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

Note: Before following this protocol, become familiar with the material safety data for all substances used, and take appropriate safety precautions. These include but are not limited to: wearing safety glasses when working with glass under vacuum, and handling dry silica gel in a fume cupboard. It is also essential that local legislation concerning working with transgenic material is checked and observed, including following appropriate containment procedures.

1. Generate A. tumefaciens Strains for Infiltration

Note: We provide here a simplified description of the steps required to generate suitable A. tumefaciens strains for transient expression. Further details of these steps are described by Sainsbury et al.⁸.

1. Amplify by PCR or synthesize the protein coding region of the gene of interest flanked by 5’ and 3’ attB sequences suitable for generation of an entry clone for use with a site-specific recombination cloning protocol^18,19.
   1. Use Forward primer: GGGGACAAGTTTGTACAAAAAAGCAGGCTTANNNNNNNNNNNNNNNNNN, Reverse primer: GGGGACCACTTTGTACAAGAAAGCTGGGTANNNNNNNNNNNNNNNNNN (NNNN… = sequence specific sequence).
   2. Use (representative) PCR conditions: Reaction mixture (25 µL, total), buffer (5x, 5 µL, see Table of Materials), dNTPs (10 mM, 0.5 µL), Forward primer (10 µM, 1.25 µL), Reverse primer (10 µM, 1.25 µL), Template DNA (variable concentration, 50 – 250 ng, 0.5 µL), DNA polymerase (2000 U/mL, 0.25 µL, see Table of Materials), Nuclease-free water (to 25 µL). Run the thermocycler as follows: initial denaturation (98 °C, 30 s); start cycle (98 °C, 30 s; 45 – 72 °C, 20 s; 72 °C, 30 s/kb) end cycle; 35 cycles; final extension (72 °C, 10 min).
   3. Follow a standard site-specific recombination cloning protocol^18,19 to generate an entry clone using any non-kanamycin-based entry vector (we use pDONR207 which is commercially available).
      Note: The integrity of the entry clone should be confirmed by sequencing, before using to generate the pEAQ-HT-DEST1 expression construct. The pEAQ-HT-DEST1 is available on request from the Lomonossoff laboratory at the John Innes Centre in Norwich, UK.
   4. Isolate the pEAQ-HT-DEST1 vector from the expression clone generated in step 1.1.3, and store at -20 °C prior to use in step 1.3.
      Note: Any commercially available convenient spin column-based plasmid purification kit designed for extracting plasmids from cloning strains of E. coli is suitable. Follow the specific instructions of the chosen plasmid purification kit (see Table of Materials).
2. Prepare chemically competent A. tumefaciens for transformation.
   1. Inoculate a selective LB (Luria-Bertani medium: 10 g/L bacto-tryptone, 10 g/L NaCl, and 5 g/L yeast extract, agar 10 g/L pH 7.0) agar plate (50 μg/mL rifampicin, 100 μg/mL streptomycin), by streaking A. tumefaciens LBA4404 from a glycerol stock culture. Incubate overnight at 28 °C in a standing incubator.
   2. Inoculate 50 mL of selective liquid LB media (50 μg/mL rifampicin, 100 μg/mL streptomycin) with a sample of A. tumefaciens LBA4404 cells from step 1.2.1. Incubate overnight at 28 °C in a shaking incubator at 200 rpm.
   3. Cool the culture from step 1.2.2 on ice for 10 min and then pellet the A. tumefaciens cells by centrifugation at 4 °C and 4500 x g for 5 min (discard the supernatant).
   4. Resuspend the pellet from step 1.2.3 in 1 mL of ice-cold 20 mM aqueous CaCl₂ solution and repeat the centrifugation step from step 1.2.3 (discard the supernatant).
   5. Resuspend the pellet from step 1.2.4 in 1 mL of ice-cold 20 mM aqueous CaCl₂ solution and aliquot the resulting suspension in to 50 µL batches. Flash freeze the aliquots in liquid N₂ and store at -80 °C prior to use.
3. Transform the prepared A. tumefaciens LBA4404 with the isolated pEAQ-HT-DEST1 vector generated in step 1.2.
   1. Thaw 50 µL of A. tumefaciens suspension generated in step 1.2 on ice, and incubate with 100 ‒ 200 ng of isolated pEAQ-HT-DEST1 vector, generated in step 1.1, at 0 °C for 5 min.
   2. Cold shock the incubated suspension from step 1.3.1 in liquid N₂ for 30 s, then allow to warm to room temperature.
   3. Add 200 μL of SOC medium (SOC: 20 g/L bacto-tryptone, 5 g/L yeast extract, 0.58 g/L NaCl, 0.19 g/L KCl, 2.03 g/L MgCl₂, 2.46 g/L magnesium sulfate 7-hydrate, 3.6 g/L glucose) to the cold shocked suspension from step 1.3.2 and incubate for 4 h at 28 °C in a shaking incubator at 200 rpm.
   4. Inoculate a selective LB agar plate (50 μg/mL rifampicin, 50 μg/mL kanamycin, 100 μg/mL streptomycin) with the SOC culture from step 1.3.3. Spread thoroughly, and incubate for 3 days at 28 °C in a standing incubator.
   5. Inoculate 5 mL of selective LB media (50 μg/mL rifampicin, 50 μg/mL kanamycin, 100 μg/mL streptomycin) with a single colony from step 1.3.4. Incubate overnight at 28 °C in a shaking incubator at 200 rpm.
   6. Add 200 μL of glycerol to 1 mL of the liquid culture from step 1.3.5, mix thoroughly and store at -80 °C.
      Note: This culture can be revived for future experiments by streaking onto LB agar plates with appropriate antibiotics (described below).

