Name: expression of recombinant cellulase cel5a from trichoderma reesei in tobacco plants
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Description: Watch this Scientific Journal Video about Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants at JoVE.com

This work was supported by the BMBF Forschungsinitiative &#8220;BioEnergie 2021 &#8211; Forschung f&#252;r die Nutzung von Biomasse&#8221; and the Cluster of Excellence &#8220;Tailor-made Fuels from Biomass&#8221;, which is funded through the Excellence Initiative by the German federal and state governments to promote science and research at German universities.

1. Growth of Wild Type N. tabacum&#160;Plants
<ol>
	<li>To grow N. tabacum L. cv. Petit Havana SR1 plants on soil, place tobacco seeds on appropriate pots filled with normal potting soil for germination. After 2 weeks, transfer seedlings to individual pots. Incubate plants in a greenhouse with constant 22 &#176;C (dark period) or 25 &#176;C (light period) and 70% relative air humidity. Provide illumination in a 16/8&#160;hr light/dark cycle (e.g. Philips IP65 400 W lamps; 180 &#956;mol&#183;sec-1&#183;m-2; &#955;= 400-700 nm) and water daily.</li>
</ol>
2. Transient Expression of TrCel5A and TrCel5A-mCherry Proteins
<ol>
	<li>Under sterile conditions, pick single colonies of Agarobacterium tumefaciens (GV3101::pMP90RK GmR, KmR, RifR) containing either pTRAkc-ER-TrCel5A or pTRAkc-ER -TrCel5A-mCherry and transfer to an Erlenmeyer flask containing 10 ml&#160;yeast extract broth (YEB, prepared as per manufacturers specifications) medium and kanamycin (50 mg/ml), rifampicin (50 mg/ml), and carbenicillin (100 mg/ml) for selection. Incubate for 36-40 hr at 26 &#176;C and 180 rpm. Note: The design of appropriate constructs for protein expression is outside the scope of this paper, but for guidance, the methods of Maclean et al.13 and Munro and Pellham14 can be followed for construct design.</li>
	<li>Take 200 &#181;l&#160;of each culture to inoculate 20 ml&#160;of YEB with the antibiotic concentrations mentioned above and incubate under the same conditions as before.</li>
	<li>After 36-40 hr, preinduce the cultures by adding 2 &#181;l&#160;acetosyringon (200 &#181;M final concentration), 100 &#181;l&#160;40% (w/v) glucose solution and 200 &#181;l&#160;of 1 M MES buffer pH 5.6. Incubate overnight.</li>
	<li>Prepare 100 ml&#160;of a 2x infiltration buffer (100 g/L sucrose, 3.6 g/L glucose, 8.6 g/L Murashige and Skoog basal salts, adjust pH to 5.6 using potassium hydroxide).</li>
	<li>Measure OD600 of the cultures with 1:5 dilutions until it reaches an OD600 of 5-6. Calculate culture volume needed for 30 ml&#160;infiltration medium at OD600. Add 15 ml&#160;of 2x infiltration buffer, then add deionized H2O to make the buffer up to 30 ml.</li>
	<li>Take plants from greenhouse and use a pipette tip to nick the abaxial side of the 3rd to 5th leaf from the top to ease infiltration.</li>
	<li>Use a pipette tip to nick the abaxial side of the leaves to ease infiltration.</li>
	<li>Fill a syringe with infiltration medium and put it (without needle) on one of the previously made nicks. Support syringe with a finger on the adaxial leaf side. Press the medium carefully into the tissue of interveinal leaf zones. Note: After infiltration, cultivate plants in a greenhouse under the same conditions as described above. Also, maintain an equal proportion of untreated, non-transient expression plants (NTEPs) as controls, under the same conditions as infiltrated plants.</li>
