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 1.5:1 molar ratio insert:vector and 1 &#181;l of salt solution (0.2 M NaCl and 0.01 MgCl2). Incubate for 5 min at room temperature (RT).</li>
		<li>Transform the entire reaction into chemically competent E. coli cells according to directional cloning kit specifications and screen obtained colonies by colony PCR3, using M13 forward and reverse primers included in the directional cloning kit. Isolate the plasmid from a positive colony according to plasmid DNA preparation kit specifications and sequence the insert using M13 forward and reverse primers to ensure the absence of mutations3.</li>
		<li>Use the obtained entry clone to perform the recombination reaction by the Lambda Recombinase Clonase II Enzyme (LR) with specific destination vectors: for recombinant protein expression in bacterial cells with pDEST17 (yielding pDEST17.G65mut), in plants (transient or stable) with pK7WG2 (yielding pK7WG2.G65mut), in Baculovirus/insect cell system with the linearized viral DNA (yielding Baculo.G65mut)4. Assemble the reaction, according to clonase kit specifications, with 100 ng of entry vector, 150 ng of destination vector and 2 &#181;l of enzyme mix in a final volume of 10 &#181;l. Incubate the mix at RT for 1 hr.</li>
		<li>Transform the recombination product into chemically competent E. coli cells according to clonase kit specifications. Screen obtained colonies by PCR3 using for all the transformation reaction derived colonies the same reverse GAD65mut-specific primer (5&#8217;-CACACGCCGGCAGCAGGT-3&#8217;) and the following specific destination vectors forward primers: for pDEST17: 5&#8217;-TAATACGACTCACTATAGGG-3&#8217;; for pK7WG2: 5&#8217;-AAGATGCCTCTGCCGACAGT-3&#8217;; for Baculo linearized vector: 5&#8217;-AAATGATAACCATCTCGC-3&#8217;.</li>
		<li>Isolate plasmid DNA using a commercial minipreparation DNA kit and confirm the presence of the target sequence by PCR3, using the same specific primer combinations described in the previous step.</li>
		<li>Use the obtained PCR-positive plasmids to transform different target organisms, depending on the platform chosen for recombinant protein expression as described in the following steps.</li>
	</ol>
	</li>
	<li>MagnICON system5-7:
	<ol>
		<li>Clone the target gene in the 3&#8217;-module pICH31070, as previously described in detail7, yielding pICH31070.G65mut. Use the following specific reaction cycle: 30 min incubation at 37 &#176;C, 5 min at 50 &#176;C and then 5 min at 80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Recombinant Protein Expression
<ol>
	<li>Bacterial cell system:
	<ol>
		<li>Transform pDEST17.G65mut into E. coli BL21 (DE3) electrocompetent cells using standard techniques and screen the colonies grown on ampicillin-containing (100 &#181;g/ml) LB-medium, by colony PCR using the following transgene-specific primers: 5&#8217;-CTGGTGCCAAGTGGCTCAGA-3&#8217; and 5&#8217;-CACACGCCGGCAGCAGGT-3&#8217;, with an annealing temperature of 58 &#176;C and an elongation time of 20 sec. Carry out the PCR reaction in a total volume of 20 &#181;l after dissolving bacterial cells with a plastic tip in the tube.</li>
		<li>Inoculate a single colony overnight at 37 &#176;C in 3 ml of ampicillin-containing (100 &#181;g/ml) LB-medium.</li>
		<li>Dilute the bacterial culture 1:100 in 100 ml of fresh LB medium (including ampicillin) and incubate at 37 &#176;C for 1-6 hr until a final OD600 of 0.8.</li>
		<li>Induce recombinant protein expression by addition of isopropil-&#946;-D-1-tiogalattopiranoside (IPTG) at a final concentration of 1 mM and incubate culture with vigorous shaking for 3 hr at 37 &#176;C.</li>
