Introduction

The invention is an engineered protease, called a single-TEV, whose highly specific proteolytic activity is chemically inducible by the experimenter in several subcellular compartments.

Unica-TEV, when expressed in viable cells, allows to control: i) protein ripening; ii) intracellular signal pathways; iii) gene expression.

Unica-TEV can also be used for the purification of recombinant proteins.

Technical features

Unica-TEV is obtained by inserting an artificial regulatory domain within a protease amino acid chain. It overcomes the disadvantages of the systems known today. The strategy used allows, in fact, to create an inducible protease, having a single peptide chain. This solution makes unica-TEV no longer subject to variable stoichiometry as, instead, occurs in the approaches used up to now based on protein complementation assay (PCA).

In this way the proteolytic response is much less heterogeneous, with a reduced background activity. Unica-TEV is able to reach localized substrates in subcellular compartments, difficult to achieve with a PCA-based approaches. Unica-TEV is able to cut any protein into which the TEV recognition site is inserted (TEVcs, 6 amino acids).

The insertion of TEVcs in the proBDNF sequence, by replacing the site of recognition of furin/plasmin, ensures the maturation of BNDF under the control of the experimenter, through the use of unique-TEV, and release from endogenous protease regulation. Unica-TEV can also be used to control gene expression.

Possible Applications

• Control of proteolytic activity in vital cells;
• Control of gene expression;
• Control of the maturation of pro-proteins;
• Control of intracellular signalling routes;
• Purification of proteins;
• Removal of Affinity Tags;
• Therapeutic applications;
• Innovative biotechnology approaches.

Advantages

• High sequence specificity;
• Single peptide chain;
• High cell tolerability;
• Activity independent of pH;
• Independence from cofactors and second messengers;
• Complete control of activity;
• Low activity Background;
• Stoichiometry not variable;
• Activities in subcellular compartments.