</ol>
3. Assessment of TrCel5A-mCherry Expression in Plant Leaves
<ol>
	<li>After 4-6 days, take plants infiltrated with A. tumefaciens containing pTRAkc-ER-TrCel5A-mCherry from the greenhouse and place them in a dark room for fluorescence analysis.</li>
	<li>To visualize the fluorescence in leaf sections expressing TrCel5A-mCherry, light plants using a portable light source emitting green light at 515&#160;nm with a red filter (620-750&#160;nm). Note: In depth characterization of protein expression in the plant leaves can be achieved by thin-sectioning leaves with a vibratome and examination under light and fluorescence microscopy. For this follow the steps below:
	<ol>
		<li>Cut small sections of approximately 5 x 5 mm size from an infiltrated leaf and embed them in 4% agarose dissolved in 50 mM Phosphate buffer (pH 5.7).</li>
		<li>Cut section of 80-90 &#181;m size from embedded leaves using a vibratome. Place them on a microscope slide, cover with buffer or a cover slip and examine them using a microscope at 10X&#160;magnification with a fluorescence detector, using the same wavelength and filter stated above.</li>
	</ol>
	</li>
</ol>
4. TrCel5A Extraction from Tobacco Leaves
<ol>
	<li>Cut pieces from tobacco leaves to an approximate weight of 1 g from plants&#160;infected with A. tumefaciens containing pTRAkc-ER-TrCel5A and place in a mortar precooled with liquid nitrogen. Add an appropriate amount of liquid nitrogen to the samples. Grind leaves to powder using a precooled pestle.</li>
	<li>Add 2 ml&#160;of phosphate buffered saline (PBS) containing 1 mM phenylmethylsulfonyl fluoride to the ground leaf matter and mix until the majority of leaf matter is in suspension. Centrifuge extract at 15,000 x g for 20 min at 4 &#176;C and take protein-containing supernatant.</li>
	<li>Centrifuge the extract at 15,000 x g for 20 min at 4&#160;&#176;C and take protein-containing supernatant being careful not to disrupt the pellet. Discard the pellet.</li>
	<li>Partially purify TrCel5A by incubating the protein-containing supernatant at 55 &#176;C for 10 min. Allow to rest at room temperature for a further 10 min, then centrifuge at 15,000 x&#160;g for 10 min at RT. Repeat partial purification process for non-infiltrated plants to obtain control samples. Use these samples for all following experiments. Note: Protein solutions can be stored at 4 &#176;C for up to 1 week or at -20 &#176;C for up to 3 months.</li>
	<li>Repeat the partial purification process for non-infiltrated plants to obtain control samples. Use these samples for all following experiments. Note: Protein solutions can be stored at 4&#160;&#176;C for up to 1 week or at -20&#160;&#176;C for up to 3 months.</li>
</ol>
5. SDS-PAGE, Western Blotting, and Azo-CMC Zymography of TrCel5A-mCherry
<ol>
	<li>Purchase or prepare 12% (w/v) SDS-PAGE gels as per established methods15.</li>
	<li>Dissolve CMC&#160;in water to obtain a 1.5% (w/v) solution. To make CMC-SDS-PAGE gels, substitute 1 ml&#160;of 1.5% CMC for 1 ml&#160;of water in a 10 ml&#160;SDS-PAGE gel mix solution, resulting in a 0.1% (w/v) CMC + 12% (w/v) SDS-PAGE gel mix. Pour gel normally. Note: Make certain that the CMC in the original solution is completely dissolved by a combination of stirring and heating to 40 &#176;C (repeat directly prior to preparing the gel solution).</li>
