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Jul 2020

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Non-radioactive Assay to Determine Product Profile of Short-chain Isoprenyl Diphosphate Synthases
用于确定短链异戊烯基二磷酸合酶产物谱的非放射性分析   

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Abstract

Isoprenoids represent the largest class of metabolites with amazing diversities in structure and function. They are involved in protecting plants against pathogens or herbivores or involved in attracting pollinators. Isoprenoids are derived from geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15), geranylgeranyl diphosphate (GGPP; C20), and geranylfarnesyl diphosphate (GFPP; C25) that are in turn formed by sequential condensations of isopentenyl diphosphate (IPP; C5) with an allylic acceptor such as dimethylallyl diphosphate (DMAPP; C5), GPP, FPP, or GGPP in a reaction catalyzed by isoprenyl diphosphate synthases (IDSs). IDS enzyme assay for determination of prenyl diphosphate products is generally performed using radiolabelled substrates, and the products formed are identified by employing expensive instruments such as phosphor imager, radio-GC, or radioHPLC. Though a non-radioactive assay for measuring IDS activity in crude plant extract has been reported, it requires a complex methodology utilizing chromatography coupled with tandem mass spectrometry (LC/MS-MS). Here, we describe a non-radioactive and simple inexpensive assay for determining the IDS assay products using non-radiolabeled IPP and its co-allylic substrates DMAPP,GPP, and FPP. The detection of prenyl diphosphate products generated in the assay was highly efficient and spots corresponding to prenyl alcohols were visible at >40 µM concentrations of IPP and DMAPP/GPP/FPP substrates. The protocol described here is sensitive, reliable, and technically simple, which could be used for functional characterization of IDS candidates.

Keywords: Isoprenyl diphosphate synthase (异戊烯基二磷酸合酶), Non-radioactive (非放射性), Phosphatase (磷酸酶), Apyrase (腺苷三磷酸双磷酸酶), Prenyl alcohol (异戊烯醇), Thin layer chromatography (薄层色谱法)

Background

Short-chain IDSs catalyze the chain length elongation reaction (1′-4 condensations of IPP units) in which allylic isoprenoids substrates C5-DMAPP, C10-GPP, or C15-FPP couple with IPP to generate linear isoprenoids (Figure 1). For instance, Catharanthus roseus GPPS large subunit (CrGPPS.LSU) and GGPP synthase 2 (CrGGPPS2) catalyze the formation of GPP/GGPP [Figure 4 in Rai et al., 2013] and GGPP [Figure 3 in Kumar et al., 2020], respectively, by sequential incorporation of IPP units with DMAPP, GPP, or FPP as substrates. The most widely used assay for determination of IDS activity is discontinuous, time and labor intensive, and involves the use of IPP radiolabeled with either 14[C] or 3[H]. A non-radioactive and LC–MS-based method for determining IDS activity has been reported using crude plant extract utilizing chromatography coupled with tandem mass spectrometry (LC/MS-MS) (Nagel et al., 2012). The protocol described here provides an efficient approach to determine the IDS activity without the need for radioactive substrates and high-end instrumentation. The method involves thin layer chromatography (TLC) separation of IDS assay products and visualization and quantification by iodine staining. The assay products generated can be extracted from TLC plate and further verified through LC/MS-based approach as reported in Kumar et al., 2020. We have used purified CrGPPS.LSU, a bifunctional G(G)PP synthase (EC 2.5.1.29) catalyzing the formation of both GPP and GGPP (Rai et al., 2013) in this protocol. The IDS assay described here is highly specific, sensitive, and technically simple which can be useful in determining the functional activity of short-chain IDS enzymes. The efficiency of enzymatic product detection with this method is comparable to that of previously reported radioactive and non-radioactive assays in terms of substrate concentrations.



Figure 1. IPP and DMAPP are utilized in the formation of GPP (C10), FPP (C15), and GGPP (C20) in a reaction catalyzed by short-chain IDS such as geranyl diphosphate synthases (GPPS), farnesyl diphosphate synthases (FPPS), and geranylgeranyl diphosphate synthase (GGPPS), respectively. While FPPS catalyzes the formation of FPP by condensation of DMAPP with 2 IPP units or GPP with one IPP unit, GGPPS forms of GGPP by condensation of DMAPP with three IPP units, GPP with two IPP units, or FPP with one IPP unit.

Materials and Reagents

Note: The materials and reagents not provided with company and catalog number can be ordered from any qualified company for using in this experiment.

  1. Pipette tips (Axygen, USA)

  2. 1.5 ml Microcentrifuge tubes (Eppendorf, catalog number: T9661)

  3. Parafilm (Sigma-Aldrich, catalog number: P7793)

  4. Pencils

  5. Disposable latex gloves

  6. Poly-Prep chromatography columns (Bio-Rad, catalog number: 7311550)

  7. Disposable PD-10 desalting columns (GE Healthcare, catalog number: GE17-0851-01)

  8. Purified isoprenyl diphosphate synthase [Catharanthus roseus GPPS large subunit (CrGPPS.LSU) for this protocol]

  9. Rosetta 2(DE3) Competent Cells (Novagen, catalog number: 71400-M)

  10. pET-28a(+) vector (Novagen, catalog number: 69864)

