Name: Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants (Video) | JoVE Journal
Uploaded: 2014-06-13T15:00:00.000+00:00
Duration: 13 min 5 s
Description: Learn transient expression of recombinant cellulase Cel5A from Trichoderma reesei in tobacco plants using Agrobacterium infiltration. This method enables rapid plant-based enzyme production for biomass conversion research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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>

<ol><li>Garvey, M., Klose, H., Fischer, R., Lambertz, C., Commandeur, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellulases+for+biomass+degradation%3A+comparing+recombinant+cellulase+expression+platforms%5BTitle%5D)+AND+%22Trends+in+Biotechnology%22%5BJournal%5D)">Cellulases for biomass degradation: comparing recombinant cellulase expression platforms.</a> Trends in Biotechnology. 31, 581-593 (2013).</li>
<li>Klose, H., Günl, M., Usadel, B., Fischer, R., Commandeur, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ethanol+inducible+expression+of+a+mesophilic+cellulase+avoids+adverse+effects+on+plant+development%5BTitle%5D)+AND+%22Biotechnology+for+Biofuels%22%5BJournal%5D)">Ethanol inducible expression of a mesophilic cellulase avoids adverse effects on plant development.</a> Biotechnology for Biofuels. 6, 53 (2013).</li>
<li>Klose, H., Röder, J., Girfoglio, M., Fischer, R., Commandeur, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperthermophilic+endoglucanase+for+in+planta+lignocellulose+conversion%5BTitle%5D)+AND+%22Biotechnology+for+Biofuels%22%5BJournal%5D)">Hyperthermophilic endoglucanase for in planta lignocellulose conversion.</a> Biotechnology for Biofuels. 5, 63 (2012).</li>
<li>Sharma, A. K., Sharma, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plants+as+bioreactors%3A+recent+developments+and+emerging+opportunities%5BTitle%5D)+AND+%22Biotechnology+Advances%22%5BJournal%5D)">Plants as bioreactors: recent developments and emerging opportunities.</a> Biotechnology Advances. 27 (6), 811-832 (2009).</li>
<li>Tremblay, R., Wang, D., Jevnikar, A. M., Ma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tobacco%2C+a+highly+efficient+green+bioreactor+for+production+of+therapeutic+proteins%5BTitle%5D)+AND+%22Biotechnology+Advances%22%5BJournal%5D)">Tobacco, a highly efficient green bioreactor for production of therapeutic proteins.</a> Biotechnology Advances. 28 (2), 214-221 (2010).</li>
<li>Lee, T. M., Farrow, M. F., Arnold, F. H., Mayo, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+structural+study+of+Hypocrea+jecorina+Cel5A%5BTitle%5D)+AND+%22Protein+Science%22%5BJournal%5D)">A structural study of Hypocrea jecorina Cel5A.</a> Protein Science. 20, 1935-1940 (2011).</li>
<li>Saloheimo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EGIII%2C+a+new+endoglucanase+from+Trichoderma+reesei%3A+the+characterization+of+both+gene+and+enzyme%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme.</a> Gene. 63, 11-21 (1988).</li>
<li>Davidson, M. W., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+fluorescent+proteins%3A+innovations+and+applications%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Engineered fluorescent proteins: innovations and applications.</a> Nature Methods. 6, 713-717 (2009).</li>
<li>Pelham, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+retention+signal+for+soluble+proteins+of+the+endoplasmic+reticulum%5BTitle%5D)+AND+%22Trends+in+Biochemical+Sciences%22%5BJournal%5D)">The retention signal for soluble proteins of the endoplasmic reticulum.</a> Trends in Biochemical Sciences. 15, 483-486 (1990).</li>
<li>Béguin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+cellulase+activity+in+polyacrylamide+gels+using+Congo+red-stained+agar+replicas%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Detection of cellulase activity in polyacrylamide gels using Congo red-stained agar replicas.</a> Analytical Biochemistry. 131, 333-336 (1983).</li>
<li>Jørgensen, H., Mørkeberg, A., Krogh, K. B. R., Olsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+and+enzyme+production+by+three+Penicillium+species+on+monosaccharides%5BTitle%5D)+AND+%22Journal+of+Biotechnology%22%5BJournal%5D)">Growth and enzyme production by three Penicillium species on monosaccharides.</a> Journal of Biotechnology. 109, 295-299 (2004).</li>
