title: BspK, a Serine Protease from the Predatory Bacterium Bdellovibrio bacteriovorus with Utility for Analysis of Therapeutic Antibodies.
abstract: The development of therapeutic and diagnostic antibodies is a rapidly growing field of research, being the fastest expanding group of products on the pharmaceutical market, and appropriate quality controls are crucial for their application. We have identified and characterized the serine protease termed BspK (Bdellovibrio serine protease K) from Bdellovibrio bacteriovorus and here show its activity on antibodies. Mutation of the serine residue at position 230 rendered the protease inactive. Further investigations of BspK enzymatic characteristics revealed autoproteolytic activity, resulting in numerous cleavage products. Two of the autoproteolytic cleavage sites in the BspK fusion protein were investigated in more detail and corresponded to cleavage after K28and K210in the N- and C-terminal parts of BspK, respectively. Further, BspK displayed stable enzymatic activity on IgG within the pH range of 6.0 to 9.5 and was inhibited in the presence of ZnCl2BspK demonstrated preferential hydrolysis of human IgG1 compared to other immunoglobulins and isotypes, with hydrolysis of the heavy chain at position K226generating two separate Fab fragments and an intact IgG Fc domain. Finally, we show that BspK preferentially cleaves its substrates C-terminally to lysines similar to the protease LysC. However, BspK displays a unique cleavage profile compared to several currently used proteases on the market.
  
  
  
  The rapid development of novel therapeutic antibodies is partly hindered by difficulties in assessing their quality and safety. The lack of tools and methods facilitating such quality controls obstructs and delays the process of product approval, eventually affecting the patients in need of treatment. These difficulties in product evaluations indicate a need for new and comprehensive tools for such analysis. Additionally, recent concerns raised regarding the limitations of established products on the market (e.g., trypsin) further highlight a general need for a larger array of proteases with novel cleavage profiles to meet current and future needs, within both the life science industry and the academic research community.
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences Lund, Lund University, Lund, Sweden eleni.bratanis@med.lu.se.
authors.forename: Eleni
authors.initials: E
authors.lastname: Bratanis
authors.affiliation: Proteomics Resource Center, The Rockefeller University, New York, New York, USA.
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.
authors.forename: Andreas
authors.initials: A
authors.lastname: Naegeli
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.
authors.forename: Mattias
authors.initials: M
authors.lastname: Collin
authors.affiliation: Division of Infection Medicine, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.
authors.forename: Rolf
authors.identifier: http://orcid.org/0000-0002-5974-0674
authors.initials: R
authors.lastname: Lood
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000627:therapeutic use*; D000070060:Bdellovibrio bacteriovorus / Q000235:genetics* / Q000378:metabolism*; D002712:Chlorides / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000378:metabolism*; D059748:Proteolysis; D057057:Serine Proteases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017967:Zinc Compounds / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D002712:Chlorides; D007074:Immunoglobulin G; D017967:Zinc Compounds; C016837:zinc chloride; D057057:Serine Proteases
keywords: Bdellovibrio bacteriovorus; autoproteolysis; biotechnology; immunoglobulins; serine protease; therapeutic antibodies