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>
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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...

Watch this Scientific Journal Video about A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems at JoVE.com

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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23.4K 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.

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

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...