2. Prepare Infiltration Suspension

1. Inoculate a selective LB agar plate with streaks of the desired A. tumefaciens strains from the glycerol stock cultures generated in step 1. Incubate overnight at 28 °C in a standing incubator.
   Note: A strain carrying tHMGR within a pEAQ-HT-DEST1 vector may also be included.
   1. Individually inoculate 50 mL of selective LB media with a sample of each A. tumefaciens strain grown in step 2.1 and incubate overnight at 28 °C in a shaking incubator at 200 rpm.
   2. Individually transfer the 50 mL cultures from step 2.1.1 to 1000 mL of selective LB media (rifampicin 50 μg/mL, kanamycin, 50 μg/mL streptomycin 100 μg/mL) and incubate overnight at 28 °C in a shaking incubator at 200 rpm.
   3. Individually pellet the A. tumefaciens by centrifugation (4000 x g), from the liquid cultures generated in step 2.1.2 (discard the supernatant).
   4. Individually resuspend the pellets from step 2.1.3 in 50 mL of freshly prepared MMA buffer (MMA Buffer: 10 mM 2-(N-morpholino)-ethanesulphonic acid, pH 5.6 (KOH), 10 mM MgCl₂, 150 μM 3’5’-dimethoxy-4’-acetophenone) and incubate at room temperature in the dark for 1 h.
   5. Combine the appropriate volume of each A. tumefaciens MMA suspension (generated in step 2.1.4) to produce a final OD₆₀₀ of at least 0.2 for each strain in a 10 L total final volume.
   6. Add additional MMA buffer to the combined suspensions generated in step 2.1.5 to a final volume of 1 L (use immediately in step 3).
      Note: It is advisable to prepare an additional 2 L of infiltration suspension for maintaining the liquid level during the infiltration process.
   7. Prepare 1 L of 10x strength MMA buffer.

3. Perform Vacuum Infiltration

Note: See Figure 2 for a description of vacuum infiltration apparatus construction. Use 5-week-old N. benthamiana plants in 9 cm x 9 cm pots for the proceeding steps. Seedlings should be sown in F1 compost (e.g. from Levington) and grown for 2 weeks before being transferred to individual pots containing F2 compost. Plants should be grown in a greenhouse at 25 °C, with 16 h per day of light (Use supplementary lighting when required), water daily, maximum density of 100 plants/m².

1. Transfer the 1 L of infiltration suspension generated in step 2 and the 1 L of 10x strength MMA buffer to the infiltration bath, followed by an additional 8 L of water.
   1. Remove the detachable wings of the bespoke plant holder, insert 4 plants (5-week-old plants see preceding note), and reattach the wings. Invert the holder and place on top of the infiltration bath so as to submerge the leaves in the infiltration suspension.
      Note: Ensure the suspension level reaches the top surface of the plant holder. Raise the level with the addition of extra infiltration suspension if required.
   2. Transfer the infiltration bath with submerged plants to the infiltration chamber and close the door. Ensure the air intake valve is closed on the infiltrator. Turn on the vacuum pump and open the inline valve to the vacuum reservoir followed by vacuum intake valve on the infiltration chamber.
   3. Once the pressure in the infiltration chamber has reduced by 880 mbar, shut the vacuum intake valve, and open the air intake valve. Once the infiltration chamber has returned to atmospheric pressure, open the door, and remove the infiltration bath.
   4. Raise the plant holder till the plants are no longer submerged in the infiltration suspension, then gently shake the holder to allow excess suspension to run off the leaves back into the infiltration bath.
   5. Return the holder to the upright position, remove the detachable wings, and remove the plants.
   6. Repeat steps 3.1.1 to 3.1.5 until the desired number of plants have been infiltrated.
   7. Autoclave the used infiltration suspension prior to disposal.
   8. Autoclave the infiltration bath prior to reuse in subsequent experiments.
   9. Sterilize the interior of the infiltration chamber by cleaning with 70% ethanol and leave to air dry prior to reuse in subsequent experiments.

