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Purification of N-coronafacoyl Phytotoxins from Streptomyces scabies
疮痂病链霉菌N-coronafacoyl植物毒素的纯化   

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Abstract

This procedure is used for large - scale purification of N-coronafacoyl phytotoxins that are produced by the potato common scab pathogen Streptomyces scabies. The procedure employs organic extraction of S. scabies culture supernatants under alternating basic and acidic conditions in order to preferentially isolate the phytotoxin - containing carboxylic acid fraction of the supernatant. Preparative thin layer chromatography and semi-preparative reverse phase - high performance liquid chromatography are then used to further purify the individual N-coronafacoyl phytotoxins of interest.

Keywords: Streptomyces(链霉菌), Plant pathogen(植物病原体), Common scab(疮痂病), N-coronafacoyl-L-isoleucine(N-coronafacoyl-L-异亮氨酸), Phytotoxin(植物毒素), Thin layer chromatography(薄层色谱法), High performance liquid chromatography(高效液相色谱法)

Background

Potato common scab is an economically important crop disease that is caused by Gram-positive, filamentous, soil bacteria from the genus Streptomyces. The first described and best characterized scab - causing Streptomyces spp. is Streptomyces scabies (syn. S. scabiei), which has a worldwide distribution (Bignell et al., 2010). Current control practices for common scab disease management include crop rotation, irrigation and soil fumigation; however, these strategies often fail, produce inconsistent results or are environmentally unfriendly (Dees and Wanner, 2012). In order to develop better control strategies for the disease, we must first understand the molecular mechanisms used by S. scabies to infect the plant and to induce disease symptoms. Research has shown that the ability of S. scabies to cause disease is due to the production of virulence factors that play different roles during the infection process. Among the known or potential virulence factors that are produced by S. scabies is a family of plant toxins referred to as the N-coronafacoyl phytotoxins (also known as the COR-like metabolites), which resemble the plant hormone jasmonic acid and may function to suppress the plant immune response during pathogen infection (Bignell et al., 2010; Fyans et al., 2015). The primary coronafacoyl phytotoxin produced by S. scabies is N-coronafacoyl-L-isoleucine (CFA-L-Ile; Figure 1), which consists of the polyketide metabolite coronafacic acid linked via an amide bond to L-isoleucine. In addition, other N-coronafacoyl phytotoxins containing different isoleucine isomers or different amino acids (e.g., valine) can be produced in minor amounts (Fyans et al., 2015; Bown et al., 2016).


Figure 1. Structure of N-coronafacoyl-L-isoleucine (CFA-L-Ile) produced by Streptomyces scabies


The protocol described here was developed to isolate and purify N-coronafacoyl phytotoxins and their biosynthetic intermediates from large-scale cultures of S. scabies for purposes of structural and functional characterization. Previously, Fyans et al. (2015) described a protocol that was based in part on a published procedure for the isolation of the related N-coronafacoyl phytotoxin coronatine (COR) from cultures of the Gram-negative plant pathogenic bacterium Pseudomonas syringae (Palmer and Bender, 1993). As outlined by Fyans et al. (2015), strains of S. scabies are cultured in a soy flour mannitol broth (SFMB) medium, which promotes the production of the coronafacoyl phytotoxins, and then the culture supernatants are subjected to a two-step extraction with organic solvent under basic and acidic conditions in order to selectively isolate the phytotoxin - containing carboxylic acid fraction of the culture supernatants. The phytotoxins are then further purified using a combination of preparative thin layer chromatography (TLC) and semi-preparative reverse phase - high performance liquid chromatography (RP - HPLC). More recently, we described a modified version of this protocol in which we incorporated additional extraction steps using an aqueous solution of potassium bicarbonate (Bown et al., 2016). This modification was based on the procedure described by Mitchell and Frey (1986) for the isolation of P. syringae N-coronafacoyl phytotoxins, and we found that the incorporation of the additional extraction steps significantly improved the purity of the final S. scabies phytotoxin preparations. Moreover, we modified the organic solvent for the N-coronafacoyl phytotoxins by the addition of a small amount of acid, which significantly improved the solubility and yield of the purified phytotoxins for downstream structural and functional studies.

Here, we present the detailed step-by-step protocol for how we currently purify the S. scabies N-coronafacoyl phytotoxins in our laboratory.

Materials and Reagents

  1. pH test strips (VWR, BDH®, catalog number: BDH35309.606 )
  2. Hydrophilic polypropylene membrane filters, 47 mm diameter, 0.45 μm pore size (Pall, catalog number: 66548 )
  3. FisherbrandTM class B clear glass threaded vials with closures attached, 3.7 ml (Fisher Scientific, catalog number: 03-338A )
  4. Slip tip syringes, 1 ml (BD, catalog number: 309659 )
  5. PTFE membrane filters, 0.2 μm pore size, 6 mm diameter (VWR, catalog number: 28145-491 )
  6. WhatmanTM filter discs, 12.5 cm (Sigma-Aldrich, catalog number: WHA1113125 )
  7. Conical centrifuge tubes, 50 ml (Corning, Falcon®, catalog number: 352098 )
  8. Silica gel GF preparative TLC plates with pre-adsorbent zone, 20 x 20 cm, 1,000 μm (Analtech/iChromatography, catalog number: P32013 )
  9. DMSO mycelial freezer stock of S. scabies (Fyans et al., 2015)
  10. BactoTM tryptic soy broth medium (BD, BactoTM, catalog number: 211825 )
  11. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-212 )
  12. Chloroform, ACS grade (VWR, BDH®, catalog number: BDH1109 )
  13. Hydrochloric acid (HCl), ACS grade (Avantor® Performance Materials, catalog number: 638801 )
  14. Sodium sulfate anhydrous (Na2SO4), ACS grade (Fisher Scientific, catalog number: S421-500 )
  15. Ethylene glycol (VWR, BDH®, catalog number: BDH1125 )
  16. Potassium bicarbonate (KHCO3), ACS grade (Sigma-Aldrich, catalog number: 237205 )
  17. Methanol (MeOH), HPLC grade (Sigma-Aldrich, catalog number: 34860 )
  18. Formic acid, reagent grade (Sigma-Aldrich, catalog number: F0507 )
  19. HiPerSolv CHROMANOR® Acetonitrile (ACN) for HPLC (VWR, BDH®, catalog number: BDH83639.400 )
  20. Soy flour, defatted (MP Biomedicals, catalog number: 960024 )
  21. D-mannitol (AMRESCO, catalog number: 0122 )
  22. Ethyl acetate, ACS grade (Sigma-Aldrich, catalog number: 319902 )
  23. 2-propanol, HPLC grade (Fisher Scientific, catalog number: A451SK-4 )
  24. Acetic acid, ACS grade (Sigma-Aldrich, catalog number: 695092 )
  25. Water, HPLC grade (EMD Millipore, catalog number: WX0008-1 )
  26. Soy flour mannitol broth (SFMB) medium (see Recipes)
  27. Preparative TLC mobile phase (see Recipes)

