Fine‐tuning levels of heterologous gene expression in plants by orthogonal variation of the untranslated regions of a nonreplicating transient expression system

A transient expression system based on a deleted version of Cowpea mosaic virus (CPMV) RNA‐2, termed CPMV‐HT, in which the sequence to be expressed is positioned between a modified 5′ UTR and the 3′ UTR has been successfully used for the plant‐based expression of a wide range of proteins, including heteromultimeric complexes. While previous work has demonstrated that alterations to the sequence of the 5′ UTR can dramatically influence expression levels, the role of the 3′ UTR in enhancing expression has not been determined. In this work, we have examined the effect of different mutations in the 3′UTR of CPMV RNA‐2 on expression levels using the reporter protein GFP encoded by the expression vector, pEAQexpress‐HT‐GFP. The results showed that the presence of a 3′ UTR in the CPMV‐HT system is important for achieving maximal expression levels. Removal of the entire 3′ UTR reduced expression to approximately 30% of that obtained in its presence. It was found that the Y‐shaped secondary structure formed by nucleotides 125–165 of the 3′ UTR plays a key role in its function; mutations that disrupt this Y‐shaped structure have an effect equivalent to the deletion of the entire 3′ UTR. Our results suggest that the Y‐shaped secondary structure acts by enhancing mRNA accumulation rather than by having a direct effect on RNA translation. The work described in this paper shows that the 5′ and 3′ UTRs in CPMV‐HT act orthogonally and that mutations introduced into them allow fine modulation of protein expression levels.     

Predictive models for the accumulation of a fluorescent marker protein in tobacco leaves according to the promoter/5′UTR combination

The promoter and 5′‐untranslated region (5′UTR) play a key role in determining the efficiency of recombinant protein expression in plants. Comparative experiments are used to identify suitable elements but these are usually tested in transgenic plants or in transformed protoplasts/suspension cells, so their relevance in whole‐plant transient expression systems is unclear given the greater heterogeneity in expression levels among different leaves. Furthermore, little is known about the impact of promoter/5′UTR interactions on protein accumulation. We therefore established a predictive model using a design of experiments (DoE) approach to compare the strong double‐enhanced Cauliflower mosaic virus 35S promoter (CaMV 35SS) and the weaker Agrobacterium tumefaciens Ti‐plasmid nos promoter in whole tobacco plants transiently expressing the fluorescent marker protein DsRed. The promoters were combined with one of three 5′UTRs (one of which was tested with and without an additional protein targeting motif) and the accumulation of DsRed was measured following different post‐agroinfiltration incubation periods in all leaves and at different leaf positions. The model predictions were quantitative, allowing the rapid identification of promoter/5′UTR combinations stimulating the highest and quickest accumulation of the marker protein in all leaves. The model also suggested that increasing the incubation time from 5 to 8 days would reduce batch‐to‐batch variability in protein yields. We used the model to identify promoter/5′UTR pairs that resulted in the least spatiotemporal variation in expression levels. These ideal pairs are suitable for the simultaneous, balanced production of several proteins in whole plants by transient expression. Biotechnol. Bioeng. 2013; 110: 471–482. © 2012 Wiley Periodicals, Inc.