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Liposome Disruption Assay to Examine Lytic Properties of Biomolecules
采用脂质体破碎测定法检测生物分子的裂解性质   

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本实验方案简略版
eLIFE
Dec 2016

Abstract

Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we describe an in vitro assay to evaluate the membrane lytic properties of proteins and biomolecules. Large unilamellar vesicles (liposomes) containing carboxyfluorescein at fluorescence-quenching concentrations are treated with the biomolecule of interest. A resulting increase in fluorescence due to leakage of the dye from liposomes and subsequent dilution in the buffer demonstrates that the biomolecule is sufficient for disrupting liposomes and membranes. Additionally, since liposome disruption may occur via pore-formation or via general solubilization of lipids similar to detergents, we provide a method to distinguish between these two mechanisms. Pore-formation can be identified and evaluated by examining the blockade of carboxyfluorescein release with dextran molecules that fit the pore. The methods described here were used to determine that the malaria vaccine candidate CelTOS and proapoptotic Bax disrupt liposomes by pore formation (Saito et al., 2000; Jimah et al., 2016). Since membrane lipid binding by a biomolecule precedes membrane disruption, we recommend the companion protocol: Jimah et al., 2017.

Keywords: Membrane (膜), Liposome (脂质体), Lipid (脂质), Disruption (破碎), Lysis (裂解), Pore (孔), Liposome leakage (脂质体渗漏), Carboxyfluorescein (羧基荧光素), Dextran (右旋糖酐)

Background

This protocol presents the procedure to evaluate the membrane lytic properties of proteins and other biomolecules. This protocol aims to provide a clear description of the various experimental steps necessary to study the membrane disrupting properties of biomolecules. Finally, the protocol describes a quantitative measurement of membrane disruption and can be applied to provide insight into the kinetics and mechanism of liposome disruption. This protocol was successfully used to study the malaria vaccine candidate CelTOS that provided a clear description of the first in vitro functional assay for CelTOS. The application of this protocol revealed that CelTOS (cell traversal protein for ookinetes and sporozoites) disrupts membranes containing phosphatidic acid. This insight suggests that CelTOS is secreted by malaria parasites within invaded host cells to disrupt the host cell membrane and enable the exit of parasites (Jimah et al., 2016). Finally, this assay can be readily applied to investigate the inhibition of CelTOS-mediated liposome disruption by small molecules, antibodies or peptides. While designed for CelTOS, this protocol is readily generalizable and applicable to any other biomolecule of interest.

Materials and Reagents

  1. Centrifuge tubes, 50 ml (Genesee Scientific, catalog number: 21-106 )
  2. Microcentrifuge tubes, 1.7 ml (Midsci, catalog number: AVSS1700RA )
  3. Parafilm (Bemis, catalog number: PM996 )
  4. Glass Pasteur pipets (Fisher Scientific, catalog number: 13-678-20C )
  5. Gravity sizing column (Bio-Rad Laboratories, catalog number: 7371032 )
  6. Glass tubes with plain end (Fisher Scientific, catalog number: 14-961-27 )
  7. Ring stand (Fisher Scientific, catalog number: S13747 )
  8. Clamps (Fisher Scientific, catalog number: 02-217-005 )
  9. Whatman filter (GE Healthcare, catalog number: 230300 )
  10. 200 nm polycarbonate Track-Etched filters (GE Healthcare, catalog number: 800281 )
  11. Carboxyfluorescein (Sigma-Aldrich, catalog number: C0662 )
  12. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  13. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958 )
  14. Sephadex G® G-25 (Sigma-Aldrich, catalog number: G25300 )
  15. Phospholipids (dissolved in chloroform, as supplied by manufacturer). Commonly used phospholipids are:
    1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, catalog number: 850375C )
    1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Avanti Polar Lipids, catalog number: 850457C )
    1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) (Avanti Polar Lipids, catalog number: 840857C )
    1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) (Avanti Polar Lipids, catalog numbers: 840034C )
  16. Nitrogen gas tank (Airgas, catalog number: NI HP300 )
  17. Triton-X 100 (Sigma-Aldrich, catalog number: X100 )
  18. Dextran molecules of different molecular weights and radii of gyration:
    5 kDa (Sigma-Aldrich, catalog number: 31417 )
    9 kDa (Sigma-Aldrich, catalog number: D9260 )
    39 kDa (Sigma-Aldrich, catalog number: D4133 )
    Note: This product has been discontinued.
    66.9 kDa (Sigma-Aldrich, catalog number: D1537 )
    Note: This product has been discontinued.
    148 kDa (Sigma-Aldrich, catalog number: D4876 )
    500 kDa (Sigma-Aldrich, catalog number: D5251 )
    Note: This product has been discontinued.
    1,500-2,800 kDa (Sigma-Aldrich, catalog number: D5376 )
  19. HEPES (Sigma-Aldrich, catalog number: H3375 )
  20. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  21. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  22. Ethyl ether (Fisher Scientific, catalog number: E138-500 )
  23. Ferric(III) chloride hexahydrate (Sigma-Aldrich, catalog number: 236489 )
  24. Ammonium thiocyanate (Sigma-Aldrich, catalog number: 221988 )
  25. Buffer-KCl (see Recipes)
  26. Buffer-NaCl (see Recipes)
  27. 0.1 M ammonium ferrothiocyanate (see Recipes)

