Studies on the Structure and Properties of Membrane Phospholipase A<sub>1</sub> Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy.

Bakholdina, Svetlana I; Stenkova, Anna M; Bystritskaya, Evgenia P; Sidorin, Evgeniy V; Kim, Natalya Yu; Menchinskaya, Ekaterina S; Gorpenchenko, Tatiana Yu; Aminin, Dmitry L; Shved, Nikita A; Solov'eva, Tamara F

Studies on the Structure and Properties of Membrane Phospholipase A₁ Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy.

Bakholdina, Svetlana I; Stenkova, Anna M; Bystritskaya, Evgenia P; Sidorin, Evgeniy V; Kim, Natalya Yu; Menchinskaya, Ekaterina S; Gorpenchenko, Tatiana Yu; Aminin, Dmitry L; Shved, Nikita A; Solov'eva, Tamara F.

Afiliación

Bakholdina SI; G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Prospekt 100-let Vladivostoku 159, 690022 Vladivostok, Russia.
Stenkova AM; Department of Medical Biology and Biotechnology, FEFU Campus, School of Biomedicine, Far Eastern Federal University, Russky Island Ajax Bay 10, 690922 Vladivostok, Russia.
Bystritskaya EP; G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Prospekt 100-let Vladivostoku 159, 690022 Vladivostok, Russia.
Sidorin EV; G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Prospekt 100-let Vladivostoku 159, 690022 Vladivostok, Russia.
Kim NY; G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Prospekt 100-let Vladivostoku 159, 690022 Vladivostok, Russia.
Menchinskaya ES; G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Prospekt 100-let Vladivostoku 159, 690022 Vladivostok, Russia.
Gorpenchenko TY; Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of the Russian Academy of Sciences, Prospect 100-let Vladivostoku, 159, 690022 Vladivostok, Russia.
Aminin DL; G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Prospekt 100-let Vladivostoku 159, 690022 Vladivostok, Russia.
Shved NA; Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, 100, Shih-Chuan 1st Road, Kaohsiung 80708, Taiwan.
Solov'eva TF; Department of Medical Biology and Biotechnology, FEFU Campus, School of Biomedicine, Far Eastern Federal University, Russky Island Ajax Bay 10, 690922 Vladivostok, Russia.

Molecules ; 26(13)2021 Jun 28.

Article en En | MEDLINE | ID: mdl-34203222

RESUMEN

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of Yersinia pseudotuberculosis phospholipase A1 (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs. The size, stability in denaturant solutions, and enzymatic and biological activity of PldA-GFP IBs expressed at 18 °C, as well as the secondary structure and arrangement of protein molecules inside the IBs, were studied. Solubilization of the chimeric protein from IBs in urea and SDS is accompanied by its denaturation. The obtained data show the structural heterogeneity of PldA-GFP IBs. It can be assumed that compactly packed, properly folded, proteolytic resistant, and structurally less organized, susceptible to proteolysis polypeptides can coexist in PldA-GFP IBs. The use of GFP as a fusion partner improves the conformational quality of PldA, but negatively affects its enzymatic activity. The PldA-GFP IBs are not toxic to eukaryotic cells and have the property to penetrate neuroblastoma cells. Data presented in the work show that the GFP-marker can be useful not only as target protein folding indicator, but also as a tool for studying the molecular organization of IBs, their morphology, and localization in E. coli, as well as for visualization of IBs interactions with eukaryotic cells.

Asunto(s)

Proteínas Bacterianas/química; Proteínas Fluorescentes Verdes/química; Cuerpos de Inclusión/química; Fosfolipasas A1/química; Proteínas Recombinantes de Fusión/química; Yersinia pseudotuberculosis/genética; Proteínas Bacterianas/biosíntesis; Proteínas Bacterianas/genética; Escherichia coli/genética; Escherichia coli/metabolismo; Proteínas Fluorescentes Verdes/biosíntesis; Proteínas Fluorescentes Verdes/genética; Cuerpos de Inclusión/genética; Cuerpos de Inclusión/metabolismo; Fosfolipasas A1/biosíntesis; Fosfolipasas A1/genética; Proteínas Recombinantes de Fusión/biosíntesis; Proteínas Recombinantes de Fusión/genética; Yersinia pseudotuberculosis/enzimología

Palabras clave

Yersinia pseudotuberculosis; fusion protein; green fluorescent protein; inclusion bodies; recombinant phospholipase A1

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Bacterianas / Proteínas Recombinantes de Fusión / Yersinia pseudotuberculosis / Cuerpos de Inclusión / Proteínas Fluorescentes Verdes / Fosfolipasas A1 Idioma: En Revista: Molecules Asunto de la revista: BIOLOGIA Año: 2021 Tipo del documento: Article País de afiliación: Rusia Pais de publicación: Suiza

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