  11. Ni2+-charged nitrilotriacetic acid (NTA) Agarose (Bio-Rad, catalog number: 7800800)

  12. Luria Bertani Broth, Miller (HIMEDIA, catalog number: M1245)

  13. Luria Bertani Agar (HIMEDIA, catalog number: M1151F)

  14. Kanamycin sulfate (Sigma-Aldrich, catalog number: 10106801001)

  15. Chloramphenicol (Sigma-Aldrich, catalog number: C0378)

  16. Imidazole (Sigma-Aldrich, catalog number: I5513-25G)

  17. Lysozyme from hen egg white (Sigma-Aldrich, catalog number: 10837059001)

  18. Isopentenyl diphosphate (Echelon Biosciences, catalog number: I-0050)

  19. Dimethylallyl diphosphate (Echelon Biosciences, catalog number: I-0051)

  20. Geranyl diphosphate (Echelon Biosciences, catalog number: I-0100)

  21. Farnesyl diphosphate (Echelon Biosciences, catalog number: I-0150)

  22. Apyrase from potatoes (Sigma-Aldrich, catalog number: A6132)

  23. Alkaline phosphatase from calf intestine (CIP) (Sigma-Aldrich, catalog number: P4978)

  24. Geraniol (Sigma-Aldrich, catalog number: 48798)

  25. Farnesol (Sigma-Aldrich, catalog number: 43348)

  26. Geranylgeraniol (Sigma-Aldrich, catalog number: G3278)

  27. TLC Silica gel 60 RP-18 F254s (Merck, catalog number: 105560)

  28. Glycine (Sigma-Aldrich, catalog number: G7126-100G)

  29. 10% Mini-PROTEAN® TGXTM precast protein gels (Bio-Rad, catalog number: 4561033)

  30. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771-100G)

  31. Bradford Protein Assay Dye Reagent (Bio-Rad, catalog number: 5000006)

  32. Coomassie brilliant blue (CBB-R-250) (Sigma-Aldrich, catalog number: B-7920-10G)

  33. Bromophenol blue (Sigma-Aldrich, catalog number: B5525-5G)

  34. Ethanol (Emsure, catalog number: 1.00983.0511)

  35. Water HPLC grade (Sigma-Aldrich, catalog number: 270733)

  36. Glycerol (Sigma-Aldrich, catalog number: G5516)

  37. 3-Morpholino-2-hydroxypropanesulfonic acid (MOPSO) (Sigma-Aldrich, catalog number: M8389)

  38. 1,4-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 10197777001)

  39. Magnesium chloride (MgCl2) (HIMEDIA, catalog number: GRM4340)

  40. Iodine (I2) crystals (Sigma-Aldrich, catalog number: 229695)

  41. n-Hexane (Merck, catalog number: 104369)

  42. TRIS hydrochloride (Tris-HCl) (HIMEDIA, catalog number: MB030)

  43. Methanol (Merck, catalog number: 60600905001730)

  44. Isopropyl-1-thio-β-D-galactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I5502-5G)

  45. Lysis buffer (see Recipes)

  46. Wash buffer (see Recipes)

  47. Elution buffer (see Recipes)

  48. Storage buffer (see Recipes)

  49. Assay buffer (see Recipes)

  50. Dephosphorylation buffer (see Recipes)

  51. Mobile phase (see Recipes)

  52. TG buffer (see Recipes)

Equipment

Note: The equipment not provided with company and catalog number can be ordered from any qualified company for using in this experiment.

  1. Pipettes

  2. Eppendorf Thermo Mixer® F1.5 (Eppendorf, catalog number: 5384000012)

  3. Glass rectangular developing chamber 20 x 20 cm (with lid)

  4. Measuring cylinders

  5. Scale ruler

  6. Tweezers

  7. Scissors

  8. Borosilicate beakers, capacity 500 ml

  9. Rocker (GeNei, catalog number: 107106GB)

  10. Variable volume pipettes (Eppendorf)

  11. Magnetic stirrer (GeNei, catalog number: 117795GB)

  12. Adsorbent TLC scraper (Sigma-Aldrich, catalog number: Z265268)

  13. Vacuum concentrator (Savant Speed Vac, catalog number: SPD131DDA)

  14. Fume hood

  15. Sonicator (PRO Scientific, catalog number: H-1021-2)

  16. Circulating refrigerated water bath

  17. ThermoMixer C (Eppendorf, catalog number: 5382000015)

  18. Vortex mixer (GeNei, catalog number: 106887GB)

  19. Centrifuge 5424 R (Eppendorf, catalog number: 5404000014)

  20. Digital camera or scanner

Software

  1. ImageJ (National Institutes of Health, USA, https://imagej.nih.gov/ij/)

Procedure

  1. Expression and purification of recombinant isoprenyl diphosphate synthase protein

    1. Transform Escherichia coli rosetta-2 cells with plasmid harboring IDS of interest (Here, we have used pET28a:CrGPPS.LSU). The GeneBank loci IDs for nucleotide and protein sequences are JX417183 and AGL91645, respectively. For more information on enzyme characteristics of CrGPPS.LSU, please refer to Rai et al., 2013.

    2. Inoculate a single colony in 25 ml Luria-Bertani (LB) medium with 37 mg/ml chloramphenicol and 50 mg/ml kanamycin.

    3. Grow overnight (16 h) at 37 °C with 200 rpm and transfer 5 ml of overnight grown culture to 1,000 ml of LB medium with 37 mg/ml chloramphenicol and 50 mg/ml kanamycin.

    4. Place the culture flask in an incubator shaker set at 37 °C and 200 rpm and grow the cells until the OD600 reaches 0.5.

    5. Add isopropyl-1-thio-β-D-galactopyranoside (IPTG) to a final concentration of 0.4 mM and continue growing cultures for an additional 18 h at 18 °C.

    6. Centrifuge cultures at 6,000 x g for 10 min at 4 °C to obtain cell pellets.

    7. Proceed to the next step or store the pellet at -80 °C until further use.

    8. Resuspend cell pellet in lysis buffer with 2-5 ml per gram cell pellet.

    9. Add 1 mg/ml lysozyme to the cell suspension and keep on ice for 30 min.

    10. Sonicate the cell suspension using a sonicator equipped with microtip and give six 10 s bursts with a 10 s cooling period.