		<li>Collect cells by centrifugation at 4,000 x g for 20 min and use bacterial pellet for protein extraction (step 3.1.1.1).</li>
	</ol>
	</li>
	<li>Baculovirus/insect cell system:
	<ol>
		<li>Seed Spodoptera frugiperda (Sf9) cells into 6-well plates (8 x 105 cells per well) and wash twice with 2 ml of non-supplemented Grace&#8217;s Insect Medium.</li>
		<li>Remove and replace the medium drop-wise with transfection mixture (5 &#181;l LR recombinant reaction, 6 &#181;l Celfectin solution and 200 &#181;l non-supplemented Grace&#8217;s Insect Medium).</li>
		<li>Incubate plates at 27 &#176;C for 5 hr.</li>
		<li>Remove and replace transfection mixture with 2 ml of fresh Sf-900 medium, supplemented with 10% fetal bovine serum, 10 &#181;g/ml gentamycin and 100 &#181;M ganciclovir to select recombinant Baculovirus clones.</li>
		<li>After incubation for 96 hr at 27 &#176;C, collect medium (V1 viral stock), centrifuge at 4,000 x g to remove cells and large debris and store in the dark at 4 &#176;C.</li>
		<li>Seed 1 x 106 Sf9 cells per well in 2.5 ml Sf-900 medium containing 10% fetal bovine serum, 10 &#181;g/ml gentamycin and 100 &#181;M ganciclovir and infect with 100 &#181;l of V1 stock in every well.</li>
		<li>Incubate cells for 3 days at 27 &#176;C.</li>
		<li>Collect and centrifuge medium at 4,000 x g.</li>
		<li>Store the supernatant (V2 high-titer stock) at 4 &#176;C.</li>
		<li>Seed 1 x 106 Sf9 cells per well in 2.5 ml Sf-900 medium containing 10% fetal bovine serum, 10 &#181;g/ml gentamycin and 100 &#181;M ganciclovir and infect with 25 &#181;l of V2 stock in every well.</li>
		<li>Incubate cells for 3 days at 27 &#176;C.</li>
		<li>Collect cells by centrifuge at 4,000 x g and use insect cell pellet for protein extraction (step 3.1.2.1).</li>
	</ol>
	</li>
	<li>Nicotiana benthamiana transient expression systems:
	<ol>
		<li>Grow N. benthamiana plants in a greenhouse, under natural light within the temperature range 18-23 &#176;C. For agroinfection use 4-5 week old plants.</li>
		<li>Keep agroinfected plants in a climate chamber at 22 &#176;C with a 13 hr day/11 hr night photoperiod.</li>
		<li>Commercial recombination cloning system:
		<ol>
			<li>Introduce pK7WG2.G65mut in Agrobacterium tumefaciens strain EHA105 electrocompetent cells as previously described8 and screen the colonies, grown on LB medium containing rifampicin (50 &#181;g/ml), streptomycin (300 &#181;g/ml) and spectinomycin (100 &#181;g/ml) after 2 days of incubation at 28 &#176;C, by colony PCR8 using the following transgene-specific primers: 5&#8217;-CATGGTGGAGCACGACACGCT-3&#8217; and 5&#8217;-CACACGCCGGCAGCAGGT-3&#8217;, with an annealing temperature of 58 &#176;C and an elongation time of 50 sec. Carry out the PCR reaction in a total volume of 20 &#181;l.</li>
			<li>Inoculate bacteria in 30 ml of LB medium containing rifampicin (50 &#181;g/ml), streptomycin (300 &#181;g/ml) and spectinomycin (100 &#181;g/ml) for two days at 28 &#176;C.</li>
			<li>Collect bacteria by centrifugation at 4,000 x g for 20 min and resuspend pellet in 10 ml of infiltration buffer (10 mM MES, 10 mM MgCl2, 100 &#181;M acetosyringone, pH 5.6). Measure the OD600 value and then adjust it to 0.9 by diluting in the same buffer. 
			NOTE: the total volume needed for infiltrating three N. benthamiana plants is 30 ml.</li>
			<li>Use a 2.5 ml needleless syringe to infiltrate the Agrobacterium suspension in N. benthamiana leaves. Carefully and slowly inject leaf panels with the suspension from the syringe. Infiltrate three expanded leaves per plant and use three plants as triplicates. 
