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The Effect of Conditions of the Expression of the Recombinant Outer Membrane Phospholipase А1 from Yersinia pseudotuberculosis on the Structure and Properties of Inclusion Bodies

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Abstract

The effect of the concentration of an inducer (IPTG) and the time of induction at 37°С on the heterologous synthesis of the mature membrane protein phospholipase А1 (PldA) from Yersinia pseudotuberculosis in the form of inclusion bodies (IBs) and on the physicochemical and structural characteristics of IBs has been studied. The sizes, shape, stability (solubility in urea and detergents, resistance against proteolysis), the secondary structure of the protein of IBs, and the presence of amyloid structures have been determined by electron microscopy, dynamic light scattering, and optical spectroscopy. It was found that IBs have a shape close to spherical and a rough surface and are cleaved by proteinase K. The protein contained in IBs has an ordered secondary structure with a high content of β-structure. As the inducer concentration and the time of expression increase, the conformation of the recombinant protein in IBs undergoes changes, as indicated by an increase in the stability of IBs and a decrease in the enzymatic activity of the protein. When IBs are dissolved in 0.06% SDS and 5 M urea, the recombinant protein retains the secondary structure in a partially modified form, and the addition of a zwitterionic detergent at a micellar concentration does not transform the protein conformation into the native one.

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Abbreviations

IB:

inclusion body

DLS:

dynamic light scattering

SB3-12:

N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate

PBS:

phosphate-buffered saline, рН 7.5

IPTG:

isopropyl β-D-1-thiogalactopyranoside

LPS:

lipopolysaccharides

PldA:

detergent-resistant phospholipase A1

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Authors and Affiliations

  1. Yelyakov Pacific Institute of Bioorganic Chemistry, Far East Branch, Russian Academy of Sciences, Vladivostok, 690022, Russia

    S. I. Bakholdina, E. V. Sidorin, V. A. Khomenko, M. P. Isaeva, N. Yu. Kim, E. P. Bystritskaya, E. A. Pimenova & T. F. Solov’eva

  2. National Scientific Center of Marine Biology, Far East Branch, Russian Academy of Sciences, Vladivostok, 690041, Russia

    E. A. Pimenova

Authors
  1. S. I. Bakholdina
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  2. E. V. Sidorin
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  3. V. A. Khomenko
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  4. M. P. Isaeva
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  5. N. Yu. Kim
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  6. E. P. Bystritskaya
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  7. E. A. Pimenova
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  8. T. F. Solov’eva
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Correspondence to S. I. Bakholdina.

Additional information

Original Russian Text © S.I. Bakholdina, E.V. Sidorin, V.A. Khomenko, M.P. Isaeva, N.Yu. Kim, E.P. Bystritskaya, E.A. Pimenova, T.F. Solov’eva, 2018, published in Bioorganicheskaya Khimiya, 2018, Vol. 44, No. 2, pp. 163–174.

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Bakholdina, S.I., Sidorin, E.V., Khomenko, V.A. et al. The Effect of Conditions of the Expression of the Recombinant Outer Membrane Phospholipase А1 from Yersinia pseudotuberculosis on the Structure and Properties of Inclusion Bodies. Russ J Bioorg Chem 44, 178–187 (2018). https://doi.org/10.1134/S1068162018020061

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  • DOI: https://doi.org/10.1134/S1068162018020061

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