    11. Recover the lysate by centrifugation at 12,000 x g for 20 min at 4 °C and transfer to a 50 ml tube.

    12. Take 1 ml of 50% Ni2+-NTA agarose slurry and remove the storage solution by brief centrifugation at 1,000 x g for 15 s and resuspend the Ni2+-NTA agarose in lysis buffer. Add 1 ml of resuspended Ni2+-NTA resin to the recovered lysate in the previous step and keep the tube on rotor shaker for 1 h at 4 °C.

    13. Transfer Ni2+-NTA resin (containing captured His-tagged protein) to the 0.8 x 4 cm Poly-Prep chromatography column and allow to settle by gravity.

    14. Equilibrate the column with 5 column volumes of lysis buffer containing 10 mM Imidazole.

    15. Wash the Ni2+-NTA resin with 5 column volumes of wash buffer containing 20 mM Imidazole to remove the unbound protein.

    16. Elute the bound protein and collect the fractions with 3 ml of elution buffer containing 250 mM Imidazole.

    17. Equilibrate PD-10 desalting column with 4 ml of storage solution and transfer protein eluant to the column.

    18. Add 2 ml storage buffer and collect eluant in 0.5 ml fractions (E1-E4).

    19. Load 30 µl protein sample on 10% SDS gel and perform electrophoresis in Tris-Glycine (TG) buffer.

    20. Determine the concentration of purified protein using the Bradford method (Bradford, 1976).


  2. Isoprenyl diphosphate synthase assay

    1. Perform IDS assay by adding equimolar concentrations (10 µM to 80 µM) of IPP and DMAPP, IPP and GPP, and IPP and FPP in a final volume of 200 µl of assay buffer in separate 1.5 ml centrifuge tubes.

    2. Add 5 µg of purified IDS enzyme (CrGPPS.LSU in this assay) to each tube to initiate the reaction.

    3. Take three separate 1.5 ml tubes and add 5 µg of heat inactivated (95 °C for 10 min) CrGPPS.LSU for negative controls in a final volume of 200 µl of assay buffer containing equimolar concentrations (40 µM) of IPP and DMAPP, IPP and GPP, and IPP and FPP.

    4. Incubate all tubes containing IDS assay reaction mixture at 30 °C in a circulating water bath or ThermoMixer for 6 h.

    5. Add 200 µl of dephosphorylation buffer and continue to incubate for 16 h at 30 °C in a ThermoMixer to hydrolyze all diphosphate esters (unreacted substrates as well as prenyl products).

    6. Add 1 ml n-hexane and vortex vigorously for 30 s to extract the hydrolyzed reaction products and substrates (prenyl alcohols).

    7. Centrifuge for 30 s at 12,000 x g, 25 °C.

    8. Carefully transfer the upper hexane fraction (approximately 800 µl) containing the prenyl alcohols to a new 1.5 ml centrifuge tube using a 1 ml pipette.

    9. Concentrate the collected hexane fraction containing the prenyl alcohols to 25 µl using a SpeedVac (at 30 °C for 30 min) and immediately use it for TLC analysis.


  3. Determination of IDS product profile

    1. Take a 500 ml beaker and add mobile phase solvent to a depth of about 0.5 cm. Seal the beaker with parafilm, swirl it gently and allow it to stand for 2 to 5 min for saturation of the chamber.

    2. Take a reverse phase TLC silica gel plate with a dimension of 5.0 x 7.5 cm. Draw a horizontal line with a pencil, 1 cm above from both edges of the bottom.

    3. Mark five spots along the line on TLC silica gel plates at equidistance (approximately 5-7 mm apart) for applying hexane fraction containing prenyl alcohols.

    4. Prepare prenyl alcohol standard solution in hexane containing 40 µM each of geraniol, farnesol, and geranylgeraniol.

    5. Use a 10 µl pipette to carefully spot the samples on the marked spots of TLC silica gel plate. After spotting each sample, blow gently on the TLC plate to evaporate the solvent.

      1. Spot the standard solution containing GOH, FOH, and GGOH in lane S.

      2. Spot 20 µl of the concentrated hexane fraction of negative control at lane number 1.

      3. Spot 20 µl hexane of the extracted prenyl alcohol products from different enzymatic reactions on the corresponding TLC plates (Figure 2).

    6. Use tweezers to place the prenyl alcohols spotted TLC silica gel plate in the beaker containing the mobile phase.

    7. Perform TLC chromatographic separation by sealing the beaker with parafilm and allow solvent front to rise upward.

    8. Take out the TLC silica gel plate from the beaker just before the solvent front reaches the top end (about 15 min), mark the solvent front with a pencil and allow the TLC silica gel plate to dry for 5-10 min.

    9. Stain the TLC plate by exposing it to iodine vapors for 3 to 5 min in a rectangular glass chamber containing iodine crystals (Figure 3b, Kumar et al., 2020).

    10. Visualize the prenyl alcohol spots corresponding to product and substrates by comparing with reference standards (Rai et al., 2013).

    11. Document the TLC image with prenyl alcohol spots using a digital camera or scanner.

    12. The documented product spots can be relatively quantified using ImageJ software.


  4. Verification of prenyl alcohol products

    1. After documenting the TLC images, circle the spots and scrape the corresponding spots of prenyl diphosphate products parallel to the reference compounds using TLC scraper.

    2. Elute the prenyl diphosphate product and reference compound in methanol.

    3. Concentrate the eluted compounds under N2 gas stream.

    4. Perform standard liquid chromatography mass spectrometry (LC-ESI-MS) according to the protocol described in Kumar et al. (2020) in a positive mode to confirm prenyl diphosphate products and to rule out any possible contamination from acid hydrolysis reactions (Figure 3c, Kumar et al., 2020).