Equipment

  1. Glass Erlenmeyer flask, 125 ml (Corning, PYREX®, catalog number: C4980125 )
  2. Glass Erlenmeyer flask, 4 L (Corning, PYREX®, catalog number: C49804L )
  3. Glass filter holder assembly with funnel, fritted base, stopper and clamp, 47 mm (EMD Millipore, catalog number: XX1004700 )
  4. NalgeneTM PPCO centrifuge bottles with sealing closure, 250 ml (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 31410250 )
  5. SorvalTM ST 16R benchtop refrigerated centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvalTM ST 16R , catalog number: BCT25)
  6. Innova® 42R refrigerated incubator shaker, orbit diameter 1.9 cm (Eppendorf, New BrunswickTM, model: Innova® 42R , catalog number: M1335-004)
  7. 2 L plastic container
  8. NalgeneTM TeflonTM FEP separatory funnel with closure, 2 L (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4301-2000 )
  9. IKA® rotary evaporator system (IKA®, model: RV 10 digital V )
  10. VWR® refrigerated circulating bath with programmable temperature controller (VWR, model: VWR® Refrigerated Circulating Baths , catalog number: 89202-982)
  11. KIMAX® Squibb separatory funnel with PTFE stopcock and glass stopper, 125 ml (Kimble Chase Life Science and Research Products, catalog number: 29048F-125 )
  12. DryFast diaphragm vacuum pump (Welch Vacuum – Gardner Denver, catalog number: 2044 )
  13. Biohit mLINE® single-channel mechanical pipettor, 2-20 μl (VWR, catalog number: 47745-545 )
  14. Aldrich® rectangular TLC developing tank (Sigma-Aldrich, model: Z126195 )
  15. UV lamp with portable cabinet (Analtech/iChromatography, catalog number: A93-04 )
  16. Metal spatula
  17. Thermo ScientificTM dry block heater (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88-860-021 )
  18. ZORBAX StableBond 80Å C18 semi-preparative HPLC column, 9.4 x 250 mm, 5 μm (Agilent Technologies, catalog number: 880975-202 )
  19. Chemical safety hood
  20. KIMAX® graduated filtering flask, 1 L (Kimble Chase Life Science and Research Products, catalog number: 27060-1000 )
  21. GeneMate vortex mixer (VWR, catalog number: 490000-094 )
  22. Agilent 1260 Infinity analytical-scale LC purification system with quaternary pump, autosampler, diode array detector and fraction collector (Agilent Technologies, model: 1260 Infinity )

Procedure

  1. Culture growth
    1. Prepare a seed culture of S. scabies by inoculating 1 ml of the DMSO freezer stock into 25 ml of tryptic soy broth medium in a 125 ml Erlenmeyer flask, and incubate at 28 °C with shaking (200 RPM) for 48 h.
    2. Subculture 10 ml of the seed culture into 1 L of SFMB medium in a 4 L Erlenmeyer flask, and incubate at 25 °C with shaking (200 RPM) for 10 days (see Notes 1 and 2).
    3. Divide the culture into four 250 ml centrifuge bottles and centrifuge at 3,005 x g for 15 min at 4 °C to pellet the cell material. Decant the supernatants into a 2 L plastic container and store at -20 °C until needed. Even if the supernatant is going to be extracted immediately, it is useful to freeze it for a minimum of 24 h (see Note 3).

  2. Organic extraction (see Figure 2 and Note 4)


    Figure 2. Schematic outline of principle steps performed during the extraction of coronafacoyl phytotoxins from S. scabies culture supernatants

    1. Measure the pH of the culture supernatant using a pH strip, and adjust the pH to 11-12 using a 5 N solution of NaOH (~4-5 ml).
    2. Decant the supernatant into a 2 L separatory funnel and extract with 0.5 volumes (~450 ml) of chloroform. To perform the extraction, add the chloroform to the separatory funnel, close the funnel stopper, and shake the funnel to mix the contents well. Open the stopcock to release any expressed gas, then repeat the shaking until no more gas is expressed. Allow the organic (bottom) and aqueous (top) layers to separate as much as possible by gravity (~15-20 min; Figure 3A), then open the stopcock and collect the organic layer. If an emulsion layer is present, this can be collected and separated by centrifugation at 3,005 x g for 10 min at 4 °C, and the aqueous layer returned to the separatory funnel (Figure 3B). Discard the organic layer, and repeat the extraction of the aqueous supernatant with another 0.5 volumes of fresh chloroform. Perform the extraction a total of three times, discarding the organic layer after each extraction (see Note 5).


      Figure 3. Extraction of S. scabies culture supernatants with chloroform. A. The extraction is performed using a 2 L separatory funnel. After mixing the supernatant and chloroform, the phases are allowed to separate as much as possible by gravity. The upper dark brown layer is the aqueous phase, and the lower layer is the organic phase. B. When an emulsion layer is present, it is collected into centrifuge bottles and the phases are separated by centrifugation.   

    3. Acidify the aqueous supernatant to a pH of 1-2 by adding 1 N HCl(aq) (~5 ml).
    4. Decant the supernatant back into the 2 L separatory funnel and extract with 0.5 volumes (~450 ml) of chloroform. Use the same extraction procedure as described under 2 except that now the organic layer should be collected and saved. Perform the extraction a total of three times, after which the collected organic layers can be pooled and the aqueous layer discarded (see Note 6).
    5. Remove any residual water from the organic extract by adding ~30 g of anhydrous Na2SO4 and gently swirling the mixture. The anhydrous Na2SO4 is added until it no longer forms clumps, indicating that all of the residual water has been absorbed.
    6. Filter the organic extract using a 0.45 μm polypropylene membrane filter and a glass filter assembly attached to a vacuum pump. Wash the beaker that contained organic extract as well as the filter assembly with fresh chloroform to recover all of the residual extract.
    7. Concentrate the organic extract to ~100 ml using a rotary evaporator. The evaporator heating bath (filled with water) should be set to 50 °C, and the condenser should be chilled with a circulating solution of 50% v/v ethylene glycol (chilled to -10 °C using a refrigerated circulating bath). 
    8. Transfer 50 ml of the organic concentrate into each of two 125 ml separatory funnels. Extract each concentrate with 0.5 volumes (25 ml) of a 0.5 M aqueous solution of KHCO3. After mixing, allow ~5 min for the phases to separate by gravity, then collect each phase separately. Repeat the extraction of the organic concentrates two more times, then pool the collected aqueous phases (to give two sets of aqueous extracts, ~75 ml each) and discard the organic extracts (see Note 7).
    9. Wash the aqueous extracts twice with 0.5 volumes (35-40 ml) of chloroform. To wash, add the chloroform to a separatory funnel containing the aqueous extract, mix, then leave for 5 min to allow the phases to separate. Collect and discard the organic layer, and repeat. After washing, combine the aqueous extracts together (total volume ~140-150 ml).
    10. Adjust the pH of the aqueous extract to 1-2 using 1 N HCl(aq) (~12-15 ml). The acid will cause a neutralization reaction with the KHCO3 that will produce heat and a large amount of CO2. The acid must be added slowly or the reaction can become violent and will cause the acid to splash. After each 1 ml of acid has been added, gently mix the solution until the CO2 dissipates.
    11. Pour ~40 ml of the aqueous extract into each of four 125 ml separatory funnels. Extract each aliquot three times with 0.5 volumes (20 ml) of chloroform. Collect and combine all of the organic extracts together, and discard the aqueous layers. Evaporate the organic extract to dryness overnight in an uncovered beaker in a chemical safety hood (see Note 8).