			NOTE: for health and safety reasons wear eye protection during infiltration process.</li>
			<li>Collect agroinfiltrated leaves 2 days post infection (dpi) and freeze in liquid nitrogen. Store plant tissue at -80 &#176;C.</li>
		</ol>
		</li>
		<li>MagnICON system:
		<ol>
			<li>Introduce the MagnICON vectors - the 5&#8217; module (pICH20111), the 3&#8217; module (pICH31070.G65mut), and the integrase module (pICH14011) - in A. tumefaciens GV3101 strain using standard techniques. Screen the colonies, grown on LB medium containing 50 &#181;g/ml rifampicin and appropriate vector-specific antibiotic (50 &#181;g/ml carbenicillin for pICH20111 and pICH14011, 50 &#181;g/ml kanamycin for pICH31070), by colony PCR using specific primers for each vector.</li>
			<li>Inoculate separately the three A. tumefaciens strains in 5 ml of LB medium containing 50 &#181;g/ml rifampicin and appropriate vector-specific antibiotic and shake overnight at 28 &#176;C.</li>
			<li>Pellet overnight bacterial cultures by centrifugation at 4,000 x g for 20 min and resuspend them in two volumes of the initial bacterial culture of 10 mM MES (pH 5.5) and 10 mM MgSO4.</li>
			<li>Mix equal volumes of bacterial suspensions containing the three vectors and use the suspension mix for syringe infiltration of N. benthamiana leaves. Infiltrate three expanded leaves per plant.</li>
			<li>Collect agroinfiltrated leaves at 4 dpi and freeze in liquid nitrogen.</li>
			<li>Store plant tissue at -80 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Nicotiana tabacum stable expression system:
	<ol>
		<li>Grow and maintain in vitro plantlets of N. tabacum (var. Sr1) on solid plant culture medium (4.4 g/L Murashige and Skoog - MS - medium including vitamins, 30 g/L sucrose, pH 5.8, 7 g/L plant agar) under controlled conditions in climate chamber at 25 &#176;C with 16 hr/8 hr day/night regime.</li>
		<li>Start a pre-culture of A. tumefaciens EHA105 harboring the expression vector pK7WG2.G65mut in 5 ml of liquid YEB medium with appropriate antibiotics (rifampicin 50 &#181;g/ml, streptomycin 300 &#181;g/ml, spectinomycin 100 &#181;g/ml) and grow overnight at 28 &#176;C in an orbital shaker set at 200 rpm.</li>
		<li>Use 1 ml of the pre-culture to inoculate a 50 ml Agrobacterium culture in YEB medium plus antibiotics (rifampicin 50 &#181;g/ml, streptomycin 300 &#181;g/ml, spectinomycin 100 &#181;g/ml) and grow for 24 hr, until the bacterial culture is saturated (OD600 value of 0.5-1.0).</li>
		<li>Collect bacteria by centrifugation at 4,000 x g for 20 min and resuspend pellet in 50 ml of liquid plant culture medium. Repeat this step twice to completely remove antibiotics.</li>
		<li>Take first healthy fully expanded leaves from in vitro tobacco plants and cut them into approximately 1 cm squares.</li>
		<li>Transfer leaf pieces to deep Petri dishes containing bacterial suspension and leave in the dark for 20 min.</li>
		<li>Remove leaf pieces from suspension and blot dry on sterile filter paper.</li>
		<li>Place leaf pieces with adaxial side (upper leaf surface) on solid plant culture medium containing 1.0 &#181;g/ml 6-benzylaminopurine (BAP) and 0.1 &#181;g/ml naphthalene acetic acid (NAA) and incubate plates for two days in a climate chamber at controlled conditions (25 &#176;C, 16 hr day/8 hr night).</li>
		<li>Transfer leaf pieces onto solid culture medium (including 1.0 &#181;g/ml BAP, 0.1 &#181;g/ml NAA, 500 &#181;g/ml cefotaxime, 100 &#181;g/ml kanamycin) with abaxial surface (lower surface of leaf) in contact with medium.</li>
		<li>Incubate plates for 2-3 weeks in the climate chamber at 25 &#176;C with 16 hr/8 hr day/night regime to induce shoot formation. Subculture every 2 weeks by transferring leaf explants to fresh solid plant culture medium containing 1.0 &#181;g/ml BAP, 0.1 &#181;g/ml NAA, 500 &#181;g/ml cefotaxime and 100 &#181;g/ml kanamycin.</li>