4. Grow the Infiltrated Plants

1. Arrange the infiltrated plants from step 3 at a maximum density of 100 plants/m² in a greenhouse.
2. Grow for 5 days at 25 °C and 16 h per day of light (use supplementary lighting when required), and water daily.

5. Harvest the Infiltrated Leaves

1. After 5 days of growth, harvest the leaves.
2. Autoclave waste plant material, pots, and soil prior to disposal.

6. Dry the Harvested Leaves

Note: The exact lyophilization procedure described below depends on the nature of the available lyophilization apparatus available.

1. Lyophilize the harvested leaves until dry.
   1. Place the harvested leaves in a 2 mm-thick polypropylene bag. Freeze the harvested leaves by storing in a -80 °C freezer for 30 min.
   2. Perforate the bag with many small holes using a pin. Place the bagged frozen leaves in the central chamber of the lyophilizer. Ensure all flask attachment taps are closed and then switch on the lyophilizer. Ensure the condenser reaches at least -50 °C and the pressure reduces to at least 0.15 mbar, and then leave overnight.
   3. Switch off the lyophilizer and vent the vacuum by opening a flask attachment tap. Remove the dried leaves from the central chamber, and proceed to step 7.

7. Pressurized Solvent Extraction

Note: The proceeding steps refer to use of a commercially available PSE instrument (see Table of Materials). Please refer to the manufacturer’s literature for more detailed information on operation. The instrument carries out 4 extractions in parallel.

1. Grind the dried leaves into a course powder via a convenient method (e.g., for most triterpene compounds of interest use a domestic food processor, or manually crush the leaves while contained within a bag).
   Note: Grinding with a pestle and mortar under liquid nitrogen is not usually necessary.
   1. Mix the leaf powder with quartz sand (0.3 ‒ 0.9 mm, 20% by volume) in a suitable container; this acts as a dispersant and improves the efficiency of the extraction process.
2. Prepare 4 extraction cells (120 mL) for filling. For this, invert the extraction cells and insert the glass fiber filter, followed by the metal frit and holding plug. Return the extraction cells to the upright position and add a 1 cm deep layer of quartz sand (0.3 ‒ 0.9 mm).
3. Fill the extraction cells.
   1. Fill the extraction cells with the prepared leaf powder generated in step 7.1 up to the fill line. If needed, gently compress the powder during this process to facilitate addition of a greater amount in each extraction cell.
   2. Add a small layer of quartz sand (0.3 ‒ 0.9 mm) to the top of the packed powder and insert the top cellulose filter.
   3. Insert the packed extraction cells in to the PSE instrument and run the desired method. Use the following settings and material; Solvent: 100% ethyl acetate; temperature: 100 °C, pressure: 130 bar. Run 3 cycles as follows: cycle 1: 0 min hold time, cycle 2 and 3: 5 min hold time, end with a 2 min solvent flush, and 12 min nitrogen gas flush.
      Note: It is advisable to first optimize the extraction method on a small scale. However, the following method is often sufficient for most oxidized triterpene aglycones. The liquor from each extraction cell can be combined in the same external collection vessel by specifying the waste line as the extraction destination, instead of the standard individual collection lines. Be aware the amount of solvent used is dependent on the packing of the extraction cells and the set temperature and pressure. The above method typically generates approximately 800 mL of extraction liquor for 4 parallel extractions.
   4. Repeat steps 7.2 to 7.3.3 until all of the leaf powder has been processed.
   5. Concentrate the combined extraction liquor to dryness, via rotary evaporation under vacuum. Check for the expected output (i.e., dark green slurry).
      Note: The exact procedure may vary depending on the set up of the available rotary evaporator. Always wear eye protection when performing rotary evaporation, and check glassware for damage before subjecting to vacuum conditions.
      1. Turn on the chiller recycler, and allow the heat transfer liquid to cool to at least 5 °C. Turn on the water bath, and set the temperature to 35 °C.
      2. Transfer the extraction liquor to an evaporation flask (do not fill more than half the volume of the flask). Concentrate the liquor batch-wise (in to the same flask) if the total volume exceeds this. Attach the filled evaporation flask to the vapor-duct of the rotary evaporator (secure with a joint clip).
      3. Adjust the flask lift position and angle, so as to submerge the bottom third of the evaporation flask in the water bath at an approximately 45° angle. Start the flask spinning at a speed which begins to spread the extraction liquor up the walls of the flask.
      4. Ensure the vent tap is closed and begin to reduce the pressure (using the controller of the vacuum pump) until a moderate drip is observed between the condenser and receiver flask.
         Note: Do not allow the extraction liquor to start to boil. If this occurs reduce the strength of the vacuum to bring the distillation under control.
      5. Monitor and adjust the vacuum strength and spin speed as appropriate until all the solvent is evaporated.