  3. Preparative TLC (see Note 4)
    1. Re-dissolve the dried organic extract in 2-3 ml of MeOH containing 0.1% v/v formic acid by pipetting up and down. Transfer the extract to a clean screw-capped glass vial. Rinse the beaker with an additional 1 ml of MeOH + 0.1% formic acid to recover residual amounts of the extract. Filter the extract into a new screw-capped vial using a 1 ml syringe and a 0.2 μm PTFE membrane filter.
    2. Pour the TLC mobile phase (see Recipes) into a TLC developing tank and fill to a depth of ~2 cm. Saturate the air in the TLC chamber by placing two pieces of 12.5 cm filter paper into the tank and covering the tank for at least 30 min.
    3. Mark a 5 x 20 cm analytical TLC plate with a pencil to indicate the submersion point of the mobile phase solution (see Note 9).
    4. Apply at least ~30-50 μg of a standard of the molecule of interest (or of a comparative analogue; see Note 10) dissolved in 100% MeOH + 0.1% formic acid onto the TLC plate. Apply the standard at least 1 cm from the edge of the plate and at least 1 cm above the submersion mark on the plate.
    5. Apply ~30-50 μl of the organic extract along ~2 cm of the plate width. Ensure that the applied extract is at least 1 cm from the edge of the plate, 1 cm above the submersion mark on the plate and 1 cm away from the previously applied standard (see Note 11).
    6. Remove the filter paper discs from the TLC tank and place the TLC plate into the tank and cover. Allow the mobile phase to migrate to within 1 cm of the top of the plate (~20-40 min). Remove the TLC plate and allow to air dry (~15 min). Repeat the process.
    7. Place the air dried TLC plate under a UV lamp and use a pencil to mark the positions of the applied standard and the N-coronafacoyl phytotoxin of interest in the organic extract (Figure 4).


      Figure 4. TLC analysis of the S. scabies organic extract containing CFA-L-Ile. A pure sample (50 µg) of CFA-L-Ile (i) and a small portion (30-50 µl) of the organic extract (ii) containing CFA-L-Ile is spotted onto a 5 x 20 cm silica gel GF analytical TLC plate. Following separation, the plate is visualized under UV light, and the position of the pure standard is marked (blue circle) along with the expected position of the phytotoxin within the extract (blue rectangle). The black arrow indicates the direction of migration of the samples on the plate.

    8. Mark the preparative TLC plate with a pencil to indicate the area of the plate that will be submersed in the mobile phase solution.
    9. Apply the organic extract along the entire width of the TLC plate, 10 μl at a time. Do not place any sample within 1 cm of either edge of the plate or within 1 cm of the marked submersed area (see Note 11).
    10. Remove the filter paper discs from the TLC tank and place the TLC plate into the tank and cover. Allow the mobile phase to migrate to within 1 cm of the top of the plate (~1-1.5 h). Remove the plate from the tank and allow to air dry (~20-30 min). Return the plate to the tank and repeat the separation.
    11. Place the air dried TLC plate under a UV lamp and use a pencil to mark the region of the plate where the N-coronafacoyl phytotoxin migrated to (Figure 5; see Note 12).


      Figure 5. Preparative TLC analysis of the S. scabies organic extract containing CFA-L-Ile. The CFA-L-Ile will be found within the dark smear as indicated by the analytical TLC reference plate (see Figure 4). To ensure complete recovery of the CFA-L-Ile, a large area (indicated by the blue rectangle) of the silica is scraped from the plate. The white arrow indicates the direction of migration of metabolites on the plate.

    12. Scrape the marked region using the flat edge of a metal spatula to remove the silica gel - bound metabolites. Transfer the silica gel to a Falcon conical tube and add 10 ml of MeOH + 0.1% v/v formic acid. Vortex the mixture, and then incubate at room temperature for 1 h, vortexing for 1 min every 10 min.
    13. Centrifuge the mixture at 3,803 x g for 5 min at room temperature to pellet the silica gel. Transfer the MeOH extract to a clean Falcon tube. Repeat the centrifugation of the silica gel mixture to recover as much of the MeOH extract as possible.
    14. Filter the MeOH extract using a 1 ml syringe and a 0.2 μm PTFE membrane filter. Concentrate the extract to a final volume of 1-1.5 ml using a dry heating block set to 60 °C (see Note 13).

  4. Semi-preparative RP - HPLC
    1. Begin running the HPLC mobile phase (30% ACN:70% H2O, with 0.1% formic acid) through the C18 semi-preparative HPLC column (held at a constant temperature of 50 °C) at a constant flow rate of 4 ml/min until the column is equilibrated.
    2. Load 50 μl of the sample onto the column, and use the following mobile phase running conditions for sample separation: hold at 30% ACN:70% H2O for 7.5 min, then increase linearly to 50% ACN:50% H2O over a period of 12.5 min. Hold at 50% ACN:50% H2O for 5 min, then return to initial conditions using a linear gradient over 7.5 min. Hold at the initial conditions for at least 12 min before beginning the next injection (see Note 14).
    3. Monitor the separated metabolites using a detection wavelength of 230 nm, and collect fractions of the peak that corresponds to the desired N-coronafacoyl phytotoxin (see Note 15).
    4. Repeat the sample injection, metabolite separation and fraction collection until all of the initial extract has been used.
    5. Pool the collected fractions for the N-coronafacoyl phytotoxin of interest, and evaporate to dryness using a rotary evaporator. The evaporator heating bath (filled with water) should be set to 70 °C, and the condenser should be chilled with a circulating solution of 50% v/v ethylene glycol (chilled to -10 °C using a refrigerated circulating bath).
    6. Re-dissolve the pure metabolite in 3 ml of MeOH + 0.1% v/v formic acid. Transfer the solution to a pre-weighed screw cap vial, and dry down in a heating block set to 60 °C.
    7. Weigh the vial + dried sample, and calculate the weight of the purified sample. Store the dried sample at -20 °C until needed (see Notes 16 and 17).