	<li>Denature samples by boiling in the presence of SDS, as per established methods12. As a positive control, use a 1:500 dilution of a commercially available cellulase mixture from T. reesei, such as T. reesei ATCC 26921. As a negative control use a similarly prepared protein solution from non-infiltrated leaves (NTEPs).</li>
	<li>Carry out the electrophoresis as per normal protocols15, e.g.&#160;use 200 V for ~50 min with the electrophoresis halted once the dye front reached the bottom of the gel.</li>
	<li>Stain one SDS-PAGE gel using 0.1% (w/v) Coomassie brilliant blue and destain using a methanol/glacial acetic acid mixture. Note: Rapid staining and destaining can be performed by gently heating the staining and destaining solutions, i.e. by using a microwave for ~15 sec to warm (but not boil) first the staining and then the destaining solutions, changing the destaining solution after 10 min, or when cooled, and repeat with new destaining solution.</li>
	<li>Perform Western blotting of one SDS-PAGE gel using established protocols16,17. Use the following antibodies: a polyclonal rabbit antibody against a&#160;His-tag (primary) and a polyclonal goat anti-rabbit antibody Fc region which has been conjugated to an alkaline phosphatase (secondary). Note: secondary antibodies should be selected to enable visualization using nitroblue tetrazolium chloride/5-bromo-4-chloro-3&#8242;-indolyphosphate p-toluidine salt (NBT/BCIP), as described18.</li>
	<li>Perform in-gel protein refolding on the 0.15% (w/v) CMC SDS-PAGE gel by incubating the gel in 1.5% (w/v) &#946;-cyclodextrin, 20 mM phosphate-citrate buffer, pH 6.5, at room temperature with gentle shaking for 15 min19.</li>
	<li>Discard the &#946;-cyclodextrin solution and briefly wash the gel with H2O. Incubate at RT in 50mM sodium acetate buffer (pH 4.8) for a further 15 min. Perform CMC zymography using the refolded 0.15% CMC SDS-PAGE gel by incubating the gel in 30 mM sodium acetate buffer pH 4.8 for 20 min at 50 &#176;C.</li>
	<li>Perform CMC zymography using the refolded 0.15% (w/v) CMC SDS-PAGE gel by incubating the gel in 30 mM sodium acetate buffer&#160;(pH 4.8) for 20 min at 50&#160;&#176;C.</li>
	<li>Stain the gel for 10&#160;min using a 0.1% w/v Congo red solution and destain for 30 min or until clear bands are observed using 1 M NaCl. Wash the gel with 0.3% (v/v) acetic acid for clearer imaging.</li>
</ol>
6. 4-MUC Activity Assay of TrCel5A
<ol>
	<li>Prepare a stock solution of 10 mM 4-MUC in 50% (v/v) methanol, and store it in the dark at RT</li>
	<li>Immediately prior to the assay, prepare a 4-MUC 2x working solution, composed of 1 mM 4-MUC and 60 mM sodium acetate, pH 4.8.</li>
	<li>Perform this test on samples containing 100 &#956;l of leaf protein (with or without TrCel5A inducibly expressed), 100 &#956;l a commercially available cellulase mixture from T. reesei 1:1,000, and pure H2O, respectively. Run each sample in triplicate.</li>
	<li>Pipette 100 &#956;l of the 4-MUC working solution into separate wells of a 96-well plate, then add 100 &#956;l of each sample.</li>
	<li>Determine 0 min timepoint of 4-MU fluorescence through fluorescent spectroscopy, using an excitation wavelength of 360 nm and an emission wavelength of 465 nm.</li>
	<li>Incubate plates for 20 min at 50 &#176;C, covered with adhesive lids to prevent evaporation. Stop the reaction by freezing (or by combining&#160;100 &#956;l 0.15 M glycine, pH 10.0, and 100 &#956;l of sample in a separate plate).</li>
	<li>Determine the endpoint of 4-MU fluorescence through fluorescent spectroscopy, using an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Subtract 0 min from endpoint (20 min) timepoint to obtain the change in fluorescence values.</li>