8. Basic Ion Exchange Resin Treatment for Removal of Chlorophylls

Note: The quantities quoted in the following steps assume an original working scale of 100 ‒ 150 plants. Step 8 is not suitable for products containing carboxylic acid groups, or functional groups that would be hydrolyzed under basic conditions such as esters.

1. Dissolve the crude extract generated in step 7 in a minimal volume of ethanol.
2. Add 50 mL of basic ion exchange resin beads (see Table of Materials) to the output of step 7.1.1, and shake at room temperature for 30 min.
   Note: Do not substitute shaking for the use of stirring apparatus. Magnetic or mechanical overhead stirring can compromise the integrity of the resin beads. Suitable agitation can also be conveniently achieved by slow rotation of reaction flask on a rotary evaporator with the vacuum set at atmospheric pressure or disconnected.
3. Add an additional 50 mL of basic ion exchange resin beads every 30 min until the reaction has gone to completion; the basic ion exchange resin beads will change from pink to green in color, and the liquid phase will change from green to orange or a murky brown color depending on the scale.
   1. If in doubt that the reaction has gone to completion, sample 0.5 mL of the liquid. Filter the sample through a small column of diatomaceous earth and observe the color of the filtrate; the reaction is usually complete within 1.5 h.
4. Filter the reaction mixture through a short column of diatomaceous earth.
   1. Perform this via vacuum filtration, or via a glass flash chromatography column using hand bellows to apply pressure. If filtration rate slows, disturb the top of the diatomaceous earth pad with a stirring rod. Collect the filtrate.
5. Rinse the collected resins beads with ethanol, followed by 1:1 ethanol:hexane, and finally hexane. Use a rinse volume sufficient to resuspend the beads on the top of the diatomaceous earth column.
6. Combine the filtrate from step 8.4, and the rinses from step 8.5, and concentrate to dryness by rotary evaporation under vacuum.

9. Initial Rough Fractionation

Note: The following method is usually appropriate for typical oxidized triterpene aglycones, and employs an automated flash chromatography instrument. Refer to the manufacturer’s literature for more details on operation.

1. Adsorb the crude product generated in step 8 onto flash grade silica gel (pore size: 60 Å, particle size: 35 ‒ 75 μm) for application to a flash chromatography cartridge via dry loading methodology.
   1. Dissolve the crude product generated in step 8 in a minimal volume of suitable organic solvent. Ensure complete dissolution.
      Note: Dichloromethane with the addition of a few drops of methanol is usually an appropriate choice of solvent.
   2. Unscrew the top of a 100 g prefilled flash chromatography cartridge (see Table of Materials) and remove the insert to reveal the dry loading head space. Use the head space to measure the appropriate volume of silica gel for dry loading.
   3. Transfer the measured volume of silica gel for dry loading to the solution of crude product, then remove the solvent by rotary evaporation under vacuum.
      Note: The silica gel should return to a free-flowing powder. Towards the end of the evaporation process gentle scraping may be required to free the silica gel from the side of the evaporation flask.
   4. Equilibrate the 100 g flash chromatography cartridge at 100 mL/min with at least 5 column volumes of 100% hexane, but ideally until the cartridge content is fully and uniformly wetted.
   5. Transfer the prepared dry loading silica gel generated in step 9.1.3 to the dry loading head space of the equilibrated 100 g flash chromatography cartridge, by pouring. Suspension in hexane and a wide mouthed pipette can be used to aid transfer of any remaining dry loading silica gel.
   6. Wet the transferred dry loading silica gel bed with 100% hexane to remove air from the dry loading headspace.
   7. Run the following chromatography method: Solvent [A]: hexane, Solvent [B]: ethyl acetate, Flow rate: 100 mL/min, Gradient: 0% [B] to 100% [B] over 3000 mL, Collection: Collect All, Fraction size: 250 mL.
   8. Analyze the fractions by thin layer chromatography (TLC)⁸ or gas chromatography-mass spectrometry (GC-MS)⁸, and concentration to dryness the fraction(s) containing the compound of interest via rotary evaporation under vacuum.
2. Perform downstream fine purification until the desired level of purity is reached.
   Note: Downstream fine purification is entirely compound specific and it is not possible to provide standardized steps (see the discussion section).