Notes

  1. We have observed variations in the production levels of the N-coronafacoyl phytotoxins depending on the strain of S. scabies that is used, and therefore the volume of culture that is grown may need to be altered depending on the production efficiency of the particular strain.
  2. The large-scale cultures can be left to incubate for up to 14 days; however, the levels of metabolite present in the culture will not increase significantly after 10 days.
  3. The freezing process forces proteins and any other dissolved material out of solution, which will then be removed during the first chloroform extraction. This will make the subsequent extractions cleaner and will allow for faster and more complete phase separation. Use of a plastic container to freeze/thaw the supernatant is recommended, since a glass beaker may crack during the freezing process.
  4. All steps (with the exception of the centrifugation in steps B2 and C8) should be carried out in a chemical safety hood or a fume hood.
  5. This step serves as a purification step since many non-carboxylic acid compounds will move into the organic phase under the basic pH conditions used. Carboxylic acids such as the N-coronafacoyl phytotoxins will be in the unprotonated form at the high pH and therefore will remain in the aqueous phase.
  6. Under the pH conditions used, the N-coronafacoyl phytotoxins are in the pronated form and will therefore be more soluble in the chloroform than in the aqueous culture supernatant.
  7. The pH of the KHCO3 solution promotes deprotonation of the N-coronafacoyl phytotoxins and their subsequent movement into the aqueous phase.
  8. This mixing stage produces a large amount of gas. Do not fill the 125 ml funnel with more than 75 ml of aqueous and organic solvent in total. If the funnel contains more than that volume, the expression of gas will force open the stopper or stopcock and the extract will be lost.
  9. Steps C3-C7 are performed in order to determine the migration point of the N-coronafacoyl phytotoxin of interest. A pure sample of the molecule (~50 μg) and a small portion of the organic extract (~30-50 μl) are applied to a 5 x 20 cm analytical TLC plate, and separation is carried out using the same conditions that will be employed for the preparative TLC steps.
  10. If a pure standard of the particular N-coronafacoyl phytotoxin of interest is not available, then the N-coronafacoyl phytotoxin coronatine (COR) can be used instead. This phytotoxin is available for purchase from Sigma-Aldrich Canada (catalog number: C8115).
  11. For best results, make sure that the spotted extract has air dried before new extract is applied to the same area.
  12. The position of the N-coronafacoyl phytotoxin of interest on the reference analytical TLC plate will not correspond exactly to the phytotoxin position on the preparative TLC plate (as demonstrated in Figures 4 and 5). However, by comparing the relative position of the standard and extract bands on the reference plate, the location of the phytotoxin on the preparative TLC plate can be estimated. As the N-coronafacoyl phytotoxin of interest will not appear as a distinct band or fragment on the preparative TLC plate but as a dark smear, it is recommended that an area of at least 1 cm past the edge of this smear is marked.
  13. The sample should not be concentrated to a volume lower than this as otherwise the metabolites may precipitate out of solution.
  14. The HPLC running conditions described are suitable for separation of the CFA-L-Ile coronafacoyl phytotoxin from other metabolites in the extract. However, if minor N-coronafacoyl phytotoxins containing other isoleucine isomers (e.g., D-isoleucine) are produced, they will not be separated from the CFA-L-Ile metabolite and will be co-purified. Also, if other N-coronafacoyl phytotoxins or biosynthetic intermediates are being targeted for purification, the HPLC mobile phase running conditions may need to be adjusted accordingly in order to get good separation of those metabolites.
  15. Using the HPLC column and running conditions described, the CFA-L-Ile coronafacoyl phytotoxin has a retention time of 18-19 min (Figure 6).
  16. The purified metabolite can also be re-dissolved in MeOH + 0.1% formic acid to a final concentration of 1 mg/ml, and then stored at -20 °C.
  17. To confirm the purity of the metabolite, the sample can be analyzed using liquid chromatography-mass spectrometry (LC-MS) as described elsewhere (Bown et al., 2016).


    Figure 6. Purification of CFA-L-Ile by semi-preparative RP - HPLC. Following purification by preparative TLC, the CFA-L-Ile - containing extract is loaded onto a C18 semi-preparative HPLC column, and the metabolite is monitored by measuring the absorbance at 230 nm. The peak corresponding to CFA-L-Ile is indicated by the asterisks, and it is this peak that is targeted for collection by the HPLC fraction collector. Note that due to the high amount of CFA-L-Ile in the extract, the absorbance reading exceeds the maximum level measured by the detector, and thus the peak appears cut off in the chromatogram. Also, as N-coronafacoyl phytotoxins containing different isoleucine isomers can sometimes be produced by S. scabies in SFMB, the collected peak may contain a mixture of the different isomers since they will not separate out during the HPLC purification (Bown et al., 2016).

Recipes

  1. Soy flour mannitol broth (SFMB) medium (Kieser et al., 2000)
    20 g/L soy flour
    20 g/L D-mannitol
    Dissolve mannitol in water
    Add soy flour
    Sterilize by autoclaving
  2. Preparative TLC mobile phase (Fyans et al., 2015)
    195 ml ethyl acetate
    4 ml 2-propanol
    0.5 ml acetic acid
    0.5 ml water

Acknowledgments

This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), and the Newfoundland and Labrador Research & Development Corporation (RDC) to D.R.D.B. L.B. was supported by a fellowship from the Memorial University School of Graduate Studies. This protocol is based on work originally reported in Bown et al. (2016) and Fyans et al. (2015).

References

  1. Bignell, D. R., Huguet-Tapia, J. C., Joshi, M. V., Pettis, G. S. and Loria, R. (2010). What does it take to be a plant pathogen: genomic insights from Streptomyces species. Antonie van Leeuwenhoek 98(2): 179-194.
  2. Bown, L., Altowairish, M. S., Fyans, J. K. and Bignell, D. R. (2016). Production of the Streptomyces scabies coronafacoyl phytotoxins involves a novel biosynthetic pathway with an F420 -dependent oxidoreductase and a short-chain dehydrogenase/reductase. Mol Microbiol 101(1): 122-135.
  3. Dees, M. W. and Wanner, L. A. (2012). In search of better management of potato common scab. Potato Research 55(3): 249-268.
  4. Fyans, J. K., Altowairish, M. S., Li, Y. and Bignell, D. R. (2015). Characterization of the coronatine-like phytotoxins produced by the common scab pathogen Streptomyces scabies. Mol Plant-Microbe Interact 28(4): 443-454.
  5. Mitchell, R. E. and Frey, E. J. (1986). Production of N-coronafacoyl-L-amino acid analogues of coronatine by Pseudomonas syringae pv. atropurpurea in liquid cultures supplemented with L-amino acids. Microbiology 132(6): 1503-1507.
  6. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. and Hopwood, D. A. (2000). Practical Streptomyces genetics. The John Innes Foundation.
  7. Palmer, D. A. and Bender, C. L. (1993). Effects of environmental and nutritional factors on production of the polyketide phytotoxin coronatine by Pseudomonas syringae pv. glycinea. Appl Environ Microbiol 59(5): 1619-1626.