Equipment

  1. Glass beaker (Fisher Scientific, catalog number: FB1012000 )
  2. pH meter (Fisher Scientific, model: AccumetTM AE150, catalog number: 13-636- AE153 )
  3. Stir bar
  4. BenchRockerTM variable 2D rocker (Benchmark Scientific, catalog number: BR2000 )
  5. Water sonicator (Laboratory Supplies, model: G112SP1T )
  6. Gas pressure regulator (Linde, catalog number: UPE-3-75-580 )
  7. Freeze-dry system for lyophilisation (Labconco, catalog number: 7750021 )
  8. Hi-vac flask (Labconco, catalog number: 7544400 )
  9. Vortexer (Fisher Scientific, catalog number: 02-215-414 )
  10. Rotary flask evaporator: Rotovap (IKA, model: RV 8 FLEX , catalog number: 0010002178)
  11. Round bottom flask, for rotovap (IKA, catalog number: 0003743200 )
  12. Mini extruder (Avanti Polar Lipids, catalog number: 610023 )
  13. Centrifuge (Beckman Coulter, model: J6-MI )
  14. Cary Eclipse fluorescence spectrophotometer (Varian Inc./Agilent, Santa Clara, CA, USA)
  15. UV/Vis/NIR spectrophotometer (capable of measuring optical density at 488 nm for Stewart assay) (Beckman Coulter, model: DU-640 )

Software

  1. Microsoft Excel
  2. GraphPad Prism

Procedure

  1. Preparing 20 mM carboxyfluorescein
    1. Weigh out appropriate mass of carboxyfluorescein to make 20 mM carboxyfluorescein in a desired volume.
      Note: 40 ml is enough to make 80 preparations of liposomes. Each preparation is sufficient for 500 experimental assays.
    2. Add ~70% of the target volume of buffer to the carboxyfluorescein powder.
      Note: Commonly used buffers are buffer-KCl and buffer-NaCl (see the Recipes section). Identical buffers should also be used in all experiments, including all liposome and protein preparation steps.
    3. While stirring the solution with a stir bar, titrate with 1 N KOH or NaOH a drop at a time.
      Note: Be sure to not go above a pH of 8 as increasing the pH causes carboxyfluorescein to precipitate, and pH also affects the fluorescence spectra.
    4. After the precipitate dissolves and the solution becomes dark, adjust the final pH to 7.4.
      Note: The concentrated dark solution is fluorescence quenched.
    5. Add appropriate volume of buffer to obtain a final concentration of 20 mM carboxyfluorescein.

