Abstract
Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity- and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9- and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture.
Highlights
The broad-spectrum physiological function of proteases includes maturation of proteins and their ultimate destruction [1]
To detect the AprBp and GseBp serine proteases expressed in B. pumilus and by the LIKE expression system in B. subtilis, specific proteotypic peptides and their fragment ions must primarily be selected
Label - free quantification does not reduce popularity implementation of this methodology has become possible considering the availability of software tools for automatic peptide spectra prediction, as well as the optimization of chromatography-mass spectrometry and instrument tuning, in order to achieve the highest sensitivity, reproducibility and throughput for hundreds or thousands of samples
Summary
The broad-spectrum physiological function of proteases includes maturation of proteins and their ultimate destruction [1]. Due to their panoramic applications, proteases have become the most important industrial enzymes involved in nearly 60% of the total worldwide enzyme sales [2,3]. Different expression systems have been developed for B. subtilis, which facilitate well-controlled protein production and secretion [4,5]. These systems ensure strict gene regulation using inducible promoters, such as xylose-inducible or isopropyl β-galactopyranoside (IPTG)-inducible or autoinducible promoters; a typical example being the SigB-dependent promoter [6,7,8,9,10,11]. PliaI is induced by a wide range of compounds including antibiotics (bacitracin, vancomycin and nisin), as well as different stress conditions, such as alkaline shock and stationary phase of the bacterial life cycle
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