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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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><tbody><tr><td>Yeast extract</td><td>Sigma</td><td>Y1333</td><td></td></tr><tr><td>Tryptone</td><td>Formedium</td><td>TRP03</td><td></td></tr><tr><td>Agar Bacteriological Grade</td><td>Applichem</td><td>A0949</td><td></td></tr><tr><td>Sf-900 II SFM medium</td><td>Gibco</td><td>10902-088</td><td></td></tr><tr><td>Grace&amp;rsquo;s Insect Medium, unsupplemented</td><td>Gibco</td><td>11595-030</td><td></td></tr><tr><td>Cellfectin II Reagent</td><td>Invitrogen</td><td>10362-100</td><td></td></tr><tr><td>MS medium including vitamins</td><td>Duchefa Biochemie</td><td>M0222</td><td></td></tr><tr><td>Sucrose</td><td>Duchefa Biochemie</td><td>S0809</td><td></td></tr><tr><td>Plant agar</td><td>Duchefa Biochemie</td><td>P1001</td><td></td></tr><tr><td>Ampicillin sodium</td><td>Duchefa Biochemie</td><td>A0104</td><td>Toxic</td></tr><tr><td>Gentamycin sulphate</td><td>Duchefa Biochemie</td><td>G0124</td><td>Toxic</td></tr><tr><td>Ganciclovir</td><td>Invitrogen</td><td>I2562-023</td><td></td></tr><tr><td>Carbenicillin disodium</td><td>Duchefa Biochemie</td><td>C0109</td><td>Toxic</td></tr><tr><td>Kanamycin sulfate</td><td>Sigma</td><td>K4000</td><td>Toxic</td></tr><tr><td>Rifampicin</td><td>Duchefa Biochemie</td><td>R0146</td><td>Toxic &amp;ndash; 25 mg/ml stock in DMSO</td></tr><tr><td>Streptomycin sulfate</td><td>Duchefa Biochemie</td><td>S0148</td><td>Toxic</td></tr><tr><td>Spectinomycin dihydrochloride</td><td>Duchefa Biochemie</td><td>S0188</td><td></td></tr><tr><td>IPTG (isopropil-&amp;beta;-D-1-tiogalattopiranoside)</td><td>Sigma</td><td>I5502</td><td>Toxic</td></tr><tr><td>MES hydrate</td><td>Sigma</td><td>M8250</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biochemical</td><td>436994U</td><td></td></tr><tr><td>Acetosyringone</td><td>Sigma</td><td>D134406</td><td>Toxic &amp;ndash; 0.1 M stock in DMSO</td></tr><tr><td>Syringe (1 ml)</td><td>Terumo</td><td></td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fluka</td><td>63136</td><td></td></tr><tr><td>BAP (6-Benzylaminopurine)</td><td>Sigma</td><td>B3408</td><td>Toxic</td></tr><tr><td>NAA (Naphtalene acetic acid)</td><td>Duchefa Biochemie</td><td>N0903</td><td>Irritant</td></tr><tr><td>Cefotaxime</td><td>Mylan Generics</td><td></td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1503</td><td>Adjust pH with 1 N HCl to make Tris-HCl buffer</td></tr><tr><td>HCl</td><td>Sigma</td><td>H1758</td><td>Corrosive</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014</td><td>1 M stock</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>PMSF (Phenylmethanesulfonylfluoride)</td><td>Sigma</td><td>P7626</td><td>Corrosive, toxic</td></tr><tr><td>Urea</td><td>Sigma</td><td>U5378</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M3148</td><td>Toxic</td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P5927</td><td></td></tr><tr><td>Hepes</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>DTT (Dithiothreitol)</td><td>Sigma</td><td>D0632</td><td>Toxic &amp;ndash; 1 M stock, store at -20 &amp;deg;C</td></tr><tr><td>CHAPS</td><td>Duchefa Biochemie</td><td>C1374</td><td>Toxic</td></tr><tr><td>Plant protease inhibitor cocktail</td><td>Sigma</td><td>P9599</td><td>Do not freeze/thaw too many times</td></tr><tr><td>SDS (Sodium dodecyl sulphate)</td><td>Sigma</td><td>L3771</td><td>Flammable, toxic, corrosive &amp;ndash; 10% stock</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>Brilliant Blue R-250</td><td>Sigma</td><td>B7920</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>24137</td><td>Flammable</td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>27221</td><td>Corrosive</td></tr><tr><td>Anti-Glutamic acid decarboxylase 65/67</td><td>Sigma</td><td>G5163</td><td>Do not freeze/thaw too many times</td></tr><tr><td>Horseradish peroxidase (HRP)-conjugate anti-rabbit antibody</td><td>Sigma</td><td>A6154</td><td>Do not freeze/thaw too many times</td></tr><tr><td>Sf9 Cells</td><td>Life Technologies</td><td>11496</td><td></td></tr><tr><td>BL21 Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>New England Biolabs</td><td>C2530H</td><td></td></tr><tr><td>Protein A Sepharose</td><td>Sigma</td><td>P2545</td><td></td></tr><tr><td>Cell culture plates</td><td>Sigma</td><td>CLS3516</td><td></td></tr><tr><td>Radio Immuno Assay kit</td><td>Techno Genetics</td><td>12650805</td><td>Radioactive material</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 | Biology | Video

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 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.

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

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Genetics

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Biochemistry

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Bioengineering

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

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 ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

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

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Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

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

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension