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PEDS Advance Access originally published online on September 1, 2008
Protein Engineering Design and Selection 2008 21(11):653-658; doi:10.1093/protein/gzn044
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Hetero- and auto-activation of recombinant glutamyl endopeptidase from Bacillus intermedius

E.V. Gasanov1, I.V. Demidyuk1,4, A.V. Shubin1, V.I. Kozlovskiy2, O.G. Leonova3 and S.V. Kostrov1

1 Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182 2 Institute of Energy Problems for Chemical Physics (Branch), Russian Academy of Sciences, Chernogolovka, Moscow 142432 3 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119992, Russia

4 To whom correspondence should be addressed. E-mail: duk{at}img.ras.ru

Glutamyl endopeptidase from Bacillus intermedius (BIGEP) is a secretory serine proteinase specifically hydrolyzing peptide bonds involving {alpha}-carboxyl groups of glutamic and aspartic acids. In this work, different BIGEP forms (full-length precursor, precursor without signal peptide and mature part) were expressed in Escherichia coli and the process of enzyme maturation was studied in vitro. BIGEP precursor renaturation leads to autocatalytic hydrolysis of the propeptide at Glu(–16). At the same time, the enzyme activation requires the complete removal of the prosequence by other proteinases. The mature part of BIGEP cannot be activated, which indicates that the propeptide is required for the active protein formation. The data obtained allowed us to apply directed mutagenesis of the processing site to obtain a BIGEP form that matured autocatalytically. This approach makes it possible to produce the enzyme without extrinsic proteinases, which is a prerequisite for using it in limited hydrolysis of proteins and peptides.

Keywords: activation/gene expression/glutamyl endopeptidase/precursor maturation/protein renaturation

Received May 8, 2008; revised July 21, 2008; accepted July 29, 2008.


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