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

Abstract

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.

Comparison of microbial and transient expression (tobacco plants and plant‐cell packs) for the production and purification of the anticancer mistletoe lectin viscumin

“…In the visFL and visA + visB PCP samples, we also detected a ~65 kDa band corresponding to the anticipated size of an N ‐glycosylated viscumin heterodimer or an A chain homodimer, as previously reported in E. coli (Kourmanova et al, ). Apart from the gene design (full‐length vs. separate chains), the difference in signal peptides and 5′‐UTRs may have also affected the A chain yields observed for the three expression setups (Jansing & Buyel, ; Meshcheriakova, Saxena, & Lomonossoff, ). Low absolute protein concentrations may have prevented dimer detection in the visA samples.…”

In Planta Synthesis of Designer-Length Tobacco Mosaic Virus-Based Nano-Rods That Can Be Used to Fabricate Nano-Wires

“…This synthetic DNA was cloned into pEAQ- HT ( Figure 1A ), a plant-based expression vector that produces abundant quantities of highly translatable RNA ( Sainsbury et al, 2009 ) to yield pEAQ- HT -TMV CP/OAS. The predicted length of the RNA transcribed from this construct is 1560 nucleotides, including sequences derived from the pEAQ vector ( Meshcheriakova et al, 2014 ), approximately one quarter of the length of genomic TMV RNA. Subsequently an additional stretch of nucleic acid from the unrelated plant virus, cowpea mosaic virus, was placed between the TMV coat protein and OAS elements, to give construct pEAQ- HT -TMV-CP/VP60/OAS which is predicted to give a transcript of 3375 nucleotides, approximately twice as long as that from pEAQ- HT -TMV CP/OAS.…”

“… Schematic representation of the infiltrated coat protein gene and OAS element containing gene constructions for (A) In cis and (B) in trans infiltrations. The predicted transcript length for each construct is listed calculated from the start of transcription at the 35S promoter to its termination by the nos terminator, Meshcheriakova et al (2014) . TMV CP, green box; the OAS element, pink box and the nos terminator, brown box are indicated.…”

Plant Virus Expression Vectors: A Powerhouse for Global Health

“…The CPMV deconstructed vector system pEAQ involves the expression of foreign proteins without the need for viral replication [ 68 , 69 ], thus relieving the cell from any hindrance created from viral cycle progression that negatively impacts protein accumulation. This involves the positioning of the foreign gene between the 5′ leader sequence and 3′ untranslated region (UTR) of RNA-2, as well as the deletion of an in-frame initiation codon found upstream of the main translation initiation site of RNA-2.…”