Assembly and Purification of Polyomavirus-Like Particles from Plants

Abstract

Polyomaviruses are small DNA viruses that have a history of use in biotechnology. The capsids of a number of species have been developed into experimental prophylactic and therapeutic virus-like particle (VLP) vaccines. In order to explore plants as a host for the expression and purification of polyomavirus-like particles, we have transiently expressed the major capsid protein, VP1, in Nicotiana benthamiana leaves. Deletion of a polybasic motif from the N-terminal region of VP1 resulted in increased expression as well as reduced necrosis of leaf tissue, which was associated with differences in subcellular localisation and reduced DNA binding by the deletion variant (ΔVP1). Self-assembled VLPs were recovered from tissue expressing both wild-type VP1 and ΔVP1 by density gradient ultracentrifugation. VLPs composed of ΔVP1 were more homogenous than wtVPLs and, unlike the latter, did not encapsidate nucleic acid. Such homogenous, empty VLPs are of great interest in biotechnology and nanotechnology. In addition, we show that both MPyV VLP variants assembled in plants can be produced with encapsidated foreign protein. Thus, this study demonstrates the utility of plant-based expression of polyomavirus-like particles and the suitability of this host for further developments in polyomavirus-based technologies.

Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease

“…There are a variety of ways to produce NK in plant systems. A rapid method is the utilization of a transient expression system, which can produce high levels of recombinant protein within a relatively short period of time (four to seven days) [65,66,67,68,69]. Various virus-based transient vectors have been studied for use in PMF systems.…”

Plants as Factories for Human Pharmaceuticals: Applications and Challenges

“…The time needed to utilize a standard PMF approach is unsuitable for addressing sudden viral epidemics, such as an outbreak of Ebola, severe acute respiratory syndrome (SARS), or MERS-CoV. Transient expression systems can be used as an alternative approach to produce recombinant protein within three to five days [ 60 , 61 , 62 , 63 ] (see Figure 2 ).…”

Murine polyomavirus VLPs as a platform for cytotoxic T cell epitope-based influenza vaccine candidates

“…The dimer could also be due to interaction via the hydrophobic VP2C motifs as it persists under denaturing conditions. There was also an antiVP1stained band in the 90 kDa region, probably incompletely denatured VP1 in the form of dimers, which sometimes occurs on SDS-PAGE (Catrice and Sainsbury, 2015). The 70 kDa band was not identified by either antibody, indicating that this is a co-purified host protein contaminant.…”

“…The hollow conical interior has a diameter of 50 Å at its base and 12 Å at its upper neck and the top of the capsomere contains a dimple 20 Å wide and 12 Å deep (Liddington et al 1991). The VP1 subunit alone can form VLPs both in vivo when expressed in insect cells (Montross et al 1991), yeast (Salunke et al 1986), or plants (Catrice and Sainsbury 2015), and in vitro when capsomeres are dialysed in optimised assembly buffer conditions (Salunke et al 1986;Schmidt et al 2000). In vitro dialysis experiments demonstrated that capsomere assembly into VLPs depends on calcium and disulphide bridges, and thus, capsomere form can be maintained in buffers containing ethylenediaminetetraacetic acid (EDTA) to chelate calcium, and dithiothreitol (DTT) to prevent the formation of disulphide bridges (Salunke et al 1986;Schmidt et al 2000).…”

“…Arrow point to truncation site. shown to permit this in plants (Catrice and Sainsbury 2015). GFP was fused at the C-terminus of VP2C and tagged with 6His.…”