Data analysis

In this methodology, a non-radioactive IDS assay has been employed to measure the activity of the CrGPPS.LSU which provides metabolic flux for both primary and specialized metabolites in C. roseus (Rai et al., 2013). CrGPPS.LSU has been well characterized and therefore, serves as an appropriate reference enzyme to validate the prenyl alcohol product profile generated using this non-radioactive assay. IDS assay was performed in the presence of CrGPPS.LSU and the formation and visualization of prenyl alcohols (GOH and GGOH) was carried out (Figure 2). The optimal conditions for the prenyl alcohol product identification were explored by probing the intensities of product spots arising after using different concentrations of substrates. The spots corresponding to GOH, and GGOH prenyl alcohol products were visible at ≥20 μM concentration of IPP and DMAPP substrates (Figure 2A). However, the spots corresponding to GGOH were detectable in a linear range and clearly visible at ≥10 μM concentration of IPP and GPP substrates (Figure 2B) and ≥40 μM concentration of IPP and FPP substrates (Figure 2C). Therefore, the results from the non-radioactive IDS assay demonstrate that about 40-80 μM of IPP and DMAPP/GPP/FPP substrate concentration was ideal to achieve the optimal and detectable level of prenyl alcohol product from the assay (Figure 2).



Figure 2. In vitro IDS assays of recombinant (His)6-CrGPPS.LSU. Spots on the TLC plates correspond to reaction products from CrGPPS.LSU assays using IPP and DMAPP (plate A), IPP and GPP (plate B), and IPP and FPP (plate C). Products formed are indicated by arrows and the spots in dashed boxes are unreacted substrates. The products were confirmed by comparing the spots with authentic geraniol (GOH), farnesol (FOH), and geranylgeraniol (GGOH) standards (lane S). Lane 1 in A, B, and C TLC plates represents negative control in which boiled CrGPPS.LSU protein was assayed with 40 µM each of IPP and DMAPP, IPP and GPP, and IPP and FPP, respectively. Lanes 2-5 represent reaction products of CrGPPS.LSU protein assayed with 10 µM, 20 µM, 40 µM, and 80 µM each of IPP and DMAPP (plate A), IPP and GPP (plate B), and IPP and FPP (plate C), respectively. Lane S: Authentic prenyl alcohol standard mix.

Notes

  1. The standards geraniol, farnesol, and geranylgeraniol were not used as carriers in all chromatographic separations of prenyl alcohol products.

  2. Overnight incubation at 30 °C after adding dephosphorylation buffer is necessary to completely hydrolyze diphosphate esters.

  3. To minimize background smear and for better chromatographic separation, carefully remove the hexane fraction, the organic layer (in the B8 step).

  4. Wear gloves while handling the TLC plates contamination.

  5. Sample spotting diameter should not be more than 3-4 mm.

  6. Mark the TLC plates very gently with a pencil to avoid damaging the silica gel.

  7. The level of mobile phase should not cover spots when the TLC plate is placed in the beaker.

  8. Always use freshly prepared mobile phase solvents for better resolution of the spots and reproducibility of the results.

  9. The product and substrate spots should be circled with a pencil upon removal of TLC plates from iodine vapor chamber as they fade away after a few minutes.

Recipes

  1. Lysis buffer (10 ml)

    50 mM NaH2PO4

    300 mM NaCl

    10 mM Imidazole, pH-8.0

  2. Wash buffer (100 ml)

    50 mM NaH2PO4

    300 mM NaCl

    20 mM Imidazole, pH-8.0

  3. Elution buffer (5 ml)

    50 mM NaH2PO4

    300 mM NaCl

    250 mM Imidazole, pH-8.0

  4. Storage buffer (50 ml)

    25 mM MOPSO, pH to 7.0

    15% [v/v] glycerol

  5. Assay buffer (10 ml)

    25 mM MOPSO, pH to 7.0

    10% [v/v] glycerol

    2 mM DTT

    10 mM MgCl2

  6. Dephosphorylation buffer (2 ml)

    0.2 M Tris-HCl, pH-9.5

    2 units of CIP (stock 18 units/mg)

    2 units of potato apyrase (stock 25.2 units/mg)

  7. Mobile phase solvent (50 ml)

    Methanol:water [95:5 v/v]

  8. TG buffer

    25 mM Tris

    192 mM Glycine

    0.1% SDS

Acknowledgments

This protocol was developed by modifying methods from Orlova et al. (2009) and Rai et al. (2013). This work was supported by the Department of Biotechnology supported projects BT/HRD/35/24/2006 and BT/PR6109/AGII/106/857/2012 to DAN. AR was supported by a Research Fellowship of University Grants Commission, New Delhi, India. The institutional communication number for this article is CIMAP/PUB/2020/APR/21.

Competing interests

The authors declare no competing interests.

References

  1. Bradford, M. M., (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Kumar, S. R., Rai, A., Bomzan, D. P., Kumar, K., Hemmerlin, A., Dwivedi, V., Godbole, R. C., Barvkar, V., Shanker, K., Shilpashree, H. B., Bhattacharya, A., Smitha, A. R., Hegde, N. and Nagegowda, D. A. (2020). A plastid-localized bona fide geranylgeranyl diphosphate synthase plays a necessary role in monoterpene indole alkaloid biosynthesis in Catharanthus roseus. Plant J 103(1): 248-265.
  3. Nagel, R., Gershenzon, J. and Schmidt, A. (2012). Nonradioactive assay for detecting isoprenyl diphosphate synthase activity in crude plant extracts using liquid chromatography coupled with tandem mass spectrometry. Anal Biochem 422(1): 33-38.
  4. Orlova, I., Nagegowda, D. A., Kish, C. M., Gutensohn, M., Maeda, H., Varbanova, M., Fridman, E., Yamaguchi, S., Hanada, A., Kamiya, Y., Krichevsky, A., Citovsky, V., Pichersky, E. and Dudareva, N. (2009). The small subunit of snapdragon geranyl diphosphate synthase modifies the chain length specificity of tobacco geranylgeranyl diphosphate synthase in planta. Plant Cell 21(12): 4002-4017.
  5. Rai, A., Smita, S. S., Singh, A. K., Shanker, K. and Nagegowda, D. A. (2013). Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis. Mol Plant 6(5): 1531-1549.

