title: Self-processing of a barley subtilase expressed in E. coli.
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)).
authors.affiliation: Division of Plant Protection, Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: stephan.plattner@gmail.com.
authors.forename: Stephan
authors.initials: S
authors.lastname: Plattner
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: clemens.gruber@boku.ac.at.
authors.forename: Clemens
authors.initials: C
authors.lastname: Gruber
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: friedrich.altmann@boku.ac.at.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Division of Plant Protection, Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: holger.bohlmann@boku.ac.at.
authors.forename: Holger
authors.initials: H
authors.lastname: Bohlmann
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D020224:Expressed Sequence Tags; D015870:Gene Expression; D001467:Hordeum / Q000201:enzymology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D013381:Subtilisins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D013381:Subtilisins; C119297:subtilase, plant
keywords: Proprotein; Subtilase; Thionin