</ol>
7. Azo-carboxymethyl Cellulose Activity Assay of TrCel5A
<ol>
	<li>Prepare a stock of 1.5% (w/v) Azo-CMC which is stored at RT, and a precipitation solution containing 18 mM zinc acetate, 0.5 M sodium acetate, pH 5 and 80% ethanol (v/v), store at room temperature.</li>
	<li>Immediately prior to the assay, prepare an Azo-CMC 2x working solution, composed of 1% (w/v) Azo-CMC (in H2O) and 60 mM sodium acetate, pH 4.8.</li>
	<li>Combine 50 &#956;l of sample and 50 &#956;l of the working solution in 1.5 ml&#160;tubes.</li>
	<li>Test samples containing 50 &#956;l of leaf protein (with or without TrCel5A transiently expressed); 100 &#956;l of a commercially available cellulase mixture from T. reesei diluted 1:1,000; and pure H2O. Run samples and standards in triplicate.</li>
	<li>Incubate samples for 20 min at 50 &#176;C. Stop the reaction by adding 250 &#956;l of precipitation solution. Vortex each sample vigorously for 10 sec to ensure solutions were completely combined.</li>
	<li>Incubate samples at RT for 10 min and then vortex again. Centrifuge samples at 1,000 x g for 10 min. Remove 200 &#956;l&#160;of each sample supernatant to a 96-well plate, taking care not to disrupt the pellet.</li>
	<li>Assay using absorbance spectroscopy at a wavelength of 590 nm.</li>
</ol>
8. PAHBAH Assay of TrCel5A
<ol>
	<li>Use a hole-punch to cut a circle (~5.5 mm diameter) of filter paper. Add 100 &#956;l&#160;60 mM NaAc, pH 4.8, and 100 &#956;l&#160;of samples, either containing leaf protein (with or without TrCel5A inducibly expressed) or a commercial T. reesei cellulase mixture, diluted 1:1,000, and incubate with filter paper circles for 20 hr at 50 &#176;C with 300 rpm shaking. For each sample incubated with filter paper, also incubate a duplicate solution without filter paper as individual sample-controls.</li>
	<li>Prepare a 330 mM p-hydroxy benzoic acid hydrazide (PAHBAH) solution A by dissolving PAHBAH in H2O with the addition of 5 ml&#160;concentrated HCl for a total of 100 ml&#160;solution, and store at RT. Note: Best results are achieved by adding the HCl to a smaller volume of PAHBAH slurry, i.e. 30 ml, which is stirred under temperature to help dissolution before making the volume to 100 ml&#160;with H2O.</li>
	<li>Prepare PAHBAH solution B, containing 50 mM sodium citrate, 10 mM calcium chloride, and 500 mM NaOH and&#160;store&#160;at RT.</li>
	<li>Immediately prior to the assay, prepare a 1.5x working solution of 1 ml&#160;PAHBAH reagent to 9 ml&#160;buffer solution, store on ice until use (to be used within 4 hr).</li>
	<li>Prepare samples in thermostable 0.2 or 0.5 ml&#160;tubes. Combine 5 &#956;l of each solution incubated with filter paper (step 8.1) 45 &#956;l of H2O and 100 &#956;l of PAHBAH working solution. Sample controls (samples incubated without filter paper) should be run under the same concentrations (5 &#956;l sample, 45 &#956;l H2O, 100 &#956;l PAHBAH working solution). Also test 5 standards (5 &#956;l) containing between 0 and 0.2 g/L of glucose, and pure H2O. Run samples and standards in triplicate.</li>
	<li>Incubate samples for exactly 10 min at 100 &#176;C, then cool at RT for exactly 10 min. Pipette solutions into a 96 well plate and assay spectroscopically at a wavelength of 410 nm. Subtract individual sample-controls (incubated without filter paper) from samples to obtain final values.</li>
</ol>