简介

[Abstrac吨]类异戊二烯代表最大的一类代谢物在结构和功能惊人多样性。它们参与保护植物免受病原体或草食动物侵害,或参与吸引传粉媒介。(;çGPP类异戊二烯是从牻牛儿基二磷酸衍生的10 ),法呢基二磷酸(FPP;Ç 15 ),香叶基香叶基二磷酸(GGPP;Ç 20 ),和geranylfarnesyl二磷酸(GFPP; C ^ 25 ),它们又通过的顺序缩合形成异戊烯基二甲基磷酸酯(IPP; C 5 )与烯丙基受体,例如二磷酸二甲基烯丙酯(DMAPP; C 5),GPP,FPP或GGPP)由异戊二烯基二磷酸合酶(IDS)催化的反应。用于确定异戊二烯基二磷酸酯产物的IDS酶测定法通常是使用放射性标记的底物进行的,并且所形成的产物是通过使用昂贵的仪器(例如磷光成像仪,radio-GC或radio-HPLC)来鉴定的。尽管已经报道了一种用于测量粗植物提取物中IDS活性的非放射性测定方法,但它需要使用色谱结合串联质谱(LC / MS-MS)的复杂方法。在这里,我们描述了用于确定使用非放射性标记的IPP及其共同烯丙基底物DMAPP,GPP的IDS分析产物非放射性和简单廉价的测定法,和FPP。在测定中生成的异戊二烯基二磷酸产物的检测非常高效,并且在浓度大于40 µM的IPP和DMAPP / GPP / FPP底物时,可以看到对应于异戊二烯醇的斑点。此处描述的协议灵敏,可靠且技术简单,可用于IDS候选者的功能表征。


[背景]短链的IDS催化链长度延伸反应(1 ' -4的IPP单元缩合),其中烯丙基类异戊二烯基板Ç 5 -DMAPP,C 10 -GPP ,或C 15 -FPP夫妇与IPP以产生线性类异戊二烯(图1)。例如,长春花GPPS大亚基(CrGPPS.LSU)和GGPP合酶2(CrGGPPS2)催化GPP / GGPP [Rai等人,2013年的图4 ]和GGPP [Kumar等人,2020年的图3]的形成。 ],分别由的IPP单位DMAPP,GPP顺序掺入,或FPP作为底物。用于确定IDS活性的最广泛使用的测定法是不连续的,费时费力的,并且涉及使用放射性同位素标记为14 [C]或3 [H ]的IPP 。据报道,使用粗植物提取物通过色谱和串联质谱(LC / MS-MS)结合使用的一种非放射性和基于LC-MS的方法来测定IDS活性(Nagel等,2012)。在这里描述的协议提供了一种有效的方法来确定IDS活性瓦特ithout需要对放射性底物和高端仪器。该方法涉及IDS分析产物的薄层色谱(TLC)分离,以及碘染色的可视化和定量分析。该测定法的产品生成可以从TLC板被提取,并进一步通过LC / MS证实为基础的方法作为库马尔报道等人。,2020年w ^ E具有用过纯化CrGPPS.LSU,双官能G(G)PP合酶(EC 2.5 1.29)在该协议中催化GPP和GGPP的形成(Rai等人,2013)。本文所述的IDS分析具有高度的特异性,敏感性和技术上的简便性,可用于确定短链IDS酶的功能活性。就底物浓度而言,用这种方法检测酶产物的效率与以前报道的放射性和非放射性a- says相当。

图1. IPP和DMAPP在地层GPP(C被利用10 ),FPP(C 15 ),和GGPP(C 20在由短链催化的反应)IDS如牻牛儿基二磷酸合成酶(GPPS),法尼基二磷酸合酶(FPPS) ,和香叶基香叶基二磷酸合酶(GGPPS)表示。尽管FPPS通过DMAPP与2个IPP单元缩合或GPP与一个IPP单元缩合来催化FPP的形成,但是GDMAS通过DMAPP与3个IPP单元,GPP与2个IPP单元缩合或FPP与一个IPP单元的缩合形成GGPP形式。