简介

该方法用于大规模纯化由马铃薯常见的痂病原体耻骨链球菌sc疮产生的N, - 海胆酰基植物毒素。 该方法采用有机萃取方法。 在交替的碱性和酸性条件下培养上清液以优先分离含有上清液的含有毒素的羧酸部分。 然后使用制备型薄层色谱法和半制备型反相 - 高效液相色谱法进一步纯化所需的单独的N, - 焦油酰甲酰植物毒素。

马铃薯普通痂病是由革兰氏阳性,丝状,链霉菌属的土壤细菌引起的经济上重要的作物病。第一个描述和最好的特征是痂病引起链霉菌属(Streptomyces spp。)是具有全球分布(Bignell等人,2010)的Streptomyces scabies (syn。 S。scabiei 。目前常见的痂病治理方法包括作物轮作,灌溉和土壤熏蒸;然而,这些策略经常失败,产生不一致的结果或者环境不友好(Dees和Wanner,2012)。为了开发更好的疾病控制策略,我们必须首先了解 S使用的分子机制。 sc疮感染植物并诱发疾病症状。研究表明,S的能力。引起疾病的sc疮是由于在感染过程中产生不同作用的毒力因子。在由s生产的已知或潜在的毒力因子中。 sc疮是植物毒素的家族,被称为具有类似于植物激素茉莉酸的可能起到抑制作用的 N - 角鲨烯酰植物毒素(也称为COR-样代谢产物)病原体感染期间的植物免疫应答(Bignell等人,2010; Fyans等人,2015)。由S生产的主要冠醚酰基植物毒素。 sc疮是由通过酰胺键与L-异亮氨酸连接的聚酮化合物代谢物冠心酸组成的抗坏血酸酰基-L-异亮氨酸(CFA-L-Ile;图1)。此外,可以少量生产含有不同异亮氨酸异构体或不同氨基酸(例如缬氨酸)的其它 N' - 角鲨烯酰植物毒素(Fyans等,。,2015; Bown 等人,2016)。


图1.由耻骨链球菌镰刀菌 产生的N 非焦油酰-L-异亮氨酸(CFA-L-Ile)的结构

这里描述的方案被开发用于分离和纯化来自大规模培养物的N,N-二十氢酰基植物毒素及其生物合成中间体。用于结构和功能表征的sc疮。以前,Fyans等人。 (2015)描述了一种方案,其部分基于用于从革兰氏阴性植物致病菌假单胞菌(Pseudomonas)的培养物中分离相关的重组核糖油酰基植物毒素冠状病毒(COR)的公开程序丁香(Palmer和Bender,1993)。如Fyans等人所概述的。 (2015),株系。在大豆粉甘露醇肉汤(SFMB)培养基中培养sc疮,促进冠状酰基油酰化植物毒素的生成,然后将培养上清液在有机溶剂中在碱性和酸性条件下按顺序进行两步萃取以选择性地分离培养物上清液中含有植物毒素的羧酸部分。然后使用制备薄层色谱(TLC)和半制备型反相 - 高效液相色谱(RP-HPLC)的组合进一步纯化植物毒素。最近,我们描述了该方案的修改版本,其中我们使用碳酸氢钾水溶液(Bown等人,2016)并入了另外的提取步骤。该修改是基于Mitchell和Frey(1986)描述的用于分离的程序。丁香 N N。。oyl oyl oyl oyl。。。,。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。 sc疮植物毒素制剂。此外,我们通过加入少量的酸改性了有机溶剂,这种有机溶剂显着提高了纯化的植物毒素在下游结构和功能研究中的溶解度和产率。
   这里,我们将介绍一下我们目前如何净化 S的详细分步协议。 sc疮 N - 我们实验室中的角质酰甲酰植物毒素。

关键字:链霉菌, 植物病原体, 疮痂病, N-coronafacoyl-L-异亮氨酸, 植物毒素, 薄层色谱法, 高效液相色谱法

材料和试剂

  1. pH测试条(VWR,BDH ®,目录号:BDH35309.606)
  2. 亲水性聚丙烯膜过滤器,直径47mm,孔径0.45μm(Pall,目录号:66548)
  3. Fisherbrand TM B类透明玻璃螺纹小瓶,带有封闭件,3.7 ml(Fisher Scientific,目录号:03-338A)
  4. 滑动注射器1毫升(BD,目录号:309659)
  5. PTFE膜过滤器,孔径0.2μm,直径6 mm(VWR,目录号:28145-491)
  6. Whatman TM滤光片,12.5厘米(Sigma-Aldrich,目录号:WHA1113125)
  7. 锥形离心管,50ml(Corning,Falcon ®,目录号:352098)
  8. 硅胶GF制备TLC板,预吸附剂区,20 x 20厘米,1,000微米(Analtech/iChromatography,目录号:P32013)
  9. DMSO的菌丝体冷冻库存。 sc疮(Fyans等人,2015)
  10. Bacto TM 胰蛋白酶大豆肉汤培养基(BD,Bacto TM,目录号:211825)
  11. 氢氧化钠(NaOH)(Fisher Scientific,目录号:BP359-212)
  12. 氯仿,ACS级(VWR,BDH ®,目录号:BDH1109)
  13. 盐酸(HCl),ACS级(Avantor 性能材料,目录号:638801)
  14. 无水硫酸钠(Na 2 SO 4),ACS级(Fisher Scientific,目录号:S421-500)
  15. 乙二醇(VWR,BDH ,目录号:BDH1125)
  16. 碳酸氢钾(KHCO 3),ACS级(Sigma-Aldrich,目录号:237205)
  17. 甲醇(MeOH),HPLC级(Sigma-Aldrich,目录号:34860)
  18. 甲酸,试剂级(Sigma-Aldrich,目录号:F0507)
  19. HiPerSolv CHROMANOR ®用于HPLC的乙腈(ACN)(VWR,BDH,目录号:BDH83639.400)
  20. 大豆粉,脱脂(MP Biomedicals,目录号:960024)
  21. D-甘露醇(AMRESCO,目录号:0122)
  22. 乙酸乙酯,ACS级(Sigma-Aldrich,目录号:319902)
  23. 2-丙醇,HPLC级(Fisher Scientific,目录号:A451SK-4)
  24. 乙酸,ACS级(Sigma-Aldrich,目录号:695092)
  25. 水,HPLC级(EMD Millipore,目录号:WX0008-1)
  26. 大豆面粉甘露醇肉汤(SFMB)培养基(见食谱)
  27. 制备型TLC流动相(参见食谱)

