TEV protease cleavage in generation of artificial substrate proteins bearing neo-N-termini.

The tobacco etch virus (TEV) protease is widely used in in vitro and in vivo approaches for the removal of affinity tags from fusion proteins or the generation of proteins with a desired N-terminal amino acid. Processing of fusion proteins by the TEV protease can either be achieved by encoding the TEV protease and its recognition site on one construct (self-cleavage) or on two different constructs (co-expression). Here, we compare the efficiency of the self-splitting approach to the co-expression approach.

Molecular determinants of protein half-life in chloroplasts with focus on the Clp protease system.

Proteolysis is an essential process to maintain cellular homeostasis. One pathway that mediates selective protein degradation and which is in principle conserved throughout the kingdoms of life is the N-degron pathway, formerly called the 'N-end rule'. In the cytosol of eukaryotes and prokaryotes, N-terminal residues can be major determinants of protein stability. While the eukaryotic N-degron pathway depends on the ubiquitin proteasome system, the prokaryotic counterpart is driven by the Clp protease system. Plant chloroplasts also contain such a protease network, which suggests that they might harbor an organelle specific N-degron pathway similar to the prokaryotic one. Recent discoveries indicate that the N-terminal region of proteins affects their stability in chloroplasts and provides support for a Clp-mediated entry point in an N-degron pathway in plastids. This review discusses structure, function and specificity of the chloroplast Clp system, outlines experimental approaches to test for an N-degron pathway in chloroplasts, relates these aspects into general plastid proteostasis and highlights the importance of an understanding of plastid protein turnover.

Engineering Destabilizing N-Termini in Plastids.

Studying the stability of a protein dependent on its N-terminal residue requires a mechanism, which selectively exposes the amino acid at the N-terminus. Here, we describe the use of the tobacco etch virus (TEV) protease to generate a specific N-terminal amino acid in the stroma of the chloroplast. The established molecular reporter system further allows the quantification of the reporter protein half-life dependent on the identity of the N-terminal residue.