Abstract
BackgroundThe growing field of plant molecular farming relies on expression vectors that allow high yields of recombinant proteins to be produced through transient gene expression. While numerous expression vectors currently exist for this purpose, there are very few examples of systematic efforts to improve upon these. Moreover, the current generation of expression systems makes use of naturally-occurring regulatory elements, typically selected from plant viruses, to maximise yields. This study aims to use rational design to generate synthetic sequences that can rival existing ones.ResultsIn this work, we present the rational design of novel synthetic 5′ and 3′ untranslated regions (UTRs) which can be used in various combinations to modulate accumulation levels of transiently-expressed recombinant proteins. Using the pEAQ-HT expression vector as a point of comparison, we show that pre-existing expression systems can be improved by the deployment of rationally designed synthetic UTRs. Notably, we show that a suite of short, synthetic 5′UTRs behave as expression enhancers that outperform the HT 5′UTR present in the CPMV-HT expression system. Furthermore, we confirm the critical role played by the 3′UTR of cowpea mosaic virus RNA-2 in the performance of the CPMV-HT system. Finally, we use the knowledge obtained from these results to develop novel expression vectors (named pHRE and pHREAC) that equal or outperform pEAQ-HT in terms of recombinant protein yield. These new vectors are also domesticated for the use of certain Type IIS restriction enzymes, which allows for quicker cloning and straightforward assessment of different combinations of UTRs.ConclusionsWe have shown that it is possible to rationally design a suite of expression modulators in the form of synthetic UTRs. We have created novel expression vectors that allow very high levels of recombinant protein expression in a transient expression context. This will have important consequences for future efforts to develop ever-better plant transient overexpression vectors for research or industrial applications.
Highlights
The growing field of plant molecular farming relies on expression vectors that allow high yields of recombinant proteins to be produced through transient gene expression
Creation of the Synth expression cassette A novel expression cassette was designed to allow straightforward replacement and testing of different untranslated regions (UTRs). This Synth cassette is based on the cowpea mosaic virus (CPMV)-HT expression cassette present in the pEAQHT vector: it contains the same 35S promoter and nos terminator sequences [19], but with synthetic 5′ and 3′UTRs replacing the CPMV-based UTRs present in CPMV-HT
The new vectors generated thanks to Synth were named in such a way as to highlight the UTR combination used: for example, the pEAQ backbone carrying the Synth-GFP cassette into which the GFP open reading frame (ORF) and the original Synth 5S0 5′UTR and 3S0 3′UTR were present was named pEAQ5S0-GFP-3S0
Summary
The growing field of plant molecular farming relies on expression vectors that allow high yields of recombinant proteins to be produced through transient gene expression. It is generally assumed that viral UTRs are excellent natural translational enhancers, and yet in the evolutionary context of plant RNA viruses (those most often used to construct expression systems), the UTRs have multiple roles during the virus life cycle [20]. Two of these roles are somewhat contradictory: the UTRs must allow synthesis of negativestranded RNA for viral RNA replication (which moves in the 3′ to 5′ direction along a positive-stranded viral RNA) as well as efficient translation of viral proteins (which moves in the 5′ to 3′ direction along the same positivestranded RNA). In a recombinant plant expression system, the goal is typically maximal expression and accumulation of one or a few recombinant proteins of interest which, in non-replicating systems, requires only efficient translation
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