Next Article in Journal
Anticancer Activity of 2-O-caffeoyl Alphitolic Acid Extracted from the Lichen, Usnea barbata 2017-KL-10
Next Article in Special Issue
Differential Expression of Yersinia pseudotuberculosis General Porin Genes during Short- and Long-Term Antibiotic Stresses
Previous Article in Journal
Antioxidant Effects and Phytochemical Properties of Seven Taiwanese Cirsium Species Extracts
Previous Article in Special Issue
Genome-Wide Analysis of PL7 Alginate Lyases in the Genus Zobellia
 
 
Article

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

1
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
2
Department of Medical Biology and Biotechnology, FEFU Campus, School of Biomedicine, Far Eastern Federal University, Russky Island Ajax Bay 10, 690922 Vladivostok, Russia
3
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
4
Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, 100, Shih-Chuan 1st Road, Kaohsiung 80708, Taiwan
5
A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Palchevskogo str. 17, 690041 Vladivostok, Russia
*
Authors to whom correspondence should be addressed.
Academic Editor: Carmelo Corsaro
Molecules 2021, 26(13), 3936; https://doi.org/10.3390/molecules26133936
Received: 2 May 2021 / Revised: 8 June 2021 / Accepted: 22 June 2021 / Published: 28 June 2021
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. View Full-Text
Keywords: recombinant phospholipase A1; Yersinia pseudotuberculosis; inclusion bodies; fusion protein; green fluorescent protein recombinant phospholipase A1; Yersinia pseudotuberculosis; inclusion bodies; fusion protein; green fluorescent protein
Show Figures

Figure 1

MDPI and ACS Style

Bakholdina, S.I.; Stenkova, A.M.; Bystritskaya, E.P.; Sidorin, E.V.; Kim, N.Y.; Menchinskaya, E.S.; Gorpenchenko, T.Y.; Aminin, D.L.; Shved, N.A.; Solov’eva, T.F. Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy. Molecules 2021, 26, 3936. https://doi.org/10.3390/molecules26133936

AMA Style

Bakholdina SI, Stenkova AM, Bystritskaya EP, Sidorin EV, Kim NY, Menchinskaya ES, Gorpenchenko TY, Aminin DL, Shved NA, Solov’eva TF. Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy. Molecules. 2021; 26(13):3936. https://doi.org/10.3390/molecules26133936

Chicago/Turabian Style

Bakholdina, Svetlana I., Anna M. Stenkova, Evgenia P. Bystritskaya, Evgeniy V. Sidorin, Natalya Yu. Kim, Ekaterina S. Menchinskaya, Tatiana Yu. Gorpenchenko, Dmitry L. Aminin, Nikita A. Shved, and Tamara F. Solov’eva. 2021. "Studies on the Structure and Properties of Membrane Phospholipase A1 Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy" Molecules 26, no. 13: 3936. https://doi.org/10.3390/molecules26133936

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop