotec.2015.08.026. Epub 2015 Aug 30.
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.
烟草蚀纹病毒蛋白酶（TEVp）是一种具有严格底物特异性的独特内肽酶。TEVp不仅作为纯化蛋白被广泛用于体外应用，还作为一种可在活细胞中直接表达的生物学工具。为使该蛋白酶适用于多种应用，已报道了几种具有不同稳定性和酶学性质的TEVp突变体。在此，我们描述了一种新型工程化TEVp突变体的研发，该突变体设计为在分泌途径中具有活性。虽然靶向哺乳动物细胞分泌途径的野生型TEVp被合成为一种N-糖基化且无催化活性的酶，但在两个已证实的N-糖基化位点和一个暴露的半胱氨酸处具有选定突变的TEVp突变体效率很高。该突变体在活细胞的内质网（ER）中非常活跃，可作为一种生物技术工具用于切割分泌途径中的蛋白质。作为直接的实际应用，我们报道了一种由单一多肽表达的完整功能性单克隆抗体的表达，该抗体被我们的TEVp突变体切割成两条抗体链，并作为组装好的功能性分子分泌出来。此外，我们展示了缺乏自我切割活性的活性TEVp突变体。
