This work was supported by the COST action &#8216;Molecular pharming: Plants as a production platform for high-value proteins&#8217; FA0804. The Authors thank Dr Anatoli Giritch and Prof. Yuri Gleba for providing the MagnICON vectors for research purposes.

1. Construction of Expression Vectors
<ol>
	<li>Commercial recombination cloning system:
	<ol>
		<li>Amplify the full-length sequence of the target gene (hGAD65mut) with appropriate primers allowing the addition of a CACC clamp at the 5&#8217;-end of the gene as previously described2.</li>
		<li>Clone the gel-purified amplification product, according to the directional cloning kit specifications, in the entry vector (topoisomerase bound) by assembling the reaction in a total volume of 6 &#181;l, using a 1.5:...

<ol>
	<li>Hampe, C. S., Hammerle, L. P., Falorni, A., Robertson, J., Lernmark, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+of+K396R+of+the+65+kDa+glutamic+acid+decarboxylase+active+site+obliterates+enzyme+activity+but+not+antibody+binding.">Site-directed mutagenesis of K396R of the 65 kDa glutamic acid decarboxylase active site obliterates enzyme activity but not antibody binding.</a> FEBS Lett. 488 (3), 185-189 (2001).</li><li>Avesani, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+GAD65+accumulates+to+high+levels+in+transgenic+tobacco+plants+when+expressed+as+an+enzymatically+inactive+mutant.">Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant.</a> Plant Biotechnol. J. 9 (8), 862-872 (2010).</li><li>Sambrook, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A laboratory manual. Second Edition. , (1989).</li><li>Avesani, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+different+biofactories+for+the+production+of+a+major+diabetes+autoantigen.">Comparative analysis of different biofactories for the production of a major diabetes autoantigen.</a> Transgenic Res. 23, 281-291 (2014).</li><li>Marillonnet, S., Giritch, A., Gils, M., Kandzia, R., Klimyuk, V., Gleba, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+planta+engineering+of+viral+RNA+replicons:+efficient+assembly+by+recombination+of+DNA+modules+delivered+by+Agrobacterium.">In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium.</a> Proc. Natl. Acad. Sci. (USA). 101 (18), 6852-6857 (2004).</li><li>Gleba, Y., Klimyuk, V., Marillonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+the+expression+of+proteins+in+plants).">Viral vectors for the expression of proteins in plants).</a> Curr. Opin. Biotechnol. 18, 134-141 (2007).</li><li>Engler, C., Kandzia, R., Marillonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+one+pot,+one+step,+precision+cloning+method+with+high+throughput+capability.">A one pot, one step, precision cloning method with high throughput capability.</a> PLoS One. 3 (11), (2008).</li><li>Xu, R., Li, Q. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+streamline+cloning+of+genes+into+binary+vectors+in+Agrobacterium+via+the+Gateway+TOPO+vector+system.">Protocol: streamline cloning of genes into binary vectors in Agrobacterium via the Gateway TOPO vector system.</a> Plant Methods. 4 (4), 1-7 (2008).</li><li>Fairbanks, G., Steck, T. L., Wallach, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+analysis+of+the+major+polypeptides+of+the+human+erythrocyte+membrane.">Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane.</a> Biochemistry. 10 (13), 2606-2617 (1971).</li><li>Falorni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioimmunoassay+detects+the+frequent+occurrence+of+autoantibodies+to+the+Mr+65,000+isoform+of+glutamic+acid+decarboxylase+in+Japanese+insulin-dependent+diabetes.">Radioimmunoassay detects the frequent occurrence of autoantibodies to the Mr 65,000 isoform of glutamic acid decarboxylase in Japanese insulin-dependent diabetes.</a> Autoimmunity. 19, 113-125 (1994).</li><li>Hunt, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+gene+to+protein:+a+review+of+new+and+enabling+technologies+for+multi-parallel+protein+expression.">From gene to protein: a review of new and enabling technologies for multi-parallel protein expression.</a> Protein Expr. Purif. 40 (1), 1-22 (2005).</li><li>Arzola, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+co-expression+of+post-transcriptional+silencing+suppressor+for+increased+in+planta+expression+of+a+recombinant+anthrax+receptor+fusion+protein.">Transient co-expression of post-transcriptional silencing suppressor for increased in planta expression of a recombinant anthrax receptor fusion protein.</a> Int. J. Mol. Sci. 12 (8), 4975-4990 (2011).</li><li>Merlin, M., Gecchele, E., Capaldi, S., Pezzotti, M., Avesani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+recombinant+protein+production+in+different+biofactories:+the+green+perspective.">Comparative evaluation of recombinant protein production in different biofactories: the green perspective.</a> Biomed. Res. Int. 2014, 136419 (2014).</li><li>Avesani, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+in+planta+expression+of+the+human+islet+autoantigen+glutamic+acid+decarboxylase+(GAD65).">Improved in planta expression of the human islet autoantigen glutamic acid decarboxylase (GAD65).</a> Transgenic Res. 12 (2), 203-212 (2003).</li><li>Leuzinger, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+agroinfiltration+of+Plants+for+high-level+transient+expression+of+recombinant+proteins.">Efficient agroinfiltration of Plants for high-level transient expression of recombinant proteins.</a> J Vis Exp. (77), (2013).</li></ol>

The authors declare that there is no conflict of interests regarding the publication of this paper.

In this study three different platforms were compared for the expression of a recombinant human protein: bacterial cells, Baculovirus/insect cells and plants. The plant-based platform was further explored by exploiting three widely used expression technologies (i.e., transient - MagnICON and pK7WG2 based - and stable). The target protein chosen for this experiment, hGAD65mut, has been previously expressed in different systems13, and its production and functionality are easily detectable and measurable...