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The authors have nothing to disclose.

Recombinant expression of cellulases is a field of great interest, due to the desire for more efficient biofuel production systems1. Plants offer many possibilities for successful cellulase production, with features such as economic harvesting techniques and autohydrolysis methods being very desirable properties for large-scale biofuel production. Further, the use of different localizations for producing proteins allow, if required, posttranslational modifications20. Altering plants to enable consti...

Degradation of lignocellulosic biomass and its conversion into liquid fuel has been envisioned as a method to reduce reliance on fossil fuels. One significant hurdle in establishing economically feasible biomass processing systems is in the enzymatic degradation of cellulose and hemicellulose1. Plant expression systems show great potential for the production of enzymes on an industrial scale. Agricultural systems to harvest plants economically and in volume are already established, as they have been for thousands of years. The production of cellulases in plants is desirable due to factors like the ease of autohydrolysis2, and the potential for maximizing the use of lower enzyme levels by increasing the digestibility of plant cell walls1. Finally, plant systems allow the targeting of recombinant proteins to specific areas of plant cells and are able to posttranslationally modify enzymes where required3.

Nicotiana tabacum (tobacco) is very commonly used as a model organism for heterologous protein expression studies in plants, due to its rapid growth and biomass accumulation features4. Transient expression of recombinant proteins is a technique which enables protein production in short time periods5, while maintaining flexibility as to the localization and thus posttranslational modifications of the selected proteins, i.e. cellulases. This enables the production of the cellulase(s) for basic analysis, while also laying the groundwork for further expression strategies in plants. Using such a transient expression strategy, an endoglucanase from glycosyl hydrolase family 5, Cel5A, derived from the fungal host Trichoderma reesei (Hypocrea jecorina)6 is produced (hereafter referred to as TrCel5A). TrCel5A is a 42 kDa protein which is natively glycosylated and is highly active in hydroylzing cellulose chains7.

The transient expression technique described here is based on a relatively commonly used system, infiltrating the plant leaves with Agrobacteria carrying the gene of interest in an appropriate expression vector. To allow for rapid analysis of successful in planta expression, TrCel5A can also be expressed as a fusion protein with mCherry, a monomeric fluorescent protein originally derived from Discosoma sp. protein DsRed8, with an additional six Histidine residues (His-tag) fused to the C-terminus (TrCel5A-mCherry). Expression of the heterologous fusion protein, TrCel5A-mCherry, can thus be monitored within the growing plant by using green light to examine mCherry dispersal. If desired, thin sections of the plant material can be examined under green light microscopically to establish the specific localization of the protein. For this work, signal and transit peptides were incorporated in the construct, to localize the heterologous protein export to the endoplasmic reticulum9.

To analyze the activity of plant expressed cellulases, including TrCel5A, a number of cellulase activity tests can be run. After the extraction of the total soluble plant proteins, TrCel5A can be partially purified using a thermal incubation technique. Protein size is established using SDS-PAGE followed by Western blotting. Zymography can be used to analyze activity against substrates, e.g. cellulose which has been carboxy-methylated (making carboxymethyl cellulose: CMC) for solubility10. Cellulase and glucosidase activity can be monitored using the fluorophore 4-methylumbelliferyl (4-MU) associated with &#946;-D-cellobioside (combined: 4-MUC). Another method to assess endoglucanase activity involves the spectrometric analysis of CMC&#160;which has been associated with an azo-dye (Remazolbrilliant Blue R)11. In addition, protein activity of both endoglucanases and a range of cellulases can be monitored by the use of a sugar analysis test, such as the p-hydroxy benzoic acid hydrazide (PAHBAH) assay12. These techniques can be used to elucidate and quantify the activity of the expressed cellulase of interest.

