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Recombinant protein production in Arabidopsis seeds: transgene expression variation and transcriptome analysis

Kirsten De Wilde UGent (2012)
abstract
The use of genetically engineered crops for the production of high-value recombinant proteins, i. e. ‘molecular farming’, offers a suitable alternative for the conventional production systems such as mammalian cells and microbial cultures (Faye and Gomord, 2010; Peters and Stoger, 2011). Numerous proof-of-concept studies have paved the way for plant-made-pharmaceuticals to reach the market. Among them, seed-based production is advantageous because seeds provide a high storage capacity for stable accumulation of proteins in a relatively small volume (Stoger et al., 2005b). Indeed, high accumulation levels for recombinant proteins could be obtained in seeds, more specifically, using the regulatory sequences of seed storage proteins β-PHASEOLIN (PHAS) and ARCELIN5-I (ARC), a singlechain Fv (SCFV) antibody accumulated up to 36.5 % of the total soluble protein (TSP) and a SCFV-FC up to 13,9 % of the TSP in Arabidopsis seeds (De Jaeger et al., 2002; Van Droogenbroeck et al., 2007). Moreover, also auto-antigens GAD67/65 and a full length antibody accumulated well using this expression cassette (Loos et al., 2011b; Morandini et al., 2011). These results proved that economically viable accumulation levels can be obtained in seeds of Arabidopsis. Nevertheless, it was also observed that not all transformants with a particular construct accumulated to similar high levels. These variable accumulation levels might be caused by different T-DNA integration patterns. Indeed, introduced transgenes, especially in inverted repeat orientation, are frequently silenced in leaves, resulting in different accumulation levels in the different transformants (Muskens et al., 2000). Also, the T-DNA random genome integration leads to a different position which might result in position effect (De Wilde et al., 2000). All these aspects imply that molecular farming still requires the focus on optimization of plant expression systems (Paul and Ma, 2011) and in this context, the general objective of this doctoral thesis was to investigate several aspects necessary for optimization of the Arabidopsis seed-based production platform. A first aspect was to assess the variability in transgene expression levels in seeds compared with what is known from leaves. More specifically, the seed-specific promoter PPHAS was compared with the frequently used constitutive P35S promoter. The variation in seedspecific transgene expression in different transformants, driven by PPHAS or P35S, was evaluated. Additionally, it was questioned whether a positive correlation exists between the number of integrated T-DNA copies and the recombinant protein accumulation level in seeds. Furthermore, it was examined whether the PPHAS and/or the P35S expression cassettes are susceptible to RDR6-mediated silencing in seeds. In an attempt to elucidate the role of the long ARCELIN 3’ regulatory region on transgene expression levels, the use of different 3’ regulatory regions was investigated. We compared the efficiency of the 4001 bp long 3’ ARC5-I regulatory region with the commonly used 767 bp long 3’ OCS, under control of the PPHAS. Additionally, we reduced the length of 3’ARC to obtain a so-called minimal 3’ regulatory region, by evaluating shorter fragments of 3’ ARC for their ability to still support high transgene accumulation levels. Also, we investigated whether 3’ ARC can positively affect transgene expression driven from the 35S promoter. In order to exploit Arabidopsis thaliana as bioreactor for the production of antibodies, it is extremely important to verify that high level expression does not negatively affect seed germination and plant growth. The effect of producing foreign proteins at very high levels in the seed is not known. In yeasts, it is already observed that high-level protein production induces a stress response, since the cells cannot manage the abundant misfolding of the proteins (Gasser et al., 2008). Therefore, another objective of this Ph.D. concerned the study of the influence of recombinant antibody accumulation on the Arabidopsis seed transcriptome. Therefore, we used a microarray and qPCR approach to analyse the transcriptome of developing seeds during seed filling, when the major seed storage reserves are produced. We compared WT Col-0 and transgenic plants, accumulating different levels of recombinant antibodies in their seeds. Additionally, we performed a preliminary study to examine whether recombinant protein production can influence plant growth and the ability of the transgenic seeds to germinate.
Please use this url to cite or link to this publication:
author
promoter
UGent and UGent
organization
year
type
dissertation (monograph)
subject
pages
195 + annexes pages
publisher
Ghent University. Faculty of Sciences
place of publication
Ghent, Belgium
defense location
Zwijnaarde : Technologiepark (FSVM building)
defense date
2012-03-16 16:30
language
English
UGent publication?
