Solubilisation and purification of recombinant bluetongue virus VP7 expressed in a bacterial system

• Published in: Protein expression and purification 2018-07, Vol.147, p.85-93
• Main Authors: Russell, Bonnie L., Gildenhuys, Samantha
• Format: Article
• Language: English
• Subjects: Bluetongue virus; Bluetongue virus - genetics; Bluetongue virus - metabolism; Circular Dichroism; Escherichia coli - genetics; Escherichia coli - metabolism; IMAC chromatography; Inclusion bodies; Inclusion Bodies, Viral - metabolism; Protein Unfolding; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Solubilisation; Solubility; Spectrometry, Fluorescence; Temperature; Viral Core Proteins - chemistry; Viral Core Proteins - genetics; Viral Core Proteins - metabolism; VP7
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2018.03.006

Bluetongue virus (BTV) is an Orbivirus that has a profound economic impact due to direct loss of livestock as well as movement bans in an attempt to prevent the spread of the disease to susceptible areas. BTV VP7, along with VP3, forms the inner capsid core of the virus where it acts as the barrier between the outer layer and the inner core housing the genetic material. Purification of BTV VP7 has proven to be problematic and expensive mainly due to its insolubility is several expression systems. To overcome this, in this paper we present a protocol for the solubilisation of BTV VP7 from inclusion bodies expressed in E.coli, and subsequent purification using nickel affinity chromatography. The purified protein was then characterised using native PAGE, far ultraviolet circular dichroism (far-UV CD) and intrinsic fluorescence and found to have both secondary and tertiary structure even in the presence of 5 M urea. Both tertiary and secondary structure was further shown to be to be maintained at least to 42 °C in 5 M urea. •All expression conditions resulted in large amounts of protein in inclusion bodies.•Method for solubilisation of bluetongue virus VP7 from E.coli inclusion bodies was developed.•Purification of solubilized bluetongue virus VP7 was achieved using a Nickel affinity column.•Fluorescence and circular dichroism confirmed that purified bluetongue virus VP7 possesses native-like structure.•Bluetongue virus VP7 is found to resist denaturation to at least 42 °C in 5M urea.