关键字:异戊烯基二磷酸合酶, 非放射性, 磷酸酶, 腺苷三磷酸双磷酸酶, 异戊烯醇, 薄层色谱法

材料和试剂
注意:可以从任何合格的公司订购未提供公司和目录号的材料和试剂,以用于本实验。
移液器吸头(美国阿克西根)
1.5 ml微量离心管(Eppendorf,目录号:T9661)
封口膜(Sigma-Aldrich,目录号:P7793)
铅笔
一次性乳胶手套
Poly-Prep色谱柱(Bio-Rad,目录号:7311550)
一次性PD-10脱盐柱(GE Healthcare,目录号:GE17-0851-01)
纯化的异戊二烯基二磷酸合酶[用于该方案的长春花玫瑰花GPPS大亚基(CrGPPS.LSU)]
Rosetta 2(DE3)感受态细胞(Novagen,目录号:71400-M)
pET-28a(+)载体(Novagen,目录号:69864)
含Ni 2+的次氮基三乙酸(NTA)琼脂糖(Bio-Rad,目录号:7800800)
Luria Bertani Broth,Miller(HIMEDIA,目录号:M1245)
Luria Bertani Agar(HIMEDIA,目录号:M1151F)
硫酸卡那霉素(Sigma-Aldrich,目录号:10106801001)
氯霉素(Sigma-Aldrich,目录号:C0378 )
咪唑(Sigma-Aldrich,目录号:I5513-25G )
鸡蛋清中的溶菌酶(Sigma-Aldrich,目录号:10837059001)
二磷酸异戊烯基(Echelon Biosciences,目录号:I-0050)
二磷酸二甲基烯丙基(Echelon Biosciences,目录号:I-0051)
Geranyl diphosp ha te(Echelon Biosciences,目录号:I-0100)
法呢基二磷酸酯(Echelon Biosciences,目录号:I-0150)
马铃薯中的磷酸酶(Sigma-Aldrich,目录号:A6132)
小牛肠中的碱性磷酸酶(CIP)(Sigma-Aldrich,目录号:P4978)
香叶醇(Sigma-Aldrich,目录号:48798)
法尼醇(Sigma-Aldrich,目录号:43348)
香叶基香叶醇(Sigma-Aldrich,目录号:G3278)
TLC硅胶60 RP-18 F 254 s(Merck,目录号:105560)
甘氨酸(Sigma-Aldrich,目录号:G7126-100G )
10%的Mini-PROTEAN ® TGX TM预制蛋白凝胶(Bio-Rad公司,目录号:4561033)
十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771-100G)
Bradford蛋白测定染料试剂(伯乐(Bio-Rad)公司,目录号:5000006)
考马斯亮蓝(CBB-R-250)(Sigma-Aldrich,目录号:B-7920-10G)
溴酚蓝(Sigma-Aldrich,目录号:B5525-5G)
乙醇(Emsure,目录号:1.00983.0511)
水HPLC级(Sigma-Aldrich,目录号:270733)
甘油(Sigma-Aldrich,目录号:G5516)
3-Morpholino-2-hydroxypropanesulfonic acid(MOPSO)(Sigma-Aldrich,目录号:M8389)
1,4-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:10197777001)
氯化镁(MgCl 2 )(HIMEDIA,目录号:GRM4340)
碘(I 2 )晶体(Sigma-Aldrich,目录号:229695)
Ñ正己烷(Merck公司,目录号:104369 )
盐酸TRIS(Tris-HCl)(HIMEDIA,目录号:MB030)
甲醇(Merck,目录号:60600905001730)
异丙基-1-硫代-β-D-吡喃半乳糖苷(IPTG)(Sigma-Aldrich,目录号:I5502-5G)
裂解缓冲液(请参见食谱)
洗涤缓冲液(请参见食谱)
洗脱缓冲液(请参见配方)
存储缓冲区(请参见食谱)
分析缓冲液(请参见配方)
脱磷酸缓冲液(请参见配方)
流动相(请参见食谱)
TG缓冲液(请参见食谱)

设备
注意:可以从任何合格的公司订购未提供公司和产品目录号的设备以用于本实验。
P ipettes
的Eppendorf热混合器® F1.5仪(Eppendorf,目录号:5384000012 )
玻璃矩形显影室20 x 20 cm(带盖)
测量缸
比例尺
镊子
剪刀
容量500毫升的硼硅酸盐烧杯
跷板(GeNei,货号:107106GB )
可变容量移液器(Eppendorf)
电磁搅拌器(GeNei,目录号:117795GB)
吸附式TLC刮板(Sigma-Aldrich,目录号:Z265268)
真空浓缩器(Savant Speed Vac,目录号:SPD131DDA)
通风柜
Sonicator(PRO Scientific,目录号:H-1021-2)
循环冷冻水浴
ThermoMixer C(Eppendorf,目录号:5382000015 )
涡旋混合器(GeNei,目录号:106887GB)
5424 R离心机(Eppendorf,目录号:5404000014)
数码相机或扫描仪

软件
ImageJ(美国国立卫生研究院,https://imagej.nih.gov/ij/)


程序

重组异戊二烯基二磷酸合酶蛋白的表达与纯化
用带有目标IDS的质粒转化大肠杆菌Rosetta-2细胞(此处,我们使用了pET28a:CrGPPS.LSU)。核苷酸和蛋白质序列的GeneBank位点ID分别为JX417183和AGL91645。有关CrGPPS.LSU酶特性的更多信息,请参阅Rai等人,2013年。
在装有37 mg / ml氯霉素和50 mg / ml卡那霉素的25 ml Luria-Bertani(LB)培养基中接种单个菌落。
在37°C下以200 rpm的速度过夜生长(16 h),然后将5 ml过夜生长的培养物转移到1,000 ml含37 mg / ml氯霉素和50 mg / ml卡那霉素的LB培养基中。
将培养瓶置于设置在37°C和200 rpm的恒温振荡器中,并使细胞生长直至OD 600达到0.5。
加入异丙基-1-硫代-β-D-吡喃半乳糖苷(IPTG)的终浓度为0.4 mM,并在18°C下继续培养18 h。
在4°C下以6,000 xg离心培养10分钟,以获得细胞沉淀。
继续进行下一步或将沉淀保存在-80°C直至进一步使用。
将细胞沉淀重悬于裂解缓冲液中,每克细胞沉淀2-5 ml。
向细胞悬液中加入1 mg / ml溶菌酶,并在冰上放置30分钟。
使用配备了微尖端的超声仪对细胞悬液进行超声处理,并在10 s的冷却时间内发出6个10 s的脉冲。
通过在4°C下以12,000 xg离心20分钟回收裂解物,然后转移至50 ml管中。             
取1毫升的50%的Ni 2+ -NTA琼脂糖浆液,并以1去除由短暂离心所述存储溶液,000 ×g离心15秒和重悬的Ni 2+在裂解缓冲液的琼脂糖-NTA。在上一步中,将1 ml重悬的Ni 2+ -NTA树脂添加到回收的裂解物中,并将试管在4°C下保持在转子振荡器上1 h。
将Ni 2+ -NTA树脂(包含捕获的His标记的蛋白)转移到0.8 x 4 cm的Poly-Prep色谱柱中,并通过重力沉降。
用含有10 mM咪唑的5倍柱体积的裂解缓冲液平衡柱。
用5倍柱体积的含有20 mM咪唑的洗涤缓冲液洗涤Ni 2+ -NTA树脂,以去除未结合的蛋白质。
洗脱结合的蛋白质,并用3 ml含有250 mM咪唑的洗脱缓冲液收集级分。
用4 ml储存溶液平衡PD - 10脱盐柱,然后将蛋白质洗脱液转移到该柱上。
加入2 ml的储存缓冲液,并以0.5 ml的馏分(E1-E4)收集洗脱液。
将30 µl蛋白质样品上样到10%SDS凝胶上,并在Tris-甘氨酸(TG)缓冲液中进行电泳。
确定使用纯化蛋白的浓度的Bradford法(布拉德福德,1976)。

