Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity

Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.

In Plant Activation: An inducible, hyperexpression platform for recombinant protein production in plants

In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the b-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein.

Interleukin-1β stimulates human renal fibroblast proliferation and matrix protein production by means of a transforming growth factor-β-dependent mechanism

One of the hallmarks of progressive renal disease is the development of tubulointerstitial fibrosis. This is frequently preceded by macrophage infiltration, raising the possibility that macrophages relay fibrogenic signals to resident tubulointerstitial cells. The aim of this study was to investigate the potentially fibrogenic role of interleukin-1beta (IL-1beta), a macrophage-derived inflammatory cytokine, on cortical fibroblasts (CFs). Primary cultures of human renal CFs were established and incubated for 24 hours in the presence or absence of IL-1beta. We found that IL-1beta significantly stimulated DNA synthesis (356.7% +/- 39% of control, P <.003), fibronectin secretion (261.8 +/- 11% of control, P <.005), collagen type 1 production, (release of procollagen type 1 C-terminal-peptide, 152.4% +/- 26% of control, P <.005), transforming growth factor-beta (TGF-beta) secretion (211% +/- 37% of control, P <.01), and nitric oxide (NO) production (342.8% +/- 69% of control, P <.002). TGF-beta (1 ng/mL) and the phorbol ester phorbol 12-myristate 13-acetate (PMA, 25 nmol/L) produced fibrogenic effects similar to those of IL-1beta. Neither a NO synthase inhibitor (N(G)-methyl-l-arginine, 1 mmol/L) nor a protein kinase C (PKC) inhibitor (bis-indolylmaleimide 1, 1 micromol/L) altered the enhanced level of fibronectin secretion or DNA synthesis seen in response to IL-1beta treatment. However, addition of a TGF-beta-neutralizing antibody significantly reduced IL-1beta-induced fibronectin secretion (IL-1beta + IgG, 262% +/- 72% vs IL-1beta + alphaTGF-beta 156% +/- 14%, P <.02), collagen type 1 production (IL-1beta + IgG, 176% +/- 28% vs IL-1beta + alphaTGF-beta, 120% +/- 14%, P <.005) and abrogated IL-1beta-induced DNA synthesis (245% +/- 49% vs 105% +/- 21%, P <.005). IL-1beta significantly stimulated CF DNA synthesis and production of fibronectin, collagen type 1, TGFbeta, and NO. The fibrogenic and proliferative action of IL-1beta on CF appears not to involve activation of PKC or production of NO but is at least partly TGFbeta-dependent.

Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants

Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

Development of a transient, high-level expression platform for protein production in plants

Plants are an attractive alternative to conventional expression systems for the production of recombinant proteins and useful biologics, however, the economic viability of plant made proteins is strongly yield dependent. This study aimed to improve transgene expression levels in the plant host Nicotiana benthamiana using the Agroinfiltration transient expression platform. Independent investigation of the physical, chemical and genetic features associated with Agroinfiltration identified factors that improved transformation frequencies, elevated transgene expression levels and ultimately improved protein yield. The major outcome of this research was a novel hyper-expression system for biofarming recombinant proteins in plants.

Effect of varying the proportion of molasses in the diet on intake, digestion and microbial protein production by steers

The present experiment was conducted to determine the efficiency of microbial protein production in the rumen and intake by cattle fed high-molasses diets. Intake and microbial crude protein (MCP) production were measured along with the concentration of rumen ammonia-nitrogen (N) and volatile fatty acids (VFA), pH and the rate of digestion of roughage in the rumen. Eight Brahman crossbred steers weighing 211 ± 19.3 (± s.d.) kg were used in a double 4 × 4 Latin square design. Steers were allocated to one of four total mixed rations: control (pangola hay only), 25M (25% molasses/urea mix + 75% hay), 50M (50% molasses/urea + 50% hay), and 75M (75% molasses/urea + 25% hay). The production and efficiency of production of MCP (EMCP) of the diet increased quadratically as the level of molasses in the diet increased. The EMCP from the molasses/urea mix was estimated as 166 g MCP/kg digestible organic matter (DOM), a relatively high value. Intake of dry matter (DM) and DOM increased quadratically, reaching a peak when molasses was ~50% (as fed) of the ration. Digestibility of DM increased quadratically and that of neutral detergent fibre decreased linearly with increasing level of molasses in the diet. Molasses inclusion in the diet had no effect on rumen pH, ammonia and VFA concentration in the rumen fluid, plasma urea-N, urine pH or ruminal fractional outflow rate of ytterbium-labelled particles and Cr-EDTA. It was concluded that a diet with a high level of molasses (>50%) and supplemented with adequate N had high EMCP, and that low MCP production was not a factor limiting intake or performance of cattle consuming high-molasses diets.

