Subject category: Bioprocess engineering and supporting technologies.
Predictive models for the accumulation of a fluorescent marker protein in tobacco leaves according to the promoter/5′UTR combination†
Article first published online: 18 SEP 2012
Copyright © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 110, Issue 2, pages 471–482, February 2013
How to Cite
Buyel, J. F., Kaever, T., Buyel, J. J. and Fischer, R. (2013), Predictive models for the accumulation of a fluorescent marker protein in tobacco leaves according to the promoter/5′UTR combination. Biotechnol. Bioeng., 110: 471–482. doi: 10.1002/bit.24715
- Issue published online: 20 DEC 2012
- Article first published online: 18 SEP 2012
- Accepted manuscript online: 4 SEP 2012 11:06AM EST
- Manuscript Accepted: 14 AUG 2012
- Manuscript Revised: 7 AUG 2012
- Manuscript Received: 21 JUN 2012
- transient expression;
- recombinant protein;
- promoter strength;
- predictive model;
- design of experiments;
- balanced expression
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.