A method for the quantitation of homologous endopeptidases AlpA and AlpB from Lysobacter sp. XL1. Issue 10 (October 2016)

• Record Type: Journal Article
• Authors: Karatovskaya, Anna P.; Rudenko, Natalia V.; Tsfasman, Irina M.; Guseva, Ksenya A.; Laman, Aleksandr G.; Boziev, Khanafi M.; Brovko, Fedor A.; Vasilyeva, Natalia V.
• Is Part Of: Process biochemistry. Volume 51:Issue 10(2016:Oct.)
• Page Start: 1521
• Page End: 1526
• Publication Date: 2016-10
• Subjects: Lysobacter -- Endopeptidases -- Preproproteins -- Monoclonal antibodies -- Monoclonal antibodies to peptides -- Sandwich enzyme immunoassay; EIA enzyme immunoassay -- mAbs monoclonal antibodies -- PBS phosphate-buffered saline -- PBST PBS containing 0.1% Tween 20; Biochemical engineering -- Periodicals; Biotechnology -- Periodicals; Biochemistry -- periodicals; Biotechnology -- periodicals; Chemical Engineering -- periodicals; Génie biochimique -- Périodiques; Biotechnologie -- Périodiques; Biochemical engineering; Biotechnology; Periodicals; 660.63
• DOI: 10.1016/j.procbio.2016.06.009
• Languages: English
• ISSNs: 1359-5113

Abstract

Graphical abstract: Highlights: Monoclonal antibodies to homologous proteases AlpA and AlpB were produced. A sandwich immunoassay for the quantitation of enzymes of Lysobacter sp. was developed. The detection ranges were 0.2–3.1 and 3–100 ng/ml for AlpA and AlpB, respectively. Abstract: Bacteriolytic homologous proteases AlpA and AlpB of Lysobacter sp. XL1 are the most active components of the antimicrobial lysoamidase complex. A sandwich enzyme immunoassay based on monoclonal antibodies was developed for the quantitation of these enzymes in complex protein mixtures. Non-cross-reactive monoclonal antibodies were produced using various methods of immunization with mature protein forms and synthetic peptide conjugates. A total of 15 monoclonal antibodies to AlpA, obtained after immunization with native protein, recognized the conformational epitopes, whereas six monoclonal antibodies to AlpB recognized the denatured form of the endopeptidase, and were obtained after immunization with peptide conjugates, whose amino acid sequences corresponded to AlpB mature form primary structure fragments different from AlpA. The developed method enables a highly sensitive assay of homologous proteases AlpA and AlpB with the detection ranges of 0.2–3.1 and 3–100 ng/ml for the native form of AlpA and the denatured form of AlpB at a 5% measurement error, respectively. The technique can be used to assess the production of AlpA and AlpB, quantitate these proteins in lysoamidase and study their secretion into the environment.