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Detection of propeptides of AlpA and AlpB lytic endopeptidases from Lysobacter sp. XL1 by the sandwich enzyme immunoassay based on monoclonal antibodies

Abstract

Extracellular lytic endopeptidases AlpA and AlpB of the Gram-negative bacterium Lysobacter sp. XL1 have a high degree of homology and are synthesized as preproproteins consisting of a signal peptide, a propeptide, and the mature protein. In the present work, two monoclonal antibodies against the AlpA propeptide (ProA) and eleven antibodies against the AlpB propeptide (ProB) have been obtained. The affinity constants for antibodies to ProA were 2.9 × 109 and 3.5 × 109 M−1, and those for antibodies to ProB were from 1.5 × 108 to 2.2 × 109 M−1. The antibodies showed no immune cross-reactivity with each other and with mature forms of the enzymes. On the basis of monoclonal antibodies, a sandwich enzyme immunoassay has been developed, which makes it possible to detect these propeptides in the dissolved native form. The linear range of the detection of ProA was 1.5–100.0 ng/mL with an error of measurement of 6%, and that of the determination of ProA was 0.2–6.25 ng/mL with an error of measurement of 6%. By using the assay, propeptides ProA and ProB were detected in cell lysates of Lysobacter sp. XL1 in an amount of 1.18 ± 0.03 and 0.096 ± 0.002 ng per 1 OD540 of bacterial culture, respectively. The immunochemical assay for the detection of different forms of AlpA and AlpB can be useful in solving the problems associated with their secretion into environment.

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Abbreviations

EIA:

enzyme immunoassay

MAb:

monoclonal antibody

MAb-bio:

biotinylated monoclonal antibodies

ORF:

open reading frame

PBS:

phosphate-buffered saline containing 1 mM KH2PO4, 9 mM Na2HPO4, 3 mM KCl, and 140 mM NaCl, pH 7.2

PBST:

PBS containing 0.1% Tween 20

ProA:

propetide of endopeptidase AlpA

ProB:

propetide of endopeptidase AlpB

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Correspondence to N. V. Rudenko.

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Original Russian Text © N.V. Rudenko, I.M. Tsfasman, O.R. Latypov, L.A. Ledova, L.A. Krasovskaya, A.P. Karatovskaya, F.A. Brovko, N.V. Vasileva, O.A. Stepnaya, 2014, published in Bioorganicheskaya Khimiya, 2014, Vol. 40, No. 3, pp. 297–304.

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Rudenko, N.V., Tsfasman, I.M., Latypov, O.R. et al. Detection of propeptides of AlpA and AlpB lytic endopeptidases from Lysobacter sp. XL1 by the sandwich enzyme immunoassay based on monoclonal antibodies. Russ J Bioorg Chem 40, 272–278 (2014). https://doi.org/10.1134/S1068162014030133

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  • DOI: https://doi.org/10.1134/S1068162014030133

Keywords

  • Lysobacter
  • endopeptidases
  • protein secretion
  • monoclonal antibodies
  • sandwich enzyme immunoassay