TY - JOUR
T1 - An optimised protocol for the expression and purification of adenovirus core protein VII
AU - Athukorala, Ajani
AU - Helbig, Karla J.
AU - McSharry, Brian P.
AU - Forwood, Jade K.
AU - Sarker, Subir
N1 - Publisher Copyright:
© 2024 The Authors
PY - 2024/5
Y1 - 2024/5
N2 - Adenovirus protein VII (pVII) is a highly basic core protein, bearing resemblance to mammalian histones. Despite its diverse functions, a comprehensive understanding of its structural intricacies and the mechanisms underlying its functions remain elusive, primarily due to the complexity of producing a good amount of soluble pVII. This study aimed to optimise the expression and purification of recombinant pVII from four different adenoviruses with a simple vector construct. This study successfully determined the optimal conditions for efficiently purifying pVII across four adenovirus species, revealing the differential preference for bacterial expression systems. The One Shot BL21 Star (DE3) proved favourable over Rosetta 2 (DE3) pLysS with consistent levels of expression between IPTG-induced and auto-induction. We demonstrated that combining chemical and mechanical cell lysis is possible and highly effective. Other noteworthy benefits were observed in using RNase during sample processing. The addition of RNase has significantly improved the quality and quantity of the purified protein as confirmed by chromatographic and western blot analyses. These findings established a solid groundwork for pVII purification methodologies and carry the significant potential to assist in unveiling the core structure of pVII, its arrangement within the core, DNA condensation intricacies, and potential pathways for nuclear transport.
AB - Adenovirus protein VII (pVII) is a highly basic core protein, bearing resemblance to mammalian histones. Despite its diverse functions, a comprehensive understanding of its structural intricacies and the mechanisms underlying its functions remain elusive, primarily due to the complexity of producing a good amount of soluble pVII. This study aimed to optimise the expression and purification of recombinant pVII from four different adenoviruses with a simple vector construct. This study successfully determined the optimal conditions for efficiently purifying pVII across four adenovirus species, revealing the differential preference for bacterial expression systems. The One Shot BL21 Star (DE3) proved favourable over Rosetta 2 (DE3) pLysS with consistent levels of expression between IPTG-induced and auto-induction. We demonstrated that combining chemical and mechanical cell lysis is possible and highly effective. Other noteworthy benefits were observed in using RNase during sample processing. The addition of RNase has significantly improved the quality and quantity of the purified protein as confirmed by chromatographic and western blot analyses. These findings established a solid groundwork for pVII purification methodologies and carry the significant potential to assist in unveiling the core structure of pVII, its arrangement within the core, DNA condensation intricacies, and potential pathways for nuclear transport.
KW - Adenovirus core protein
KW - Core protein VII purification
KW - DNA binding protein
KW - pVII
KW - RNase
UR - http://www.scopus.com/inward/record.url?scp=85186986510&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85186986510&partnerID=8YFLogxK
U2 - 10.1016/j.jviromet.2024.114907
DO - 10.1016/j.jviromet.2024.114907
M3 - Article
C2 - 38432358
AN - SCOPUS:85186986510
SN - 0166-0934
VL - 326
SP - 1
EP - 9
JO - Journal of Virological Methods
JF - Journal of Virological Methods
M1 - 114907
ER -