		<li>When shoots appear transfer them to Magenta boxes containing solid culture medium including 500 &#181;g/ml cefotaxime and 100 &#181;g/ml kanamycin to induce root formation. Incubate plantlets with 16 hr day/8 hr night photoperiod at 25 &#176;C for 1-2 weeks.</li>
		<li>When roots form, transfer the plants to soil in the greenhouse.</li>
		<li>Collect a leaf piece for each plant and isolate genomic DNA with a commercial kit.</li>
		<li>Detect the presence of the transgene by specific PCR. Use 30 ng of genomic DNA as template for PCR amplification using the following transgene-specific primers: 5&#8217;-CTGGTGCCAAGTGGCTCAGA-3&#8217; and 5&#8217;-CACACGCCGGCAGCAGGT-3&#8217;, with an annealing temperature of 58 &#176;C and an elongation time of 20 sec. Carry out the PCR reaction in a total volume of 50 &#181;l.</li>
		<li>Analyze the 220 bp product by electrophoresis on 1% agarose gel in Tris-acetate-EDTA (TAE) buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA).</li>
		<li>Select transgenic plants containing the target gene.</li>
		<li>Collect a leaf piece from each of the selected transgenic plant and freeze in liquid nitrogen immediately.</li>
		<li>Prepare total protein extracts from plant leaf tissue (step 3.1.3) and test each sample by western blot analysis as previously described2 to select the best recombinant protein expressing tobacco plant.</li>
		<li>Bag flowers of the selected best performing plant before blooming to prevent cross-pollination.</li>
		<li>After blooming, fruit ripening and seed drying, collect bags.</li>
		<li>Separate seeds from the chaff and store them in a dry room at controlled temperature (20-24 &#176;C).</li>
		<li>Sow the dried seeds to produce the second generation transgenic plants and subsequently select the best performing plant to be self-pollinated.</li>
		<li>Repeat the same procedure described in steps 2.4.19-2.4.21 for subsequent generations.</li>
	</ol>
	</li>
</ol>
3. Recombinant Protein Expression Analyses
<ol>
	<li>Total protein extraction:
	<ol>
		<li>Bacterial cells:
		<ol>
			<li>Resuspend the bacterial cell pellet, collected as described in step 2.1.5, in half the culture volume of Tris-buffered saline (TBS - 2 mM Tris/HCl , 500 mM NaCl) pH 7.4 supplemented with 1 mM phenylmethanesulfonylfluoride (PMSF).</li>
			<li>Sonicate resuspended cell pellet three times for 40 sec at half power, while keeping sample on ice.</li>
			<li>Clarify lysate by centrifugation at 14,000 x g for 20 min at 4 &#176;C.</li>
			<li>Transfer supernatant to a clean tube and store both supernatant and pellet separately at -80 &#176;C.</li>
			<li>Solubilize the inclusion bodies, collected in the pellet, in half cellular lysate volume of TBS pH 8.0 supplemented with 6 M urea by vigorous shaking overnight at room temperature.</li>
			<li>Centrifuge at 10,000 x g for 25 min at room temperature and collect supernatant in a clean tube.</li>
			<li>Store both supernatant and pellet at -80 &#176;C.</li>
		</ol>
		</li>
		<li>Insect cells:
		<ol>
			<li>Wash infected insect cell pellet, collected as described in step 2.2.12, with 1 ml of phosphate buffered saline (PBS - 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) pH 7.4.</li>
			<li>Resuspend cells in 200 &#181;l of lysis buffer (20 mM Tris/HCl pH 8.0, 0.5 M NaCl, 3 mM &#946;-mercaptoethanol and 1% Tween-20) and incubate on ice for 30 min.</li>
			<li>Centrifuge solubilized cells at 14,000 x g for 20 min at 4 &#176;C.</li>
			<li>Collect soluble fractions and store at -80 &#176;C and discard the pellet.</li>
		</ol>
		</li>
		<li>Plant leaf tissue:
		<ol>
			<li>Grind N. benthamiana or N. tabacum tissue to fine powder in liquid nitrogen using mortar and pestle.</li>
			<li>Homogenize 100 mg of ground tissue in 300 &#181;l of extraction buffer (40 mM Hepes pH 7.9, 5 mM DTT, 1.5% CHAPS), supplemented with 3 &#181;l of protease inhibitor cocktail and thaw on ice. 
