Elaboration of a method of lysozyme purification from Human saliva

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Abstract

Introduction. Growing antibiotic resistance dictates the need for search for alternative antimicrobial drugs. Antimicrobial polypeptides, including lysozyme, are among prospective prototypes. Existing methods of human lysozyme purification are less elaborated in comparison with methods of lysozyme purification from other species sources.

The aim of the study. Development of a simple and rapid method of lysozyme purification from human saliva, which can be performed in laboratories equipped for research of peptides and low-molecular proteins.

Material and methods. Human saliva was treated with NaCl, centrifuged, filtered through filter with pores diameter 0.45 μm, used for two-steps solid-phase extraction on C18 cartridges (elution with 20% and 60% acetonitrile), reversed-phase high-performance liquid chromatography on C18 column in acetonitrile gradient and re-chromatography. Homogeneity and molecular weight were confirmed by SDS-PAGE electrophoresis. Enzyme activity was confirmed in turbidimetric analysis in the presence of Micrococcus lysodeikticus. The presence of antimicrobial activity was confirmed by electrophoresis under acidic conditions with subsequent gel overlay against Listeria monocytogenes.

Results. Saliva treatment with 62.5—1000 mM NaCl did not affect total protein content in the supernatants after centrifugation. Treated with 0.5 M NaCl and filtered saliva was used for solid-phase extraction. After the separation of components eluted with 20% acetonitrile, components eluted with 60% acetonitrile were used for RP-HPLC. As the result, a major peak was obtained and the fractions used for further purification and analysis. The sample contained a protein with the molecular weight of lysozyme. The protein possessed muramidase activity towards Micrococcus lysodeikticus cell walls and antimicrobial activity towards L. monocytogenes.

Conclusions. In the present study, a simple method of lysozyme purification from human saliva was elaborated (about 5 μg of lysozyme from 1 mL of saliva). The protocol preserves essential biological properties of lysozyme.

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About the authors

I. A. Krenev

Institute of Experimental Medicine

Author for correspondence.
Email: il.krenevv13@yandex.ru
ORCID iD: 0000-0001-7970-3291
SPIN-code: 1411-0962

Post-graduate Student, Junior Research Scientist, Department of General Pathology and Pathological Physiology

Russian Federation, Acad. Pavlov Str., 12, Saint Petersburg, 197022

M. N. Berlov

Institute of Experimental Medicine

Email: berlov.mn@iemspb.ru
ORCID iD: 0000-0001-5191-0467
SPIN-code: 9006-6127

Ph.D. (Biol.), Senior Research Scientist, Department of General Pathology and Pathological Physiology

 

Russian Federation, Acad. Pavlov Str., 12, Saint Petersburg, 197022

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Supplementary files

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2. Fig. 1. Elution profile obtained during the solid-phase extraction of the treated saliva. Volume of each fraction is 5 mL

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3. Fig. 2. Results of RP-HPLC of the sample obtained during the solid-phase extraction of the treated human saliva

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4. Fig. 3. Results of lysozyme purification using RP-HPLC

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5. Fig. 4. Results of the analytical electrophoresis of the human lysozyme in polyacrylamide gel under denaturating conditions in the presence of SDS (SDS-PAGE electrophoresis): st – molecular weights marker; 1 – hen egg white lysozyme; 2 – the prepared sample of human saliva lysozyme

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6. Fig. 5. Results of the evaluation of the obtained lysozyme sample enzymatic activity by turbidimetric method

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7. Fig. 6. Results of the electrophoresis in polyacrylamide gel in acidic system and of the antimicrobial gel overlay assay: 1 – lane with the prepared sample of human lysozyme; 2 – agarose gel with bacteria on Petri dish containing two inhibition zones; 3 – lane on polyacrylamide gel with the prepared sample of human lysozyme after overlay; 4 – hen egg white lysozyme. The higher bands contain lysozyme dimers and the lower bans contain lysozyme monomers

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