设备

  1. 玻璃锥形瓶,125ml(Corning,PYREX ,目录号:C4980125)
  2. 玻璃锥形瓶4升(Corning,PYREX ,目录号:C49804L)
  3. 玻璃过滤器支架组件,带漏斗,烧结基座,止动器和夹具,47 mm(EMD Millipore,目录号:XX1004700)
  4. Nalgene TM 具有密封闭合的PPCO离心机瓶,250ml(Thermo Fisher Scientific,Thermo Scientific TM,目录号:31410250)
  5. Sorval TM ST16R台式冷冻离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorval TM ST 16R,目录号:BCT25) br />
  6. Innova ® 42R冷藏培养箱振荡器,轨道直径为1.9厘米(Eppendorf,New Brunswick TM,型号:Innova ® 42R,目录号:M1335- 004)
  7. 2升塑料容器
  8. Nalgene TM 具有封闭的FEP FEP分液漏斗,2L(Thermo Fisher Scientific,Thermo Scientific TM,目录号:4301-2000) br />
  9. IKA ®旋转蒸发器系统(IKA ®,型号:RV 10数字V)
  10. VWR ®带可编程温度控制器的冷冻循环浴(VWR,型号:VWR ®冷藏循环浴,目录号:89202-982)
  11. KIMAX ®具有PTFE活塞和玻璃塞的Squibb分液漏斗,125 ml(Kimble Chase Life Science and Research Products,目录号:29048F-125)
  12. DryFast隔膜真空泵(Welch Vacuum - Gardner Denver,目录号:2044)
  13. Biohit mLINE 单通道机械移液器,2-20μl(VWR,目录号:47745-545)
  14. Aldrich矩阵TLC显影槽(Sigma-Aldrich,型号:Z126195)
  15. 紫外灯带便携式机柜(Analtech/iChromatography,目录号:A93-04)
  16. 金属铲子
  17. Thermo Scientific TM干式加热器(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:88-860-021)
  18. ZORBAX StableBond80ÅC18半制备型HPLC柱,9.4 x 250 mm,5μm(Agilent Technologies,目录号:880975-202)
  19. 化学安全罩
  20. KIMAX ®分级过滤瓶,1L(Kimble Chase Life Science and Research Products,目录号:27060-1000)
  21. GeneMate涡流混合器(VWR,目录号:490000-094)
  22. Agilent 1260 Infinity分析级LC纯化系统,具有四元泵,自动进样器,二极管阵列检测器和馏分收集器(Agilent Technologies,型号:1260 Infinity)

程序

  1. 文化成长
    1. 准备种子文化。通过将1ml DMSO冷冻储存物接种到125ml锥形瓶中的25ml胰蛋白酶大豆肉汤培养基中,并在28℃下振荡(200RPM)孵育48小时。
    2. 将10ml的种子培养物在4升锥形瓶中1升SFMB培养基中培养10分钟,并在25℃下振荡(200RPM)孵育10天(参见注释1和2)。
    3. 将培养物分成四个250ml离心瓶,并在4℃下以3,005×g离心15分钟以沉淀细胞材料。将上清液倒入2升塑料容器中,储存于-20°C直到需要。即使立即提取上清液,将其冻结至少24小时也是有用的(见注3)。

  2. 有机提取(见图2和附注4)


    图2.从S提取冠醚酰基植物毒素期间执行的主要步骤的示意图。 sc疮培养上清

    1. 使用pH条测量培养上清液的pH,并使用5N NaOH溶液(〜4-5ml)将pH调节至11-12。
    2. 将上清液倒入2L分液漏斗中,并用0.5体积(〜450ml)氯仿萃取。要进行萃取,将氯仿加入分液漏斗中,关闭漏斗塞,并摇动漏斗以使内容物良好混合。打开旋塞以释放任何表达的气体,然后重复振荡,直到不再表达气体。允许有机(底部)和水(顶部)层通过重力(〜15-20分钟;图3A)尽可能分离,然后打开旋塞并收集有机层。如果存在乳剂层,则可以通过在4℃下以3,005×g离心10分钟收集并分离,并将水层返回到分液漏斗(图3B)。弃去有机层,并用另外0.5体积的新鲜氯仿重复提取含水上清液。进行提取总共三次,每次提取后丢弃有机层(见注5)

