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

• Published in: Protein engineering, design and selection design and selection, 2008-11, Vol.21 (11), p.653-658
• Main Authors: Gasanov, E.V., Demidyuk, I.V., Shubin, A.V., Kozlovskiy, V.I., Leonova, O.G., Kostrov, S.V.
• Format: Article
• Language: English
• Subjects: activation; Animals; Bacillus - enzymology; Bacillus - genetics; Bacillus - metabolism; Bacillus intermedius; Cells, Cultured; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; glutamyl endopeptidase; precursor maturation; Protein Renaturation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Serine Endopeptidases - chemistry; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; Substrate Specificity
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzn044

Glutamyl endopeptidase from Bacillus intermedius (BIGEP) is a secretory serine proteinase specifically hydrolyzing peptide bonds involving α-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.