Purification and characterization of adenovirus core protein VII

A histone-like protein that is critical for adenovirus core formation

Gaurav Sharma, Nithesh Moria, Martin Williams, Bankala Krishnarjuna, Colin W. Pouton

Research output: Contribution to journalArticleResearchpeer-review

1 Citation (Scopus)

Abstract

Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98%, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

Original languageEnglish
Article number000817
Pages (from-to)1785-1794
Number of pages10
JournalJournal of General Virology
Volume98
Issue number7
DOIs
Publication statusPublished - 1 Jul 2017

Keywords

  • Adenovirus protein VII
  • Core protein
  • DNA binding
  • Expression
  • Purification

Cite this

@article{179863190f1f42eca4d0c709673babb4,
title = "Purification and characterization of adenovirus core protein VII: A histone-like protein that is critical for adenovirus core formation",
abstract = "Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98{\%}, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.",
keywords = "Adenovirus protein VII, Core protein, DNA binding, Expression, Purification",
author = "Gaurav Sharma and Nithesh Moria and Martin Williams and Bankala Krishnarjuna and Pouton, {Colin W.}",
year = "2017",
month = "7",
day = "1",
doi = "10.1099/jgv.0.000817",
language = "English",
volume = "98",
pages = "1785--1794",
journal = "Journal of General Virology",
issn = "0022-1317",
publisher = "Microbiology Society",
number = "7",

}

Purification and characterization of adenovirus core protein VII : A histone-like protein that is critical for adenovirus core formation. / Sharma, Gaurav; Moria, Nithesh; Williams, Martin; Krishnarjuna, Bankala; Pouton, Colin W.

In: Journal of General Virology, Vol. 98, No. 7, 000817, 01.07.2017, p. 1785-1794.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Purification and characterization of adenovirus core protein VII

T2 - A histone-like protein that is critical for adenovirus core formation

AU - Sharma, Gaurav

AU - Moria, Nithesh

AU - Williams, Martin

AU - Krishnarjuna, Bankala

AU - Pouton, Colin W.

PY - 2017/7/1

Y1 - 2017/7/1

N2 - Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98%, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

AB - Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98%, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

KW - Adenovirus protein VII

KW - Core protein

KW - DNA binding

KW - Expression

KW - Purification

UR - http://www.scopus.com/inward/record.url?scp=85026645066&partnerID=8YFLogxK

U2 - 10.1099/jgv.0.000817

DO - 10.1099/jgv.0.000817

M3 - Article

VL - 98

SP - 1785

EP - 1794

JO - Journal of General Virology

JF - Journal of General Virology

SN - 0022-1317

IS - 7

M1 - 000817

ER -