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 language | English |
|---|---|
| Article number | 000817 |
| Pages (from-to) | 1785-1794 |
| Number of pages | 10 |
| Journal | Journal of General Virology |
| Volume | 98 |
| Issue number | 7 |
| DOIs | |
| Publication status | Published - 1 Jul 2017 |
Keywords
- Adenovirus protein VII
- Core protein
- DNA binding
- Expression
- Purification
Cite this
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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 journal › Article › Research › peer-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 -