异戊二酸二磷酸合酶测定
通过添加等摩尔浓度(执行IDS测定10 μM到IPP的80μM)和DMAPP,IPP和GPP ,和IPP和在200μl的测定缓冲液的单独的1.5ml离心管中,终体积FPP。
向每个试管中加入5 µg纯化的IDS酶(在此测定法中为CrGPPS.LSU)以引发反应。
取三个单独的1.5 ml管,并在最终体积为200 µl的测定缓冲液(含等摩尔浓度(40 µM)的IPP和DMAPP,IPP )中加入5 µg加热灭活(95°C,10分钟)的CrGPPS.LSU作阴性对照。和GPP ,以及IPP和FPP。
INCU在30贝特含有IDS分析反应混合物中的所有管℃的循环水浴或恒温6小时。
加入200μl脱磷酸化缓冲液中并继续incub吃16小时,在30在一个℃的恒温以水解所有二磷酸酯(未反应的底物,以及异戊烯基产品)。
加入1ml ñ -己烷和剧烈涡旋30秒以提取已水解的反应产物和底物(异戊烯醇)。
在25 °C下以12,000 xg离心30 s 。
仔细含有上部己烷级分(约800微升)转移异戊烯醇到一个使用1ml移液器新的1.5毫升离心管中。
使用SpeedVac(在30°C下30分钟)将收集的含有异戊二烯醇的己烷馏分浓缩至25 µl,并立即用于TLC分析。

确定IDS产品资料
取一个500毫升的烧杯,并加入流动相溶剂至约0.5厘米的深度。用石蜡膜密封烧杯,轻轻旋转,然后静置2至5分钟以使反应室饱和。
采取反相TLC硅胶板用一个5.0×7.5cm的尺寸。绘制的水平线一个铅笔1厘米以上从底部的两个边缘。
沿TLC硅胶板上的线等距离(相距约5-7 mm)标记五个点,以涂覆含异戊二烯醇的己烷馏分。
制备在含有40μM的每个香叶醇,金合欢醇己烷异戊二烯醇标准溶液,和香叶基香叶醇。
使用10 µl移液管将样品小心地标记在TLC硅胶板上的标记点上。发现每个样品后,在TLC板上轻轻吹动以蒸发掉溶剂。
现货含有GOH,FOH标准溶液,并在GGOH车道S.
在第1泳道点点20 µl阴性对照的浓己烷馏分。
从不同酶促反应中提取的异戊二烯醇产物中的20 µl己烷在相应的TLC板上点样(图2)。 
使用镊子放置异戊二烯基醇在含有烧杯斑TLC硅胶板的流动相。
通过用封口膜密封烧杯,进行TLC色谱分离,并使溶剂前沿向上上升。
从溶剂前沿之前到达顶端(约15分钟)的烧杯中取出TLC硅胶板,标记溶剂前沿与一个铅笔并允许TLC硅胶板上干燥5-10分钟。
在含有碘晶体的矩形玻璃室内将TLC板暴露于碘蒸气中3至5分钟,以对其染色(图3b,Kumar等,2020)。
通过与参考标准进行比较,可视化对应于产物和底物的异戊二烯醇斑点(Rai等人,2013)。
使用数码相机或扫描仪记录带有异戊二烯醇斑点的TLC图像。
可以使用ImageJ软件相对定量地记录所记录的产品斑点。

异戊烯醇机生产线的验证CTS
记录TLC图像后,圈出斑点,并使用TLC刮板刮除平行于参考化合物的异戊二烯基二磷酸异戊酯产物的相应斑点。
洗脱异戊二烯基diphosph一个在甲醇TE产品和参比化合物。
在N 2气流下浓缩洗脱的化合物。
根据Kumar等人所述的协议执行标准液相色谱质谱(LC-ESI-MS)。(2020年)以肯定的方式确认异戊二烯基二磷酸产物,并排除酸水解反应可能造成的污染(图3c,Kumar等人,2020年)。

数据分析



在这种方法中,非放射性测定法IDS已被用来测量,其提供代谢通量对于初级的CrGPPS.LSU的活性和在专门的代谢产物长春花(RAI等。,2013)。CrGPPS.LSU已被很好地表征,因此可作为一种适当的参考酶,以验证使用该非放射性测定法生成的异戊二烯醇产物谱。在CrGPPS.LSU存在下进行IDS分析,并进行异戊烯醇(GOH和GGOH)的形成和可视化(图2)。通过探测在使用不同浓度的底物后产生的产物斑点的强度,探索了异戊烯醇产物鉴定的最佳条件。的对应于GOH斑点,和GGOH异戊二烯醇产物瓦特ERE可见在≥ IPP和DMAPP基板20μM浓度(图2A)。然而,对应于GGOH斑点是在一个线性范围检测和在清晰可见≥ IPP和GPP基板10μM浓度(图2B)和≥ 40 μM浓度IPP和FPP基板(FIGUR ë2C)。因此,吨他从结果非放射性IDS分析表明,大约4 0 - 80μM的IPP和DMAPP / GPP / FPP衬底浓度是理想的,以实现所述最佳和检测水平的异戊二烯基醇产物从所述测定(图2) 。