      图3.提取。 sc ies 用氯仿培养上清。 A.使用2L分液漏斗进行提取。混合上清液和氯仿后,通过重力使相分离尽可能多。上层深棕色层为水相,下层为有机相。 B.当存在乳剂层时,将其收集到离心瓶中,并通过离心分离各相。
    3. 通过加入1N HCl(水溶液)(〜5ml)将水性上清液酸化至pH为1-2。
    4. 将上清液倒入2L分液漏斗中,并用0.5体积(〜450ml)氯仿萃取。使用与2所述相同的提取程序,除了现在应该收集和保存有机层。进行三次萃取,然后收集有机层,弃去水层(见附注6)。
    5. 通过加入〜30g无水Na 2 SO 4从有机提取物中除去残留的水,并轻轻地旋转混合物。加入无水Na 2 SO 4,直到其不再形成团块,表明所有的残余水已被吸收。
    6. 使用0.45μm聚丙烯膜过滤器和连接到真空泵的玻璃过滤器组件过滤有机萃取物。用新鲜氯仿洗涤含有有机提取物的烧杯以及过滤器组件,以回收所有残留的提取物
    7. 使用旋转蒸发器将有机萃取液浓缩至〜100ml。蒸发器加热浴(充满水)应设定为50℃,冷凝器应冷却至50%v/v乙二醇的循环溶液(使用冷藏循环浴冷至-10℃)。 ;
    8. 将50ml有机浓缩液转移到两个125ml分液漏斗中的每一个中。用0.5体积(25毫升)的0.5M KHCO 3水溶液萃取每种浓缩物。混合后,通过重力使相分离约5分钟,然后分别收集各相。重复萃取有机浓缩物两次,然后收集收集的水相(给出两组水提取物,各75ml),弃去有机萃取液(见注7)。
    9. 用0.5体积(35-40毫升)氯仿洗涤含水萃取液两次。为了洗涤,将氯仿加入到含有水提取物的分液漏斗中,混合,然后放置5分钟以使相分离。收集并丢弃有机层,并重复。洗涤后,将含水萃取液合并(总体积〜140〜150ml)
    10. 使用1N HCl(水溶液)(〜12-15ml)将水提取物的pH调节至1-2。酸会导致与KHCO 3的中和反应,产生热量和大量的CO 2。必须缓慢加入酸,否则反应会变得剧烈,会引起酸溅。加入每1ml酸后,轻轻混合溶液直至CO 2分散。
    11. 将40ml水提取物倒入四个125ml分液漏斗中的每一个中。用0.5体积(20ml)的氯仿将每个等分试样提取三次。将所有有机萃取液收集并合并在一起,并丢弃水层。在化学品安全罩中的未覆盖的烧杯中将有机萃取物蒸发干燥过夜(见附注8)
  3. 制备TLC(见注4)
    1. 通过上下移液将干燥的有机萃取物再溶解在含有0.1%v/v甲酸的2-3ml MeOH中。将提取物转移到干净的螺旋盖玻璃瓶中。用额外的1ml MeOH + 0.1%甲酸冲洗烧杯,以回收剩余量的提取物。使用1 ml注射器和0.2μmPTFE膜过滤器将提取物过滤到一个新的螺旋瓶中。
    2. 将TLC流动相(参见食谱)倒入TLC显影槽中,并填充至约2厘米的深度。通过将两块12.5厘米的滤纸放入容器中并覆盖该罐至少30分钟,使TLC室中的空气饱和。
    3. 用铅笔标记5 x 20厘米分析TLC板,以指示流动相溶液的浸没点(见注9)。
    4. 将溶于100%MeOH + 0.1%甲酸的至少30-50μg标准的目标分子(或比较类似物;参见附注10)施用到TLC板上。将标准从板的边缘至少1厘米,并在板上的浸入标记之上至少1厘米。
    5. 将约30-50μl的有机提取物沿板宽约2厘米。确保所提取的提取物距离板的边缘至少1厘米,距离板上浸入标记1厘米处,距离之前施加的标准1厘米(见注11)。
    6. 从TLC储罐中取出滤纸盘,并将TLC板放入罐中并盖住。允许流动相迁移到板顶部的1厘米(约20-40分钟)内。取出TLC板,使其干燥(约15分钟)。重复此过程。
    7. 将空气干燥的TLC板置于紫外线灯下,用铅笔标记所用标准品和有机提取物中所需的 - 焦油酰基植物毒素的位置(图4)。


      图4."S"的TLC分析。含有CFA-L-Ile的有机提取物 将纯化的样品(50μg)的CFA-L-Ile(i)和小部分(30-50μl)的有机提取物)含有CFA-L-Ile被点样到5×20cm硅胶GF分析TLC板上。分离后,将板在UV光下显现,纯标准品的位置与蓝色矩形中植物毒素的预期位置一起标记(蓝色圆圈)。黑色箭头表示样品在板上的迁移方向。

    8. 用铅笔标记制备型TLC板,以指示将浸入流动相溶液中的板的面积。
    9. 沿着TLC板的整个宽度施加有机提取物,每次10μl。不要将任何样品放置在板的任一边缘的1厘米内或标记的浸没区域的1厘米内(见附注11)。
    10. 从TLC储罐中取出滤纸盘,并将TLC板放入罐中并盖住。允许流动相迁移到板顶部的1厘米(〜1-1.5小时)内。从罐中取出板,并让其风干(约20-30分钟)。将板返回到水箱并重新分开。
    11. 将空气干燥的TLC板置于紫外线灯下,用铅笔标记转移到其中的芥子油 - 植物毒素植物的区域(图5;见附注12)。


      图5.S的制备TLC分析。含有CFA-L-Ile的sc ies> organic。。。。。>>>。。。。。。。。。。。。。。。。。。。。。(参见图4)。为了确保CFA-L-Ile的完全回收,从板上刮下二氧化硅的大面积(由蓝色矩形表示)。白色箭头表示板上代谢物的迁移方向。

    12. 使用金属刮刀的平坦边缘刮去标记的区域以除去硅胶结合的代谢物。将硅胶转移到Falcon锥形管中,加入10ml MeOH + 0.1%v/v甲酸。涡旋混合物,然后在室温下孵育1小时,每10分钟涡旋1分钟
    13. 将混合物在室温下离心分离5分钟,使硅胶沉淀。将MeOH提取物转移到干净的Falcon管中。重复硅胶混合物的离心,以尽可能多地回收MeOH提取物。
    14. 使用1 ml注射器和0.2μmPTFE膜过滤器过滤MeOH萃取液。使用设定为60℃的干燥加热块将提取物浓缩至1-1.5ml的最终体积(参见附注13)。

  4. 半制备RP-HPLC
    1. 开始通过C18半制备HPLC柱(保持在50℃的恒定温度)下运行HPLC流动相(30%ACN:70%H 2 O,0.1%甲酸)恒定流速为4 ml/min,直到色谱柱平衡
    2. 将50μl样品加入柱中,并使用以下流动相运行条件进行样品分离:保持在30%ACN:70%H 2 O下7.5分钟,然后线性增加至50% ACN:在12.5分钟的时间内为50%H 2 O。保持在50%ACN:50%H 2 O下5分钟,然后使用7.5分钟的线性梯度返回初始条件。在初始条件下保持至少12分钟,然后开始下次注射(见附注14)。
    3. 使用230nm的检测波长监测分离的代谢物,并收集对应于所需的N, - 邻 - 酰基油酰植物毒素的峰的部分(参见附注15)。
    4. 重复样品注射,代谢物分离和级分收集,直到使用所有初始提取物。
    5. 将收集到的用于所需的N, - 邻 - 酰基油酰植物毒素的馏分进行收集,并使用旋转蒸发器蒸发至干。蒸发器加热浴(充满水)应设置在70℃,冷凝器应用50%v/v乙二醇的循环溶液冷却(使用冷藏循环浴冷至-10℃)。
    6. 将纯代谢物重新溶解在3ml MeOH + 0.1%v/v甲酸中。将溶液转移到预先称重的螺旋盖小瓶中,并在设定为60℃的加热块中干燥。
    7. 称量小瓶+干燥样品,计算纯化样品的重量。将干燥的样品储存在-20°C直到需要(参见注释16和17)。