yes
classification
D1
additional info
dissertation consists of copyrighted material
copyright statement
I have transferred the copyright for this publication to the publisher
id
3005551
handle
http://hdl.handle.net/1854/LU-3005551
date created
2012-10-03 16:06:09
date last changed
2012-10-05 09:29:38
@phdthesis{3005551,
  abstract     = {The use of genetically engineered crops for the production of high-value recombinant proteins, i. e. {\textquoteleft}molecular farming{\textquoteright}, offers a suitable alternative for the conventional production systems such as mammalian cells and microbial cultures (Faye and Gomord, 2010; Peters and Stoger, 2011). Numerous proof-of-concept studies have paved the way for plant-made-pharmaceuticals to reach the market. Among them, seed-based production is advantageous because seeds provide a high storage capacity for stable accumulation of proteins in a relatively small volume (Stoger et al., 2005b). Indeed, high accumulation levels for recombinant proteins could be obtained in seeds, more specifically, using the regulatory sequences of seed storage proteins \ensuremath{\beta}-PHASEOLIN (PHAS) and ARCELIN5-I (ARC), a singlechain Fv (SCFV) antibody accumulated up to 36.5 \% of the total soluble protein (TSP) and a SCFV-FC up to 13,9 \% of the TSP in Arabidopsis seeds (De Jaeger et al., 2002; Van Droogenbroeck et al., 2007). Moreover, also auto-antigens GAD67/65 and a full length antibody accumulated well using this expression cassette (Loos et al., 2011b; Morandini et al., 2011). These results proved that economically viable accumulation levels can be obtained in seeds of Arabidopsis. Nevertheless, it was also observed that not all transformants with a particular construct accumulated to similar high levels. These variable accumulation levels might be caused by different T-DNA integration patterns. Indeed, introduced transgenes, especially in inverted repeat orientation, are frequently silenced in leaves, resulting in different accumulation levels in the different transformants (Muskens et al., 2000). Also, the T-DNA random genome integration leads to a different position which might result in position effect (De Wilde et al., 2000). All these aspects imply that molecular farming still requires the focus on optimization of plant expression systems (Paul and Ma, 2011) and in this context, the general objective of this doctoral thesis was to investigate several aspects necessary for optimization of the Arabidopsis seed-based production platform. 
A first aspect was to assess the variability in transgene expression levels in seeds compared with what is known from leaves. More specifically, the seed-specific promoter PPHAS was compared with the frequently used constitutive P35S promoter. The variation in seedspecific transgene expression in different transformants, driven by PPHAS or P35S, was evaluated. Additionally, it was questioned whether a positive correlation exists between the number of integrated T-DNA copies and the recombinant protein accumulation level in seeds. Furthermore, it was examined whether the PPHAS and/or the P35S expression cassettes are susceptible to RDR6-mediated silencing in seeds. 
In an attempt to elucidate the role of the long ARCELIN 3{\textquoteright} regulatory region on transgene expression levels, the use of different 3{\textquoteright} regulatory regions was investigated. We compared the efficiency of the 4001 bp long 3{\textquoteright} ARC5-I regulatory region with the commonly used 767 bp long 3{\textquoteright} OCS, under control of the PPHAS. Additionally, we reduced the length of 3{\textquoteright}ARC to obtain a so-called minimal 3{\textquoteright} regulatory region, by evaluating shorter fragments of 3{\textquoteright} ARC for their ability to still support high transgene accumulation levels. Also, we investigated whether 3{\textquoteright} ARC can positively affect transgene expression driven from the 35S promoter. 
In order to exploit Arabidopsis thaliana as bioreactor for the production of antibodies, it is extremely important to verify that high level expression does not negatively affect seed germination and plant growth. The effect of producing foreign proteins at very high levels in the seed is not known. In yeasts, it is already observed that high-level protein production induces a stress response, since the cells cannot manage the abundant misfolding of the proteins (Gasser et al., 2008). Therefore, another objective of this Ph.D. concerned the study of the influence of recombinant antibody accumulation on the Arabidopsis seed transcriptome. Therefore, we used a microarray and qPCR approach to analyse the transcriptome of developing seeds during seed filling, when the major seed storage reserves are produced. We compared WT Col-0 and transgenic plants, accumulating different levels of recombinant antibodies in their seeds. Additionally, we performed a preliminary study to examine whether recombinant protein production can influence plant growth and the ability of the transgenic seeds to germinate.},
  author       = {De Wilde, Kirsten},
  language     = {eng},
  pages        = {195 + annexes},
  publisher    = {Ghent University. Faculty of Sciences},
  school       = {Ghent University},
  title        = {Recombinant protein production in Arabidopsis seeds: transgene expression variation and transcriptome analysis},
  year         = {2012},
}

Chicago
De Wilde, Kirsten. 2012. “Recombinant Protein Production in Arabidopsis Seeds: Transgene Expression Variation and Transcriptome Analysis”. Ghent, Belgium: Ghent University. Faculty of Sciences.
APA
De Wilde, K. (2012). Recombinant protein production in Arabidopsis seeds: transgene expression variation and transcriptome analysis. Ghent University. Faculty of Sciences, Ghent, Belgium.
Vancouver
1.
De Wilde K. Recombinant protein production in Arabidopsis seeds: transgene expression variation and transcriptome analysis. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2012.
MLA
De Wilde, Kirsten. “Recombinant Protein Production in Arabidopsis Seeds: Transgene Expression Variation and Transcriptome Analysis.” 2012 : n. pag. Print.