Effect of varying the proportion of molasses in the diet on intake, digestion and microbial protein production by steers

The present experiment was conducted to determine the efficiency of microbial protein production in the rumen and intake by cattle fed high-molasses diets. Intake and microbial crude protein (MCP) production were measured along with the concentration of rumen ammonia-nitrogen (N) and volatile fatty acids (VFA), pH and the rate of digestion of roughage in the rumen. Eight Brahman crossbred steers weighing 211 ± 19.3 (± s.d.) kg were used in a double 4 x 4 Latin square design. Steers were allocated to one of four total mixed rations: control (pangola hay only), 25M (25% molasses/urea mix + 75% hay), 50M (50% molasses/urea + 50% hay), and 75M (75% molasses/urea + 25% hay). The production and efficiency of production of MCP (EMCP) of the diet increased quadratically as the level of molasses in the diet increased. The EMCP from the molasses/urea mix was estimated as 166 g MCP/kg digestible organic matter (DOM), a relatively high value. Intake of dry matter (DM) and DOM increased quadratically, reaching a peak when molasses was ∼50% (as fed) of the ration. Digestibility of DM increased quadratically and that of neutral detergent fibre decreased linearly with increasing level of molasses in the diet. Molasses inclusion in the diet had no effect on rumen pH, ammonia and VFA concentration in the rumen fluid, plasma urea-N, urine pH or ruminal fractional outflow rate of ytterbium-labelled particles and Cr-EDTA. It was concluded that a diet with a high level of molasses (>50%) and supplemented with adequate N had high EMCP, and that low MCP production was not a factor limiting intake or performance of cattle consuming high-molasses diets.

Spirulina (Spirulina platensis) algae supplementation increases microbial protein production and feed intake and decreases retention time of digesta in the rumen of cattle

Cattle consuming pastures low in protein have low liveweight gain due to low rumen degradable protein (RDP) supply and thus low microbial crude protein (MCP) production and efficiency of MCP production [EMCP, g MCP/kg digestible organic matter (DOM)]. Nitrogen supplements can increase MCP production and EMCP of cattle grazing low protein pastures. The objective of this study was to compare the effects of supplementation with a non-protein-N source (NPN), in this case urea and ammonium sulfate (US), with a single-cell algal protein source (Spirulina platensis), on intake, microbial protein supply and digestibility in cattle. Nine cannulated Bos indicus steers [initial liveweight 250.1 ± 10.86 (s.d.) kg] were fed Mitchell grass hay (Astrebla spp; 6.1 g N, 746 g NDF/kg DM) ad libitum and were supplied with increasing amounts of US (0, 6, 13, 19 and 33 g US DM/kg hay DM) or Spirulina 0, 0.5, 1.4, 2.5 and 6.1 g Spirulina DM/kg W.day in an incomplete Latin square design. The response of MCP production and EMCP to increasing amounts of the two supplements was different, with a greater response to Spirulina evident. The MCP production was predicted to peak at 140 and 568 g MCP/day (0.64 and 2.02 g MCP/kg W.day) for the US and Spirulina supplements, respectively. The highest measured EMCP were 92 and 166 g MCP/kg DOM for the US and Spirulina treatments at 170 and 290 g RDP/kg DOM, respectively, or a Spirulina intake of 5.7 g DM/kg W.day. Increasing RDP intake from US and Spirulina resulted in an increase in Mitchell grass hay intake and rumen NH3-N concentration and reduced the retention time of liquid and particulate markers and digesta DM, NDF and lignin in the rumen with greater changes due to Spirulina. Total DM intake peaked at a Spirulina supplement level of 4.6 g Spirulina DM/kg W.day with a 2.3-fold higher DOM intake than Control steers. Rumen NH3-N concentrations reached 128 and 264 mg NH3-N/L for the US and Spirulina treatments with a significant increase in the concentration of branched-chain fatty acids for the Spirulina treatment. The minimum retention time of liquid (Cr-EDTA; 23 and 13 h) and particulate (Yb; 34 and 22 h) markers in the rumen were significantly lower for Spirulina compared with US and lower than unsupplemented animals at 24 and 34 h for Cr-EDTA and Yb, respectively. Spirulina could be provided safely at much higher N intakes than NPN supplements. The results suggest that, at an equivalent RDP supply, Spirulina provided greater increases than US in MCP production, EMCP and feed intake of Bos indicus cattle consuming low protein forage and could also be fed safely at higher levels of N intake.