<li>Lever, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+reaction+for+colorimetric+determination+of+carbohydrates%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">A new reaction for colorimetric determination of carbohydrates.</a> Analytical Biochemistry. 47, 273-279 (1972).</li>
<li>Maclean, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+human+papillomavirus+type+16+%28HPV-16%29+L1+expression+in+plants%3A+comparison+of+the+suitability+of+different+HPV-16+L1+gene+variants+and+different+cell-compartment+localization%5BTitle%5D)+AND+%22Journal+of+General+Virology%22%5BJournal%5D)">Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization.</a> Journal of General Virology. 88, 1460-1469 (2007).</li>
<li>Munro, S., Pelham, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+C-terminal+signal+prevents+secretion+of+luminal+ER+proteins%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A C-terminal signal prevents secretion of luminal ER proteins.</a> Cell. 48, 899-907 (1987).</li>
<li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li>
<li>Burnette, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%E2%80%9CWestern+Blotting%E2%80%9D%3A+Electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate-polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">“Western Blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112, 195-203 (1981).</li>
<li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elelctrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets+-+procedure+and+some+applications%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Elelctrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets - procedure and some applications.</a> Proceedings of the National Academy of Sciences of the United States of America. 76, 4350-4354 (1979).</li>
<li>Blake, M. S., Johnston, K. H., Russell-Jones, G. J., Gotschlich, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid%2C+sensitive+method+for+detection+of+alkaline+phosphatase-conjugated+anti-antibody+on+Western+blots%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots.</a> Analytical Biochemistry. 136, 175-179 (1984).</li>
<li>Yamamoto, E., Yamaguchi, S., Nagamune, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+%CE%B2-cyclodextrin+on+the+renaturation+of+enzymes+after+sodium+dodecyl+sulfate%E2%80%93polyacrylamide+gel+electrophoresis%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Effect of β-cyclodextrin on the renaturation of enzymes after sodium dodecyl sulfate–polyacrylamide gel electrophoresis.</a> Analytical Biochemistry. 381, 273-275 (2008).</li>
<li>Fischer, R., Vaquero-Martin, C., Sack, M., Drossard, J., Emans, N., Commandeur, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Towards+molecular+farming+in+the+future%3A+transient+protein+expression+in+plants%5BTitle%5D)+AND+%22Biotechnology+and+Applied+Biochemistry%22%5BJournal%5D)">Towards molecular farming in the future: transient protein expression in plants.</a> Biotechnology and Applied Biochemistry. 30 (2), 113-116 (1999).</li>
<li>Sammond, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellulase+linkers+are+optimized+based+on+domain+type+and+function%3A+Insights+from+sequence+analysis%2C+biophysical+measurements%2C+and+molecular+simulation%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Cellulase linkers are optimized based on domain type and function: Insights from sequence analysis, biophysical measurements, and molecular simulation.</a> PLoS One. 7, (2012).</li>
<li>Boonvitthya, N., Bozonnet, S., Burapatana, V., O'Donohue, M. J., Chulalaksananukul, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+the+heterologous+expression+of+Trichoderma+reesei+endoglucanase+II+and+cellobiohydrolase+II+in+the+yeasts+Pichia+pastoris+and+Yarrowia+lipolytica%5BTitle%5D)+AND+%22Molecular+Biotechnology%22%5BJournal%5D)">Comparison of the heterologous expression of Trichoderma reesei endoglucanase II and cellobiohydrolase II in the yeasts Pichia pastoris and Yarrowia lipolytica.</a> Molecular Biotechnology. 54, 158-169 (2013).</li>
</ol>

