Vacuolar processing enzymes (VPEs) are cysteine proteases found in plants. They are localized in the vacuole, where they cleave peptide bonds after asparagine and aspartate amino acid residues. VPEs are synthesized as inactive precursors and are autocatalytically processed and activated at acidic pH. Such conditions are found in the vacuole, where VPEs are active. After autoactivation, VPEs can perform its function of processing other vacuolar proteins that are synthesized as inactive precursors. VPE is an important component of plant programmed cell death, because it has the ability of autocatalytic processing and activation of precursors of hydrolytic enzymes. VPE therefore acts as an initiator of this process.
The goal of this thesis was the expression of the vacuolar processing enzyme from the alga Chlamydomonas reinhardtii (CrVPE) in E. coli and characterization of its autocatalytic processing and activity. We hypothesized that CrVPE would autoactivate at acidic pH (below 5) and cleave peptide substrates Ac-ESEN-AMC and Z-YVAD-AFC, which are two of the characteristics of vacuolar processing enzymes.
We prepared five CrVPE constructs. Wild type CrVPE and its inactive mutant were expressed in the soluble fraction, but we were not able to isolate them, because they degraded during the process of isolation. We did not detect any relevant activity on substrates Ac-ESEN-AMC and Z-YVAD-AFC in the soluble fraction of E. coli cell lysate after expression of CrVPE; we also did not observe any autocatalytic processing. We therefore concluded that the expressed CrVPE was not active. In addition to the wild type CrVPE and its inactive mutant, we also prepared three shortened variants of CrVPE lacking the N- and/or C-terminal prodomain. We found that the mature CrVPE without the N- and C-terminal prodomain and CrVPE without the N-terminal prodomain are accumulated in inclusion bodies. We speculate that the N-terminal prodomain is important for the correct folding of CrVPE and thus functions as a chaperone.
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