Recombinant proteins are commercially mass-produced in heterologous expression systems with the aid of emerging biotechnology tools. Key factors that have to be considered when choosing the heterologous expression system include: protein quality, functionality, process speed, yield and cost.
In the recombinant protein field, the market for pharmaceuticals is expanding rapidly and, consequently, most biopharmaceuticals produced today are recombinant. Proteins can be expressed in cell cultures of bacteria, yeasts, molds, mammals, plants and insects, as well as in plant systems (either via stable- or transient-transformation) and transgenic animals; each expression system has its inherent advantages and limitations and for each target recombinant protein the optimal production system has to be carefully evaluated.
Plant-based platforms are arising as an important alternative to traditional fermenter-based systems for safe and cost-effective recombinant protein production. Although downstream processing costs are comparable to those of microbial and mammalian cells, the lower up-front investment required for commercial production in plants and the potential economy of scale, provided by cultivation over large areas, are key advantages.
We evaluated plants as bioreactors for the expression of the 65 kDa isoform of human glutamic acid decarboxylase (hGAD65), one of the major autoantigen in Type 1 autoimmune diabetes (T1D). hGAD65 is largely adopted as a marker, both for classifying and monitoring the progression of the disease and its role in T1D prevention is currently under investigation in clinical trials. If these trials are successful, the global demand for recombinant hGAD65 will increase dramatically.
Here, we focus on the expression of the enzymatically inactive counterpart of hGAD65, hGAD65mut, a mutant generated by substituting the lysine residue that binds the cofactor PLP (pyridoxal-5'-phosphate) with an arginine residue (K396R)1. 
hGAD65mut retains its immunogenicity and, in plant and insect cells, accumulates up to ten-fold higher than hGAD65, its wild-type counterpart. It was hypothesized that the enzymatic activity of hGAD65 interferes with plant cell metabolism to such an extent that it suppresses its own synthesis, whereas hGAD65mut, the enzymatically-inactive form, can be accumulated in plant cells to higher levels.
For the expression of hGAD65mut, the use of different technologies, widely used in plant biotechnology, was explored here and compared to traditional expression platforms (Escherichia coli and Baculovirus/insect cell-based).
In this work, the recombinant platforms developed for the expression of hGAD65mut comprising traditional and plant-based systems were reviewed and compared on the basis of process speed and yield, and of final product quality and functionality.

<table border="0" cellpadding="0" cellspacing="0" width="667">
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			<td dir="LTR" height="40" style="height:40px;width:191px;">Plant protease inhibitor cocktail</td>
			<td dir="LTR" style="width:111px;">Sigma</td>
			<td dir="LTR" style="width:143px;">P9599</td>
			<td dir="LTR" style="width:223px;">Do not freeze/thaw too many times</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:191px;">SDS (Sodium dodecyl sulphate)</td>
			<td dir="LTR" style="width:111px;">Sigma</td>
			<td dir="LTR" style="width:143px;">L3771</td>
			<td dir="LTR" style="width:223px;">Flammable, toxic, corrosive &#8211; 10% stock</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:191px;">Glycerol</td>
			<td dir="LTR" style="width:111px;">Sigma</td>
			<td dir="LTR" style="width:143px;">G5516</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:191px;">Brilliant Blue R-250</td>
			<td dir="LTR" style="width:111px;">Sigma</td>
			<td dir="LTR" style="width:143px;">B7920</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:191px;">Isopropanol</td>
			<td dir="LTR" style="width:111px;">Sigma</td>
			<td dir="LTR" style="width:143px;">24137</td>
			<td dir="LTR" style="width:223px;">Flammable</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:191px;">Acetic acid</td>
			<td dir="LTR" style="width:111px;">Sigma</td>
			<td dir="LTR" style="width:143px;">27221</td>
			<td dir="LTR" style="width:223px;">Corrosive</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:191px;">Anti-Glutamic acid decarboxylase 65/67</td>
			<td dir="LTR" style="width:111px;">Sigma</td>
			<td dir="LTR" style="width:143px;">G5163</td>
			<td dir="LTR" style="width:223px;">Do not freeze/thaw too many times</td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:191px;">Horseradish peroxidase (HRP)-conjugate anti-rabbit antibody</td>
			<td dir="LTR" style="width:111px;">Sigma</td>
			<td dir="LTR" style="width:143px;">A6154</td>
			<td dir="LTR" style="width:223px;">Do not freeze/thaw too many times</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:191px;">Sf9 Cells</td>
			<td dir="LTR" style="width:111px;">Life Technologies</td>
			<td dir="LTR" style="width:143px;">11496</td>
			<td dir="LTR" style="width:223px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:191px;">BL21 Competent E.coli</td>
			<td dir="LTR" style="width:111px;">New England Biolabs</td>
			<td dir="LTR" style="width:143px;">C2530H</td>
			<td dir="LTR" style="width:223px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Protein A Sepharose</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:143px;">P2545</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Cell culture plates&#160;</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:143px;">CLS3516</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Radio Immuno Assay kit</td>
			<td>Techno Genetics</td>
			<td>12650805</td>
			<td style="width:223px;">Radioactive material&#160;</td>
		</tr>
	</tbody>
</table>

a comparative analysis of recombinant protein expression in different biofactories: bacteria, insect cells and plant systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

An experimental design for the heterologous expression of a target recombinant protein in different production systems is described here. The first focus was the set-up of the different platforms by establishing the optimal conditions for the expression of the target protein in each system.
The expression of the target protein, hGAD65mut, was induced in triplicate E. coli cultures. Following 3 hr of expression at 37 &#176;C, bacterial cells were collected by centrifugation and lysed b...

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A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

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23.2K visualizações.  University of Verona, Verona, Italy. In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.