Comparative and functional genome analysis of fungi for development of the protein production host Trichoderma reesei

Filamentous fungi of the subphylum Pezizomycotina are well known as protein and secondary metabolite producers. Various industries take advantage of these capabilities. However, the molecular biology of yeasts, i.e. Saccharomycotina and especially that of Saccharomyces cerevisiae, the baker's yeast, is much better known. In an effort to explain fungal phenotypes through their genotypes we have compared protein coding gene contents of Pezizomycotina and Saccharomycotina. Only biomass degradation and secondary metabolism related protein families seem to have expanded recently in Pezizomycotina. Of the protein families clearly diverged between Pezizomycotina and Saccharomycotina, those related to mitochondrial functions emerge as the most prominent. However, the primary metabolism as described in S. cerevisiae is largely conserved in all fungi. Apart from the known secondary metabolism, Pezizomycotina have pathways that could link secondary metabolism to primary metabolism and a wealth of undescribed enzymes. Previous studies of individual Pezizomycotina genomes have shown that regardless of the difference in production efficiency and diversity of secreted proteins, the content of the known secretion machinery genes in Pezizomycotina and Saccharomycotina appears very similar. Genome wide analysis of gene products is therefore needed to better understand the efficient secretion of Pezizomycotina. We have developed methods applicable to transcriptome analysis of non-sequenced organisms. TRAC (Transcriptional profiling with the aid of affinity capture) has been previously developed at VTT for fast, focused transcription analysis. We introduce a version of TRAC that allows more powerful signal amplification and multiplexing. We also present computational optimisations of transcriptome analysis of non-sequenced organism and TRAC analysis in general. Trichoderma reesei is one of the most commonly used Pezizomycotina in the protein production industry. In order to understand its secretion system better and find clues for improvement of its industrial performance, we have analysed its transcriptomic response to protein secretion stress conditions. In comparison to S. cerevisiae, the response of T. reesei appears different, but still impacts on the same cellular functions. We also discovered in T. reesei interesting similarities to mammalian protein secretion stress response. Together these findings highlight targets for more detailed studies.

Oxidised, glycated LDL selectively influences tissue inhibitor of metalloproteinase-3 gene expression and protein production in human retinal capillary pericytes

Aims/hypothesis: Matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitor of metalloproteinases (TIMPs), regulate important biological processes including the homeostasis of the extracellular matrix, proteolysis of cell surface proteins, proteinase zymogen activation, angiogenesis and inflammation. Studies have shown that their balance is altered in retinal microvascular tissues in diabetes. Since LDLs modified by oxidation/glycation are implicated in the pathogenesis of diabetic vascular complications, we examined the effects of modified LDL on the gene expression and protein production of MMPs and TIMPs in retinal pericytes. Methods: Quiescent human retinal pericytes were exposed to native LDL (N-LDL), glycated LDL (G-LDL) and heavily oxidised and glycated LDL (HOG-LDL) for 24 h. We studied the expression of the genes encoding MMPs and TIMPs mRNAs by analysis of microarray data and quantitative PCR, and protein levels by immunoblotting and ELISA. Results: Microarray analysis showed that MMP1, MMP2, MMP11, MMP14 and MMP25 and TIMP1, TIMP2, TIMP3 and TIMP4 were expressed in pericytes. Of these, only TIMP3 mRNA showed altered regulation, being expressed at significantly lower levels in response to HOG- vs N-LDL. Quantitative PCR and immunoblotting of cell/matrix proteins confirmed the reduction in TIMP3 mRNA and protein in response to HOG-LDL. In contrast to cellular TIMP3 protein, analysis of secreted TIMP1, TIMP2, MMP1 and collagenase activity indicated no changes in their production in response to modified LDL. Combined treatment with N- and HOG-LDL restored TIMP3 mRNA expression to a level comparable with that after N-LDL alone. Conclusions/interpretation: Among the genes encoding for MMPs and TIMPs expressed in retinal pericytes, TIMP3 is uniquely regulated by HOG-LDL. Reduced TIMP3 expression might contribute to microvascular abnormalities in diabetic retinopathy. © 2007 Springer-Verlag.

