Purification of Virus-Like Particles (VLPs) from Plants
Viral coat proteins expressed in plants often form virus-like particles (VLPs) which are good vaccine candidates as they are safe and highly immunogenic and can be easily purified. The VLPs can be purified by rate-zonal density centrifugation which is bas
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Introduction Viral coat proteins expressed in plants very often self-assemble and form virus-like particles (VLPs). VLPs have been shown to make excellent vaccine candidates [1, 2]. One important factor of VLP vaccines is that they are safe as they only contain the viral capsid proteins, with no viral genomic material, and they mimic the native virion in antigenicity. Many VLP vaccine candidates such as Hepatitis B (HBV), Norwalk virus (NV) and human papillomavirus (HPV) have been produced in plants and many of those have have been shown to be safe and efficacious [3, 4]. One challenge that remains in plant-produced vaccines is purification of the expressed proteins. VLPs in general are more stable against degradation and can be isolated from plants utilizing centrifugation techniques similar to those developed for purification of plant viruses. VLPs can be separated and purified utilizing density gradient centrifugation that is either based on the buoyant density of the VLP and called isopycnic centrifugation or depends on the size and sedimentation coefficient of the VLP and is called rate-zonal centrifugation [5]. Either sucrose, which is relatively cheap and easy to handle, or iodixanol (OptiPrep™; http://www.axis-shielddensity-gradient-media.com/virusindexes.htm) can be used in density gradient centrifugation. For most post-purification analysis such as gel electrophoresis, electron microscopy, or studies on cell culture, sucrose must be dialyzed before use, but most analysis can be performed without dialysis if the VLPs are purified in iodixanol. Rate zonal centrifugation can be used to separate VLPs of different sizes by centrifugation through a steep density gradient, e.g., 5–20 % or 10–40 % sucrose, where the density at no point in the gradient exceeds that of the VLPs to be purified. Rate zonal centrifugation separation of VLPs is based on their size and rate of
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_37, © Springer Science+Business Media New York 2016
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Albertha R. van Zyl and Inga I. Hitzeroth
sedimentation and not so much their density and it is mainly used for analytical purposes where the VLPs are as much separated from other components as possible, but it also results in lower yields. VLPs of different sizes will sediment to different places in the gradient. With isopycnic centrifugation, separation of VLPs is based entirely on their buoyant density. The densest part of the gradient exceeds the density of the VLPs to be purified, and therefore the VLPs will never pellet. VLPs of a particular density will travel down the gradient until the point is reached where their density is the same as that of the gradient—an equilibrium position. Different sizes of the VLPs will only influence the rate at which the VLPs reach their equilibrium position. With this method one is able to purify VLPs of different sizes but with the same density and in general this meth
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