			NOTE: the selected ratio between plant tissue weight (g) to buffer volume (ml) is 1:3.</li>
			<li>Centrifuge extract at 30,000 x g for 30 min at 4 &#176;C.</li>
			<li>Collect supernatant in a clean tube and store at -80 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Coomassie gel staining:
	<ol>
		<li>Prepare an appropriate dilution of total protein extracts (e.g., 2 &#181;l of plant extract, 5 &#181;l of bacterial and insect extracts) to a final volume of 10 &#181;l of extraction buffer and add 5 &#181;l of 3x sample buffer (1.5 M Tris/HCl pH 6.8, 3% SDS, 15% glycerol, 4% &#946;&#8211;mercaptoethanol) to a final 1x concentration.</li>
		<li>Boil samples for 10 min.</li>
		<li>Separate proteins by 10% SDS-PAGE.</li>
		<li>After electrophoresis, heat gel in the presence of Coomassie solution A (0.05% Brilliant Blue R-250, 25% isopropanol, 10% acetic acid) in a microwave oven for about two minutes until boiling point.</li>
		<li>Cool down gel to room temperature with gentle shaking.</li>
		<li>Discard Coomassie staining solution A.</li>
		<li>Destain gel by heating in the presence of Coomassie solution B (0.05% Brilliant Blue R-250, 25% isopropanol), C (0.002% Brilliant Blue R-250, 10% acetic acid) and D (10% acetic acid) each time following the same protocol for staining.</li>
		<li>After the last heating step with solution D, leave gel destaining till background is clear9.</li>
	</ol>
	</li>
	<li>Western blot analysis:
	<ol>
		<li>After electrophoresis, transfer separated proteins onto a nitrocellulose membrane using standard techniques and block with 4% milk in PBS pH 7.4 at room temperature for 1 hr.</li>
		<li>Incubate the blot overnight at 4 &#176;C or for 4 hr at room temperature in the blocking solution containing rabbit primary antibody raised against the target protein at 1:10,000 and supplemented with 0.1% Tween-20.</li>
		<li>After primary antibody labeling, wash the membrane 3 times for 5 min each with blocking solution containing 0.1% Tween-20.</li>
		<li>Incubate with horseradish peroxidase (HRP)-conjugate anti-rabbit antibody at 1:10,000 for 1.5 hr.</li>
		<li>Wash membrane 5 times for 5 min each with PBS-T (PBS supplemented with 0.1% Tween-20).</li>
		<li>Process western blot using the chemiluminescent peroxidase substrate.</li>
	</ol>
	</li>
	<li>Radioimmuno assay (RIA):
	<ol>
		<li>Allow reagents (antiserum, tracer and protein A Sepharose) to reach room temperature and pipet 20 &#181;l of protein extract samples and standards at different concentrations (from 300-2,400 ng/ml of commercial recombinant hGAD65) into 12 x 75 mm polystyrene tubes.</li>
		<li>Add 20 &#181;l of antiserum, prepared diluting 1:30 the serum from plasmapheresis of a T1D patient.</li>
		<li>Add 50 &#181;l of tracer (125-I-GAD65) and incubate for 2 hr at room temperature. Set a tube with tracer only to estimate the total activity.</li>
		<li>Add 50 &#181;l of protein A Sepharose and incubate for 1 hr at room temperature.</li>
		<li>Dispense 1 ml of cold PBS buffer into each tube and centrifuge at 1,500 x g at 4 &#176;C for 30 min.</li>
		<li>Decant the tubes to remove supernatant and absorb residual liquid on blotting paper by gently tapping tube.</li>
		<li>Measure immunoprecipitated radioactivity in all tubes by counting for 1 min in a gamma-counter.</li>
		<li>Plot in log scale the binding rates (B/T %) of calibrators related to total activity, to establish the standard curve, and read GAD concentration of samples off the calibration curve10. 
		NOTE: Restricted areas should be designed for storage, handling and disposal of radioactive material.</li>
	</ol>
	</li>
</ol>

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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 height="20" style="height:20px;width:191px;">Cell culture plates&#160;</td>
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		<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>
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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 Views.  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.