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Interaction of Recombinant Gallus gallus SEPT5 and Brain Proteins of H5N1-Avian Influenza Virus-Infected Chickens

Septin forms a conserved family of cytoskeletal guanosine triphosphate (GTP) binding proteins that have diverse roles in protein scaffolding, vesicle trafficking, and cytokinesis. The involvement of septins in infectious viral disease pathogenesis has been demonstrated by the upregulation of SEPT5 p...

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Autores principales: Khairat, Jasmine Elanie, Balasubramaniam, Vinod, Othman, Iekhsan, Omar, Abdul Rahman, Syed Hassan, Sharifah
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620540/
https://www.ncbi.nlm.nih.gov/pubmed/28895884
http://dx.doi.org/10.3390/proteomes5030023
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author Khairat, Jasmine Elanie
Balasubramaniam, Vinod
Othman, Iekhsan
Omar, Abdul Rahman
Syed Hassan, Sharifah
author_facet Khairat, Jasmine Elanie
Balasubramaniam, Vinod
Othman, Iekhsan
Omar, Abdul Rahman
Syed Hassan, Sharifah
author_sort Khairat, Jasmine Elanie
collection PubMed
description Septin forms a conserved family of cytoskeletal guanosine triphosphate (GTP) binding proteins that have diverse roles in protein scaffolding, vesicle trafficking, and cytokinesis. The involvement of septins in infectious viral disease pathogenesis has been demonstrated by the upregulation of SEPT5 protein and its mRNA in brain tissues of H5N1-infected chickens, thus, providing evidence for the potential importance of this protein in the pathogenesis of neurovirulence caused by the avian influenza virus. In this study, cloning, expression, and purification of Gallus gallus SEPT5 protein was performed in Escherichia coli. The SEPT5 gene was inserted into the pRSETB expression vector, transformed in the E. coli BL21 (DE3) strain and the expression of SEPT5 protein was induced by IPTG. The SEPT5 protein was shown to be authentic as it was able to be pulled down by a commercial anti-SEPT5 antibody in a co-immunoprecipitation assay. In vivo aggregation of the recombinant protein was limited by cultivation at a reduced temperature of 16 °C. Using co-immunoprecipitation techniques, the purified recombinant SEPT5 protein was used to pull down host’s interacting or binding proteins, i.e., proteins of brains of chickens infected with the H5N1 influenza virus. Interacting proteins, such as CRMP2, tubulin proteins, heat-shock proteins and other classes of septins were identified using LCMS/MS. Results from this study suggest that the codon-optimized SEPT5 gene can be efficiently expressed in the E. coli bacterial system producing authentic SEPT5 protein, thus, enabling multiple host’s proteins to interact with the SEPT5 protein.
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spelling pubmed-56205402017-10-03 Interaction of Recombinant Gallus gallus SEPT5 and Brain Proteins of H5N1-Avian Influenza Virus-Infected Chickens Khairat, Jasmine Elanie Balasubramaniam, Vinod Othman, Iekhsan Omar, Abdul Rahman Syed Hassan, Sharifah Proteomes Article Septin forms a conserved family of cytoskeletal guanosine triphosphate (GTP) binding proteins that have diverse roles in protein scaffolding, vesicle trafficking, and cytokinesis. The involvement of septins in infectious viral disease pathogenesis has been demonstrated by the upregulation of SEPT5 protein and its mRNA in brain tissues of H5N1-infected chickens, thus, providing evidence for the potential importance of this protein in the pathogenesis of neurovirulence caused by the avian influenza virus. In this study, cloning, expression, and purification of Gallus gallus SEPT5 protein was performed in Escherichia coli. The SEPT5 gene was inserted into the pRSETB expression vector, transformed in the E. coli BL21 (DE3) strain and the expression of SEPT5 protein was induced by IPTG. The SEPT5 protein was shown to be authentic as it was able to be pulled down by a commercial anti-SEPT5 antibody in a co-immunoprecipitation assay. In vivo aggregation of the recombinant protein was limited by cultivation at a reduced temperature of 16 °C. Using co-immunoprecipitation techniques, the purified recombinant SEPT5 protein was used to pull down host’s interacting or binding proteins, i.e., proteins of brains of chickens infected with the H5N1 influenza virus. Interacting proteins, such as CRMP2, tubulin proteins, heat-shock proteins and other classes of septins were identified using LCMS/MS. Results from this study suggest that the codon-optimized SEPT5 gene can be efficiently expressed in the E. coli bacterial system producing authentic SEPT5 protein, thus, enabling multiple host’s proteins to interact with the SEPT5 protein. MDPI 2017-09-12 /pmc/articles/PMC5620540/ /pubmed/28895884 http://dx.doi.org/10.3390/proteomes5030023 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Khairat, Jasmine Elanie
Balasubramaniam, Vinod
Othman, Iekhsan
Omar, Abdul Rahman
Syed Hassan, Sharifah
Interaction of Recombinant Gallus gallus SEPT5 and Brain Proteins of H5N1-Avian Influenza Virus-Infected Chickens
title Interaction of Recombinant Gallus gallus SEPT5 and Brain Proteins of H5N1-Avian Influenza Virus-Infected Chickens
title_full Interaction of Recombinant Gallus gallus SEPT5 and Brain Proteins of H5N1-Avian Influenza Virus-Infected Chickens
title_fullStr Interaction of Recombinant Gallus gallus SEPT5 and Brain Proteins of H5N1-Avian Influenza Virus-Infected Chickens
title_full_unstemmed Interaction of Recombinant Gallus gallus SEPT5 and Brain Proteins of H5N1-Avian Influenza Virus-Infected Chickens
title_short Interaction of Recombinant Gallus gallus SEPT5 and Brain Proteins of H5N1-Avian Influenza Virus-Infected Chickens
title_sort interaction of recombinant gallus gallus sept5 and brain proteins of h5n1-avian influenza virus-infected chickens
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620540/
https://www.ncbi.nlm.nih.gov/pubmed/28895884
http://dx.doi.org/10.3390/proteomes5030023
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