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activation and transfer of the T-DNA containing the genes of interest,
acetosyringone is added to the agrobacteria suspension. Two to 3 weeks
post-inoculation, leaves can be harvested and analyzed, and if stably
expressed, the mutant VNPs can be extracted or passaged onto subsequent
plants (Lico
et al.
, 2008).
Figure 3.1
Agroinfection method. Adapted from Sainsbury, F., and Lomonossoff, G. P.
(2008) Extremely high-level and rapid transient protein production in plants without
the use of viral replication,
Plant Physiol
.,
148
, 1212-1218.
. heterologouS eXPreSSIon SySteMS
Heterologous expression systems are of critical importance for large-scale
production of VLPs and mutant particles. Various expression systems have
been developed for many different viruses. For many years, the study of virus
structure and assembly was the driving force for developing heterologous
expression systems. Only a heterologous expression system allows the
creation of assembly intermediates, which are essential for understanding
viral assembly. Mutations that perturb virus assembly can have dramatic
effects on virus viability;
studies of assembly intermediates in the
natural host are technically challenging. Heterologous expression systems
offer enormous flexibility for investigating protein-protein and protein-
nucleic acid interactions un-coupled from the processes of transcription,
protein expression, and replication (reviewed in Schneemann & Young,
2003).
Heterologous expression systems are of great interest as they allow the
production of VLPs. VLPs are devoid of infectious nucleic acids and thus
cannot replicate themselves. VLPs are considered safer from an agricultural
point of view and human health perspective. Heterologous expression
systems used to generate VLPs include use of bacteria, yeast, insect cells,
and mammalian cells. All these systems have advantages and disadvantages
in terms of yield, scale, time, costs, assembly efficiency, and biological
integrity (reviewed in Schneemann & Young, 2003). In the following
sections, we will discuss different systems and highlight their benefits and
pitfalls (summarized in Table 3.1).
in vivo
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