<table><tbody><tr><td>4-methylumbelliferyl &amp;beta;-D-cellobioside</td><td>SIGMA-ALDRICH</td><td>M6018</td><td></td></tr><tr><td>Acetic acid</td><td>ROTH</td><td>3738</td><td></td></tr><tr><td>Acetosyringon (concentrated)</td><td>ROTH</td><td>6003</td><td></td></tr><tr><td>Antibiotic - kanamycin</td><td>ROTH</td><td>T832</td><td></td></tr><tr><td>Antibiotic - rifampicin</td><td>ROTH</td><td>4163</td><td></td></tr><tr><td>Antibiotic - carbenicillin</td><td>ROTH</td><td>6344</td><td></td></tr><tr><td>Antibody - rabbit anti His(6) pAb</td><td>ROCKLAND</td><td>600-401-382</td><td></td></tr><tr><td>Antibody - goat anti rabbit AP, FC pAb</td><td>DIANOVA</td><td>111-055-008</td><td></td></tr><tr><td>Azo-carboxymethyl cellulose</td><td>MEGAZYME</td><td>S-ACMC</td><td></td></tr><tr><td>&amp;beta;-cyclodextrin</td><td>SIGMA-ALDRICH</td><td>C4805</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>SIGMA-ALDRICH</td><td>C5080</td><td></td></tr><tr><td>Carboxymethyl cellulose</td><td>ROTH</td><td>3333.1</td><td></td></tr><tr><td>Cellulase from &lt;em&gt;Trichoderma reesei&lt;/em&gt; ATCC 26921</td><td>SIGMA-ALDRICH</td><td>C2730</td><td></td></tr><tr><td>Congo red</td><td>SIGMA-ALDRICH</td><td>75768</td><td></td></tr><tr><td>Coomasie brilliant blue G 250</td><td>ROTH</td><td>9598.2</td><td></td></tr><tr><td>D(+)-glucose</td><td>ROTH</td><td>8337</td><td></td></tr><tr><td>Ethanol</td><td>ROTH</td><td>K928</td><td></td></tr><tr><td>Filter paper - Rotilabo blotting paper</td><td>ROTH</td><td>CL67</td><td></td></tr><tr><td>NBT/BCIP</td><td>SIGMA-ALDRICH</td><td>72091</td><td></td></tr><tr><td>MES buffer</td><td>ROTH</td><td>4256</td><td></td></tr><tr><td>Methanol</td><td>ROTH</td><td>8388</td><td></td></tr><tr><td>Sodium citrate</td><td>ROTH</td><td>3580</td><td></td></tr><tr><td>Sodium dodecylsulfate</td><td>SERVA</td><td>20765</td><td></td></tr><tr><td>Sodium hydroxide</td><td>ROTH</td><td>6771</td><td></td></tr><tr><td>Soil, Einheitserde classic&amp;nbsp;</td><td>PATZER GMBH</td><td></td><td></td></tr><tr><td>Spectrometer - Infinite M200</td><td>TECAN</td><td></td><td></td></tr><tr><td>Sucrose</td><td>SIGMA-ALDRICH</td><td>S-5390</td><td></td></tr><tr><td>4-Hydroxy benzhydrazide&amp;nbsp;</td><td>SIGMA-ALDRICH</td><td>H9882</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>ROTH</td><td>1058</td><td></td></tr><tr><td>UV light source - BLAK RAY</td><td>UVP</td><td></td><td></td></tr><tr><td>Vibratome - VT1000S</td><td>Leica</td><td></td><td></td></tr></tbody></table>

expression of recombinant cellulase cel5a from trichoderma reesei in tobacco plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

TrCel5A and TrCel5A-mCherry were expressed successfully in tobacco plants using the transient expression system (Figures 1A and 1B). Examination of the expression pattern of the TrCel5A-mCherry fusion protein revealed healthy leaves (Figure 1C), which showed widespread protein expression under green light (Figure 1D).
Extraction of the total soluble proteins was performed on tobacco plants which had leaves that contained the t...

Watch this Scientific Journal Video about Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants at JoVE.com

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

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12.8K Views.  RWTH Aachen University. Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Video: Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genetics

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Biochemistry

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 ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Bioengineering

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Chemistry

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 ...

Biology

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 ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

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.

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 ...

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Summary

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Chapters in this video

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

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

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

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

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

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

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