Culture of Candida in vinasse and molasses: Effect of acid and salt addition on biomass and raw protein production

Species of the genera Candida grown in vinasse and molasses were studied under the following conditions: agitation of containers, pH 4.6, culture time of 24 hours at 30°C. The greatest biomass production of C. krusei grown in vinasse was obtained with the addition of 0.1% H3PO4, and of C. guilliermondii and C. utilis with the addition 0.02% urea plus 0.03% H3PO4. Protein levels near 50% were found in C. utilis in vinasse supplemented either with molasses, with 0.05% MgSO4, or with 0.02% urea plus 0.03% H3PO4. © 1982 Springer-Verlag.

Heat shock protein production and immunity and altered fetal development in diabetic pregnant rats

We evaluated associations between the concentrations of heat shock proteins (hsp60 and hsp70) and their respective antibodies, alterations in maternal reproductive performance, and fetal malformations in pregnant rats with hyperglycemia. Mild diabetes (MD) or severe diabetes (SD) was induced in Sprague-Dawley rats prior to mating; non-treated non-diabetic rats (ND) served as controls. On day 21 of pregnancy, maternal blood was analyzed for hsp60 and hsp70 and their antibodies; and fetuses were weighed and analyzed for congenital malformations. Hsp and anti-hsp levels were correlated with blood glucose levels during gestation. There was a positive correlation between hsp60 and hsp70 levels and the total number of malformations (R∈=∈0.5908, P∈=∈0.0024; R∈=∈0.4877, P∈=∈0.0134, respectively) and the number of malformations per fetus (R∈=∈0.6103, P∈=∈0.0015; R∈=∈0.4875, P∈=∈0.0134, respectively). The anti-hsp60 IgG concentration was correlated with the number of malformations per fetus (R∈=∈0.3887, P∈=∈0.0451) and the anti-hsp70 IgG level correlated with the total number of malformations (R∈=∈0.3999, P∈=∈0.0387). Moreover, both hsp and anti-hsp antibodies showed negative correlations with fetal weight. The results suggest that there is a relationship between hsp60 and hsp70 levels and their respective antibodies and alterations in maternal reproductive performance and impaired fetal development and growth in pregnancies associated with diabetes. © 2012 Cell Stress Society International.

Drivers of change in animal protein production systems: changes from 'traditional' to 'modern' beef cattle production systems in Brazil

Until 1990, beef cattle production in Brazil was recognized as an enterprise of low investment, low cost of production, as well as an extensive production system that used large land areas to be economically feasible. The situation changed for the Brazilian beef market when the economy stabilized in 1994 and all sectors involved in the beef supply chain were able to predict their associated costs and profits. During the same period, purchasing power of the Brazilian population grew, and as a consequence, consumers began to demand higher quality beef products. This new scenario in the late 1990s and early 2000s has led to more intensive beef production systems in Brazil as well as the need for a greater understanding of how technologies might be applied. As consequence of this evolution, in the last five years, a necessity to increase beef production has occurred. The quality of Brazilian beef has improved compared with beef produced 15 years ago, but continued improvements are needed to achieve excellence in terms of large-scale production of high quality beef, be sustainable over a long period of time, and increase our contribution to global food security.

Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production

Anderson MIYOSHI, Daniela FREITAS, Luciana RIBEIRO, Jane E. GABRIEL, Sophie LECLERCQ, Maricê N. OLIVEIRA, and Valeria D. GUIMARÃES were recipients of a CAPES fellowship (project CAPESCOFECUB #319II). Luis BERMUDEZ and Sébastien NOUAILLE were recipients of a fellowship from the French Ministry of Education and Research. INRA and Région IledeFrance also financed L. BERMUDEZ and V. GUIMARAES. Cathy CHARLIER is recipient of a fellowship from INRA and Région Bretagne.