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Tytuł:
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Efficient matrix-assisted refolding of the recombinant anti-staphylococcal truncated endolysin LysKCA and its structural and enzymatic description.
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Autorzy:
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Alaksandr Ž; Department of Biochemistry, Faculty of Biology, Belarusian State University, Minsk, 220030, Belarus. Electronic address: .
Sergey G; Department of Microbiology Faculty of Biology, Belarusian State University, Minsk, 220030, Belarus.
Maksim P; Department of Microbiology Faculty of Biology, Belarusian State University, Minsk, 220030, Belarus.
Sergey K; A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of Russian Academy of Science, Moscow, 119071, Russia; Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Moscow, 119334, Russia.
Niyaz S; Laboratory of Biotechnology, Baku State University, Baku, AZ, 1148, Azerbaijan.
Uladzimir P; Department of Microbiology Faculty of Biology, Belarusian State University, Minsk, 220030, Belarus.
Mikhail S; Department of Biochemistry, Faculty of Biology, Belarusian State University, Minsk, 220030, Belarus.
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Źródło:
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Protein expression and purification [Protein Expr Purif] 2020 Oct; Vol. 174, pp. 105683. Date of Electronic Publication: 2020 Jun 11.
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Typ publikacji:
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Journal Article
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Język:
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English
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Imprint Name(s):
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Publication: Orlando, FL : Academic Press
Original Publication: San Diego : Academic Press, c1990-
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MeSH Terms:
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Anti-Bacterial Agents*/chemistry
Anti-Bacterial Agents*/isolation & purification
Anti-Bacterial Agents*/pharmacology
Endopeptidases*/biosynthesis
Endopeptidases*/genetics
Endopeptidases*/isolation & purification
Endopeptidases*/pharmacology
Protein Refolding*
Staphylococcus aureus/*growth & development
Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/pharmacology
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Contributed Indexing:
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Keywords: Anti-staphylococcal activity; Endolysin LysK(CA); Inclusion bodies; Matrix-assisted refolding (MAR); Recombinant proteins
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Substance Nomenclature:
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0 (Anti-Bacterial Agents)
0 (Recombinant Proteins)
EC 3.4.- (Endopeptidases)
EC 3.4.99.- (endolysin)
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Entry Date(s):
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Date Created: 20200615 Date Completed: 20210114 Latest Revision: 20210114
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Update Code:
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20240105
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DOI:
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10.1016/j.pep.2020.105683
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PMID:
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32534980
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The recombinant truncated endolysin LysK consisting of two catalytic domains, N-terminal CHAP and amidase-2 (LysK CA ) was overexpressed in E. coli in the form of inclusion bodies (IBs). These IBs were dissolved in 6 M solution of urea followed by the refolding process. The refolding efficacy of the dilution and matrix-assisted renaturation method on SP Sepharose was compared at different purification stages of LysK CA . Solubilizate of IBs, DEAE Sepharose flowthrough, and SP Sepharose elution fractions were examined. The presence of negatively charged nucleic acids (NA) in the solution has shown a decrease in the recombinant LysK CA refolding yield (less than 11.5 ± 1.3% for both renaturation methods) due to their non-specific interaction with the positively charged endolysin. The renaturation efficiency of the enzyme purified from NA (SP elution fraction) was about 29.5 ± 6.7% and 28.2 ± 3.75% for dilution and matrix-assisted methods respectively. The later approach allows conducting one-step LysK CA refolding, purification and collection, and also noticeably cuts time and material expenses. The analysis of CD spectroscopy data of LysK CA , renatured on the resin matrix, revealed alpha helices and beta strands content similar to that of the modeled 3D structure. The theoretical 3D model with two predicted domains (CHAP and amidase-2) agrees well with the differential scanning calorimetry (DSC) results of the renatured LysK CA showing two well-resolved peaks corresponding to the two calorimetrically-revealed domains with the midpoint transition temperature (T m ) of 40.1 and 65.3°С. The enzyme so obtained exhibited in vitro anti-staphylococcal activity with 2.3 ± 0.45 × 10 3 U/mg and retained it for at least one year.
(Copyright © 2020 Elsevier Inc. All rights reserved.)
