Cysteine endopeptidase CEP2 from the green alga Chlamydomonas reinhardtii belongs to papain-like cysteine proteases (PLCPs). In plants, PLCPs are associated with regulated cell death, also called programmed cell death (PCD) under physiological conditions (such as plant development). A subfamily of PLCPs from the model plant Arabidopsis thaliana, RD21, are particularly important enzymes involved in PCD, as they initiate PCD and mediate the plant-pathogen immune response. Cysteine endopeptidase CEP2 from C. reinhardtii shares many similarities with members of the RD21B subfamily of enzymes, which leads us to believe it may have a similar role in plant cell death as RD21B enzymes.
Firstly, in order to more easily predict biochemical properties of CEP2, we preformed a bioinformatic analysis of the protease. Based on its amino acid sequence and the use of an internet tool we prepared a model tertiary structure of the protein. We also compared the sequences of CEP2 and RD21B, respectively, and used that knowledge to predict the role and position of specific parts of the sequence and the domains in the polypeptide chain.
Our laboratory work began with the preparation of the recombinant protein CEP2 in Escherichia coli strain Rosetta-gami 2. This was followed by protein isolation and subsequent analysis of the protein's activation in the presence of a mildly acidic buffer. We noticed the protein gets processed during expression and isolation and that it is similarly active with and without incubation in a buffer with a lower pH value. Subsequenly, we determined the protein's acitivity pH profile which helped us establish the pH value for the protein's optimal activity which is slighly acidic, at 5.0. Lastly, based on results from an experiment during which the protein's autocatalytic activity was inhibited, we predict that the activation happens by means of another protease rather than by autoprocessing. The identitiy of said protease, which is present in the E. coli lysate, remains to be elucidated.
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