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

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...

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.

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

The overall goal of the following experiment is to express target proteins transiently in tobacco to gain an idea about the feasibility of plant-based enzyme expression in a short timeframe. This transient expression is achieved by infiltrating tobacco leaves with a strain of agrobacterium tumor fain, which contains a vector incorporating the gene of interest as a second step After incubation infiltrated plant leaves are harvested and protein extracts are partially purified to obtain a working enzyme solution. Next protein analysis is performed in order to assess the effects of plant-based expression on the enzyme and to verify its enzymatic activity.Results are obtained that show protein size possible glycosylation effects and enzymatic activity based on western blotting, zy and substrate degradation assays. This method can help answer key questions about plant biomass conversion and can also provide insight into the activity of enzymes after being transiently expressed in tobacco. Visual demonstration of this method is critical as the infiltration steps are difficult to learn due to the leaves being easily damaged by the syringe herger clothes and I will be performing the experiments Under sterile conditions.Transfer single colonies of agrobacterium toan to an erlenmeyer flask containing 10 milliliters. Yeast extract broth with antibiotics. Then incubate the inoculated broth for 36 to 40 hours at 26 degrees Celsius with 180 RPM shaking next inoculate 200 microliters of each culture in 20 milliliters of yeast extract broth with the same antibiotics as before and incubate under the same conditions after 36 to 40 hours.Induce the cultures with two microliters of acetophenone, 100 microliters of 40%glucose solution and 200 microliters of one molar MES buffer at pH 5.6 and continue to incubate overnight. Now take the plants from the greenhouse and use a pipette tip to nick the AB axial side of the third to fifth leaf from the top of the plant to ease infiltration. Then fill a syringe with infiltration medium and place it on one of the previously made nicks.Support the syringe with a finger on the axio leaf side and inject the medium carefully into the intravenous leaf zone. To visualize the fluorescence in the leaf sections expressing TR cell 5:00 AM cherry shine the plants with a portable light source emitting green light. When viewed through a red filter, fluorescence from TR cell 5:00 AM cherry will be clearly visible.To extract the expressed proteins, select tobacco leaves which have been infiltrated with AUM containing P track ERTR cell five A and cut them into one gram pieces. Next, place them in a pre-cool mortar and add an appropriate amount of liquid nitrogen to the samples. Grind the leaves to powder using a pestle.Repeat the process with non infiltrated tobacco leaves to obtain control samples. Then add two milliliters of PBS containing one millimolar phenyl methyl suen fluoride to the ground leaf matter and mix until the majority of leaf matter is in suspension. Centrifuge the extract at 15, 000 GS for 20 minutes at four degrees Celsius.Then discard the pellet, partially purified TR cell five A by incubating the protein containing S natin at 55 degrees Celsius for 10 minutes and allow it to rest at room temperature for another 10 minutes. Then centrifuge a SNAT at 15, 000 GS for 10 minutes at room temperature. Discard the pellet and use the protein containing snat for all.Further assays. Also perform the partial purification process for non infiltrated plants to obtain control samples. In this procedure, mix ethyl cellulose and water to obtain a 1.5%solution and incubate with stirring until dissolved.To make A-C-M-C-S-D-S page gel substitute one milliliter of 1.5%CMC for one milliliter of water in a 10 milliliter SDS page gel mix solution and pour the gel normally next, denature the samples by boiling them in the presence of SDS. Carry out the electrophoresis at 200 volts for 50 minutes until the dye front reaches the bottom of the gel. Now perform in gel protein refolding on the point 15%C-M-C-S-D-S page gel.Using 1.5%beta cyclodextrin in 20 millimolar phosphate citrate buffer at pH 6.5. Incubate the gel in this solution at room temperature with gentle shaking for 15 minutes. Discard the beta cyclodextrin solution and briefly wash the gel with water incubated at room temperature in 50 millimolar sodium acetate buffer at pH 4.8 for another 15 minutes.Then perform CMC zy using the refolded point 15%C-M-C-S-D-S page gel by incubating the gel in 30 millimolar sodium acetate buffer for 20 minutes at 50 degrees Celsius. To visualize where the CMC has degraded, stain the gel for 10 minutes using a 0.1%Congo red solution and detain it using one molar sodium chloride for 30 minutes or until clear bands are observed at the end, wash the gel with 0.3%acetic acid. For clearer imaging immediately prior to running the assay, prepare a four MUC two X working solution pipette 100 microliters of four MUC working solution into separate wells of a 96 well plate.Then add 100 microliters of each sample into the appropriate wells running each sample in triplicate using an excitation wavelength of 360 nanometers and an emission wavelength of 465 nanometers. Determine the zero minute time point of four mu fluorescence through fluorescent spectroscopy. Next, incubate the plates covered with adhesive lids at 50 degrees Celsius for 20 minutes.Subsequently, stop the reaction by freezing. After that, measure the