Proteases site-specific modifications

The simplest modification is to place a destabilizing amino acid at the N-terminal end according to the N-end rule (Varshavsky, 1992). This can be done by expressing the protein as a fusion protein, e.g. with maltose binding protein behind a cleavage site specific for a rare-cutting protease, such as factor Xq. In this case the N-terminal amino acid can be any amino acid except proline. 

A buccal drug delivery system is applied to a specific area on the buccal membrane. Moreover, the delivery system ean be designed to be unidirectional in drug release so that it can be protected from the loeal environment of the oral cavity. It also permits the inclusion of a permeation enhancer/protease inhibitor or pH modifier in the formulation to modulate the membrane or the tablet-mucosal environment at that particular application site. While the irritation is limited to the well-defined area, the systemic toxicity of these enhancers/inhibitors and modifiers can be reduced. The buccal mucosa is well suited for this type of modification as it is less prone to irreversible damage [9]. In the event of drug toxicity, delivery can be terminated promptly by removal of the dosage form. 

A slightly modified approach was applied in the identification of protein complexes in Schizosaccharomyces pombe [120]. The major modification involves the use of a tandem affinity purification [121]. To a protein of interest, an epitope is appended that contains two different affinity tags separated by a specific protease cleavage site. This enables isolation of the complex under milder conditions. 

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