图2.重组(His)6 -CrGPPS.LSU的体外IDS分析。在TLC板上的斑点对应于从使用IPP CrGPPS.LSU测定反应产物和DMAPP(板A),IPP和GPP(板B) ,和IPP和FPP(板C)。形成的产物用箭头表示,虚线框中的斑点是未反应的底物。通过将斑点与真实的香叶醇(GOH),法尼醇(FOH)和香叶基香叶醇(GGOH)标准品(泳道S)进行比较来确认产品。泳道1中A,B ,和C TLC板表示,其中阴性对照煮沸CrGPPS.LSU PROT EIN用各自分别IPP和DMAPP,IPP和GPP,IPP和FPP和,40μM测定。泳道2-5代表用10μM,20μM,40μM测定CrGPPS.LSU蛋白质的反应产物,和80μM每个IPP和DMAPP(板A),IPP和GPP(板B)的,和IPP和FPP(板C)。泳道S:正宗异戊二烯醇标准混合物。 

笔记



在异戊烯醇产品的所有色谱分离中,未使用标准香叶醇,法尼醇和香叶基香叶醇作为载体。
加入脱磷酸缓冲液后,必须在30 °C过夜孵育才能完全水解二磷酸酯。
为了最大程度地减少背景污迹并实现更好的色谱分离,请小心除去己烷部分(有机层)(在B8步骤中)。
处理TLC板污染时请戴手套。
样品点样直径不应超过3-4毫米。
具有非常轻轻标出TLC板一个铅笔,以避免损坏硅胶。
将TLC板放入烧杯中时,流动相的水平不应覆盖斑点。
始终使用新鲜配制的流动相溶剂,以更好地分辨斑点和结果重现性。
的产物和底物斑点应当与盘旋一个在去除从碘蒸气室TLC板的铅笔如它们消失后离开一个几分钟。

菜谱
裂解缓冲液(10毫升)
50毫米NaH 2 PO 4
300毫米氯化钠
10 mM咪唑,pH-8.0
洗涤缓冲液(100毫升)
50毫米NaH 2 PO 4
300毫米氯化钠
20 mM咪唑,pH-8.0
洗脱缓冲液(5毫升)
50毫米NaH 2 PO 4
300毫米氯化钠
250 mM咪唑,pH-8.0
储存缓冲液(50毫升)
25 mM MOPSO,pH值至7.0
15%[v / v]甘油           
测定缓冲液(10毫升)
25 mM MOPSO,pH值至7.0
10%[v / v]甘油
2毫米DTT
10毫米MgCl 2
脱磷酸缓冲液(2毫升)
0.2 M Tris-HCl,pH-9.5
2单位CIP(原液18单位/毫克)
2个单位的马铃薯腺苷三磷酸酶(原液25.2单位/毫克)
流动相溶剂(50毫升)
甲醇:水[95:5 v / v]
TG缓冲液
25 mM Tris
192 mM甘氨酸
0.1%SDS

致谢
该协议是通过修改Orlova等人的方法开发的。(2009年)和Rai等人。(2013)。这项工作得到了生物技术部支持的项目BT / HRD / 35/24/2006和DAN的BT / PR6109 / AGII / 106/857/2012的支持。AR得到了印度新德里大学教育资助委员会研究奖学金的支持。本文的机构通信号码为CIMAP / PUB / 2020 / APR / 21。

利益争夺
作者宣称没有利益冲突。

参考文献
MM,布拉德福德(1976)。利用蛋白质-染料结合原理快速,灵敏地定量蛋白质的微克量。肛门生物化学72:248-254。
库马尔(Kumar,SR),赖(Rai),A.,孟山(Bomzan),DP,库马尔(Kumar),K.,Hemmerlin,A.,Dwivedi,V.,Godbole,RC,Barvkar,V.,Shanker,K.,Shilpashree,HB,Bhattacharya,A. ,Smitha,AR,Hegde,N.和Nagegowda,DA(2020)。质体定位的真品香叶基香叶基二磷酸合酶在长春花中的单萜吲哚生物碱生物合成中起着必要的作用。植物J 103(1):248-265。
Nagel,R.,Gershenzon,J.和Schmidt,A.(2012年)。液相色谱-串联质谱法检测植物粗提物中异戊二烯基二磷酸合酶活性的非放射性分析。Anal Biochem 422(1):33-38。
I. Orlova,Nagegowda,DA,Kish,CM,Gutensohn,M。Maeda,H.,Varbanova,M.,Fridman,E.,Yamaguchi,S.,Hanada,A.,Kamiya,Y.,Krichevsky, A.,Citovsky,V.,Pichersky,E.和Dudareva,N.(2009)。金鱼草香叶基二磷酸合酶的小亚基修饰了植物中烟草香叶基香叶基二磷酸合酶的链长特异性。植物细胞21(12):4002-4017。
Rai,A.,Smita,SS,Singh,AK,Shanker,K.和Nagegowda,DA(2013)。长春花(Catharanthus roseus)的异聚和同聚香叶基二磷酸合酶及其在单萜吲哚生物碱生物合成中的作用。摩尔工厂6(5):1531-1549。
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引用:Rai, A. and Nagegowda, D. (2021). Non-radioactive Assay to Determine Product Profile of Short-chain Isoprenyl Diphosphate Synthases. Bio-protocol 11(1): e3874. DOI: 10.21769/BioProtoc.3874.
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