endpoint mu fluorescence using the same parameters as for the zero minute time point. Subtract the fluorescence value at zero minute from the fluorescence value at 20 minutes to determine the change in fluorescence caused by enzymatic activity immediately prior to running the assay.Prepare the A-O-C-M-C two X working solution. Then combine 50 microliters of the sample and 50 microliters of the working solution in a 1.5 milliliter tube. Next, incubate the samples for 20 minutes at 50 degrees Celsius.Stop the reaction by adding 250 microliters of precipitation solution. Subsequently, vortex each sample vigorously for 10 seconds to ensure complete mixing of the sample with the precipitation solution. Then incubate the samples at room temperature for 10 minutes and vortex them again before centrifuging them at 1000 Gs for 10 minutes.After that transfer 200 microliters of the SNAT from each sample to a 96 well plate without disturbing. The pellet enzymatic activity is shown by higher levels of solubilized dye. Measure this using absorbent spectroscopy at a wavelength of 590 nanometers.In this step. Place circles of filter paper into 1.5 or two milliliter tubes. Add 100 microliters of 60 millimolar sodium acetate and 100 microliters of samples to the filter paper circles and incubate for 20 hours at 50 degrees Celsius with 300 RPM shaking.In addition, incubate the individual samples without filter paper as controls immediately prior to running the assay. Prepare a 1.5 x PABA working solution containing one milliliter of PBA solution A and nine milliliters of PABA solution B.Store on ice until use. Next, prepare the samples in 0.5 milliliter thermo stable tubes.The samples are composed of 45 microliters of water, five microliters of each solution incubated with filter paper and 100 microliters of PABA working solution. After that, run the sample controls and five standards prepared in the same manner. Then incubate the samples, controls and standards for exactly 10 minutes at 100 degrees Celsius and cool at room temperature for another 10 minutes.Pipette 100 microliters of the solution into a 96 well plate an assay with a spectrophotometer at a wavelength of 410 nanometers. Subtract the individual sample control values from the filter paper sample values to determine the amount of reducing sugars released. Here is an image showing the expression cassettes as well as the protein expression in tobacco plants.This is how an infiltrated tobacco leaf appears under normal light and under green light with a red filter where expression of TR cell 5:00 AM cherry indicated by the red color is clearly visible here. The SDS page gel western lot and CMC zy that show the size and activity of T cell five A.The total soluble proteins have been separated and we can visualize them with kumasi blue staining Western blotting against the hist tag region of T cell five A confirms the protein size and additionally the cellulase activity against CMC is shown by a xenograft stained with Congo red. What can be seen here is the total soluble proteins from the tobacco plant leaves where TR cell five A was transiently expressed before and after thermal precipitation purification where TR cell five A is the largest visible band and is also visible in the western lot and the CMCs igraph.We also see the total soluble proteins from tobacco plants where Tcell five A was not also after thermal precipitation and the proteins from a commercially available mixture of tresa cellulase. This figure shows the enzyme activity quantification of TR cell five A TR cell five A was incubated with four MUC Azo CMC or filter paper, and the substrate degradation was measured by the release of the fluoro four four mu the azo dye, or by quantifying the reducing sugars from the color change of PBA Following this procedure. Other methods like protein targeting can be used to provide additional information into how the protein localization can affect the enzyme stability and activity.Having watched this video, you should now have a clear understanding of how to transiently express proteins into tobacco as well as some good knowledge into how to assess enzymatic activity. Foro cellulose biomass conversion.

expression-recombinant-cellulase-cel5a-from-trichoderma-reesei

Research

JoVE Journal

Environment

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

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.

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

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.

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

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.

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

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

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

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.

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

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.

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

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

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.

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

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.

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

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.

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

Learn transient expression of recombinant cellulase Cel5A from Trichoderma reesei in tobacco plants using Agrobacterium infiltration. This method enables rapid plant-based enzyme production for biomass conversion research.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Explore More Videos