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Self-processing of a barley subtilase expressed in E. coli.

Authors
  • Plattner, Stephan1
  • Gruber, Clemens2
  • Altmann, Friedrich3
  • Bohlmann, Holger4
  • 1 Division of Plant Protection, Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: [email protected] , (Austria)
  • 2 Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: [email protected] , (Austria)
  • 3 Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: [email protected] , (Austria)
  • 4 Division of Plant Protection, Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: [email protected] , (Austria)
Type
Published Article
Journal
Protein Expression and Purification
Publisher
Elsevier
Publication Date
Sep 01, 2014
Volume
101
Pages
76–83
Identifiers
DOI: 10.1016/j.pep.2014.05.014
PMID: 24927642
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

The barley protease BAJ93208 belongs to the subtilase family of serine proteases. We have expressed BAJ93208 in the cytoplasm of the Escherichiacoli strain SHuffle C3030 using a rhamnose-inducible promoter. The expression construct included a (His)6-tag at the N-terminus and a strep-tag at the C-terminus. Western blot analysis revealed that the protein was processed at the N- and C-terminus. To exclude that this processing was due to contaminating E. coli proteases, a mutated BAJ93208 protease was constructed. This inactive mutant was not processed, demonstrating that the processing was an autocatalytic process. To define the exact cleavage sites mass spectrometry was used which detected four differently processed versions of the protease. At the N-terminus, the self-processing removed the internal inhibitor and an additional 19 amino acids. At the C-terminus there was a cleavage site after Ala(765) which also removed the strep-tag. This explained the inability to detect the purified (His)6-BAJ93208-strep protease with an anti-strep-tag antibody. Finally, an additional alanine was removed either at the N-terminus (Ala(119)) or at the C-terminus (Ala(764)). Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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