An optimised protocol for the expression and purification of adenovirus core protein VII.

Autor: Athukorala A; Department of Microbiology, Anatomy, Physiology, and Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, VIC 3086, Australia., Helbig KJ; Department of Microbiology, Anatomy, Physiology, and Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, VIC 3086, Australia., McSharry BP; School of Dentistry and Medical Sciences Biomedical Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia., Forwood JK; School of Dentistry and Medical Sciences Biomedical Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia., Sarker S; Biomedical Sciences and Molecular Biology, College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, QLD 4811, Australia. Electronic address: subir.sarker@jcu.edu.au.
Jazyk: angličtina
Zdroj: Journal of virological methods [J Virol Methods] 2024 May; Vol. 326, pp. 114907. Date of Electronic Publication: 2024 Mar 02.
DOI: 10.1016/j.jviromet.2024.114907
Abstrakt: 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.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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