笔记

  1. 我们已经观察到取决于S的应变的N,N-二十氢酰基植物毒素的生产水平的变化。所使用的sc疮,因此可能需要根据特定菌株的生产效率改变培养物的体积。
  2. 大型文化可以孵育多达14天;然而,培养物中存在的代谢物的水平在10天后不会显着增加。
  3. 冷冻过程迫使蛋白质和任何其他溶解的物质脱离溶液,然后在第一次氯仿萃取过程中将其去除。这将使随后的提取物更清洁,并且将允许更快和更完整的相分离。建议使用塑料容器冷冻/解冻上清液,因为玻璃烧杯在冷冻过程中可能会破裂。
  4. 所有步骤(步骤B2和C8中的离心除外)应在化学品安全罩或通风橱中进行。
  5. 该步骤用作纯化步骤,因为许多非羧酸化合物将在所用的碱性pH条件下移入有机相。羧酸,例如N, - 蒽醌酰基植物毒素在高pH下将处于未质子化的形式,因此将保留在水相中。
  6. 在所使用的pH条件下,N' - 角鲨烯酰植物毒素呈旋转形式,因此在氯仿中比在水性培养上清液中更易溶。
  7. KHCO 3溶液的pH促进了N, - 邻 - 酰基油酰植物毒素的去质子化和随后的运动进入水相。
  8. 该混合阶段产生大量的气体。不要在125ml的漏斗中填充超过75ml的水和有机溶剂。如果漏斗含有超过该体积,气体的表达将强制打开塞子或旋塞,并且提取物将丢失。
  9. 执行步骤C3-C7以便确定感兴趣的N,N-二十氢酰基植物毒素的迁移点。将分子的纯样品(约50μg)和少量有机提取物(〜30-50μl)施加到5×20cm分析TLC板上,并使用相同的条件进行分离用于制备TLC步骤。
  10. 如果不能获得目标特定的N, - 邻 - 酰基油酰植物毒素的纯标准,则可以代替使用N,N-二十氢酰甲酰基植物毒素冠状病毒(COR)。该植物毒素可从Sigma-Aldrich Canada(目录号:C8115)购买。
  11. 为获得最佳效果,请确保在将新提取物应用于同一区域之前,点样提取物已经空气干燥。
  12. 目的基因分析TLC板上的目的蛋白质-NO-海胆酰基植物毒素的位置将不完全对应于制备型TLC板上的植物毒素位置(如图4和5所示)。然而,通过比较参考板上的标准品和提取物条带的相对位置,可以估计制备型TLC板上的植物毒素的位置。由于目的在于制备TLC板上不同的条带或片段,但是作为黑色涂片,所以推荐将至少1cm的面积超过这个涂片被标记。
  13. 不要将样品浓缩到低于此值的体积,否则代谢物可能沉淀出溶液
  14. 描述的HPLC运行条件适用于从提取物中的其他代谢物中分离CFA-L-Ile冠醚酰基植物毒素。然而,如果产生含有其他异亮氨酸异构体(例如,异亮氨酸)的次要的N, - 大茴香酰植物毒素,则它们不会与CFA-L-Ile代谢物分离并将被共同纯化。另外,如果其他的N -/- 巨氢酰基植物毒素或生物合成中间体被靶向用于纯化,则可能需要相应地调整HPLC流动相运行条件以便获得这些代谢物的良好分离。
  15. 使用HPLC柱和所述的运行条件,CFA-L-Ile冠醚酰基植物毒素的保留时间为18-19分钟(图6)。
  16. 纯化的代谢物也可以重新溶解在MeOH + 0.1%甲酸中,最终浓度为1 mg/ml,然后储存在-20°C。
  17. 为了确认代谢物的纯度,可以使用液相色谱 - 质谱(LC-MS)分析样品,如其他地方所述(Bown等人,2016)。


    图6.通过半制备型RP-HPLC纯化CFA-L-Ile。在通过制备型TLC纯化后,将含CFA-L-Ile的提取物加载到C18半制备型HPLC柱,并通过测量230nm处的吸光度来监测代谢物。对应于CFA-L-Ile的峰由星号表示,是由HPLC馏分收集器收集的该峰。请注意,由于提取物中CFA-L-Ile含量高,吸光度读数超过检测器测得的最大值,因此色谱图中出现峰值。另外,由于含有不同异亮氨酸异构体的N - 诺卡非酰甲基植物毒素有时可以通过S来产生。 SFMB中的sc疮,收集的峰可能含有不同异构体的混合物,因为它们在HPLC纯化期间不会分离出来(Bown等人,2016)。

食谱

  1. 大豆面粉甘露醇肉汤(SFMB)培养基(Kieser等人,2000)
    20克/升大豆粉
    20g/L D-甘露糖醇
    将甘露糖溶解在水中
    添加大豆粉
    高压消毒灭菌
  2. 制备型TLC流动相(Fyans等人,2015)
    195ml乙酸乙酯
    4毫升2-丙醇
    0.5ml乙酸
    0.5 ml水

致谢

这项工作得到加拿大自然科学与工程研究理事会(NSERC),加拿大创新基金会(CFI)以及纽芬兰和拉布拉多研究与发展研究院的资助。发展公司(RDC)到D.R.D.B.磅。得到了纪念大学研究生院研究生的支持。该协议基于最初在Bown等人中报告的工作。 (2016)和Fyans等人。 (2015)。

参考文献

  1. Bignell,DR,Huguet-Tapia,JC,Joshi,MV,Pettis,GS和Loria,R。(2010)。  作为植物病原体需要做什么:来自链霉菌种类的基因组分析安东尼·范Leeuwenhoek 98(2):179-194。
  2. Bown,L.,Altowairish,MS,Fyans,JK和Bignell,DR(2016)。  寻找更好的马铃薯常见痂的管理。土豆研究 55(3):249-268。
  3. Fyans,JK,Altowairish,MS,Li,Y.和Bignell,DR(2015)。  由常见的痂病原体耻骨链球菌痉挛产生的类冠状动植物毒素的表征。 Mol Plant-Microbe Interact 28(4 ):443-454
  4. Mitchell,RE和Frey,EJ(1986)。< a class ="ke-insertfile"href ="http://mic.microbiologyresearch.org/content/journal/micro/10.1099/00221287-132-6-1503 "靶="_ blank">通过丁香假单胞菌(Pseudomonas syringae)生产冠状病毒的n 非酰基酰基-L-氨基酸类似物。补充有L-氨基酸的液体培养物中的 atropurpurea 。微生物学 132(6):1503-1507。
  5. Kieser,T.,Bibb,MJ,Buttner,MJ,Chater,KF和Hopwood,DA(2000)。< a class ="ke-insertfile"href ="https://www.jic.ac.uk/约翰·恩斯基金会基金会。 。
  6. Palmer,DA and Bender,CL(1993)。  效果的环保和营养因素通过丁香假单胞菌生产聚酮化合物植物毒素冠状病毒。大肠杆菌。 Appl Environ Microbiol 59(5):1619-1626。
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引用:Bown, L. and Bignell, D. R. (2017). Purification of N-coronafacoyl Phytotoxins from Streptomyces scabies. Bio-protocol 7(7): e2214. DOI: 10.21769/BioProtoc.2214.
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