  2. Preparation of the size-exclusion chromatography column
    1. Suspend 1 g of Sephadex® G-25 resin with 35 ml of buffer in a 50 ml conical tube.
      Note: Commonly used buffers are buffer-KCl and buffer-NaCl.
    2. Gently shake the tube at 30 rpm for 12-16 h at room temperature.
    3. Add about 2 ml of buffer to the gravity column, and resin slurry in aliquots frequently enough to avoid layering in the packed column.
      Note: The buffer provides resistance to the resin as it settles into the column and prevents the resin from forming channels.
    4. During aliquot addition allow the column to flow slowly and continuously.
      Note: Avoid introducing air bubbles when packing the column, and maintain buffer on top of the resin to prevent it from drying or forming channels.
    5. Fill the ~30 ml gravity column about 95% to the top.
      Note: Leave room at the top to add sample (liposomes). It is important to be able to see the top and bottom meniscus of dark orange colored liposomes through the clear glass stem of the sizing column.
    6. The column can be reused indefinitely provided it has not dried out, the resin is not broken, and there are no air bubbles trapped in the resin.
      Note: The column should be washed after each use, and equilibrated 3 times with 1 column volume of buffer before use.

  3. Drying phospholipids
    1. Test glass tubes for integrity by sonicating for 30 sec in a water sonicator.
    2. Aliquot 10 mg of lipid (supplied by manufacturer with lipids dissolved in chloroform) into glass vial.
    3. Clamp the vial to a stand while it is immersed in a beaker filled with water (Figure 1).


      Figure 1. Drying phospholipids

    4. Evaporate off all of the chloroform by flowing a nitrogen stream over the phospholipid solution until there is a thin layer of lipid.
    5. Cover the test tube with Parafilm, and poke a small hole in the Parafilm.
    6. Place the tube in a hi-vac flask, and lyophilize under vacuum for 3 h.
    7. After drying the lipid, fill the vial with nitrogen and cover with Parafilm.
      Note: Dried lipids can usually be stored for 1-3 months at -20 °C.

  4. Making large unilamellar vesicles (liposomes) containing carboxyfluorescein
    1. Add 500 µl of ethyl ether and 500 µl of the 20 mM carboxyfluorescein solution to the glass vial containing the dried lipid.
    2. Cover the vial with Parafilm.
      Note: Provide excess Parafilm because the Parafilm expands as the volatile gases escape from the vial.
    3. Vortex for 5 sec to dissolve the lipids in the solution of ether and carboxyfluorescein.
      Note: A layer of ether will sit on top of the green layer of carboxyfluorescein.
    4. Sonicate the vial in a water sonicator three times for 30 sec each.
      Note: Vortex sample for 15 sec between each sonication step. For optimal sonication, the vial should be at the center of the harmonic standing wave that forms in the water sonicator.
    5. Use a Pasteur pipette to transfer the water-ether-phospholipid suspension into a round bottom flask.
    6. Attach the round bottom flask to a rotary flask evaporator to remove the ether.
      Note: Using a water pump vacuum start the vacuum low to avoid excess bubbling and to reduce bumping. Gradually increase the vacuum, for about a minute at a time as the ether is removed.
    7. Release the vacuum and remove the round bottom flask from the roto-vap spindle. Sniff to determine if all the ether has evaporated. Usually, all the ether has evaporated 3 min after the sample stops bubbling while under the highest vacuum.
      Notes:
      1. Use the ‘wafting technique’ when sniffing.
      2. At this step liposomes containing carboxyfluorescein will have typically formed as a suspension in the remaining buffer. However, it is critical to obtain liposomes of uniform size by extrusion.
    8. Assemble the extruder (Avanti mini-extruder) with a 100 nm diameter cut off filter following the manufacturer’s directions (Figure 2).


      Figure 2. Assembly of Avanti mini-extruder (Avanti Polar Lipids)
      Notes:
      1.  Image from ‘Mini-Extruder Assembly Instructions’. Avanti Polar Lipids. Web. 19 June 2017 https://avantilipids.com/divisions/equipment/mini-extruder-assembly-instructions/.
      2. All components above are included in the Avanti mini-extruder (Avanti Polar Lipids) except the ‘filter supports’ (Sigma-Aldrich) and ‘polycarbonate membrane’ (Sigma-Aldrich).

    9. Pull buffer into one syringe and place it opposite to an empty syringe on the extrusion chamber. Gently inject the buffer across the extrusion chamber and membrane into the empty syringe.
      Note: Injecting the buffer across the extruder wets and prepares it for liposomes. Empty out the receiving syringe, and place it back into the extruder.
    10. Fill the injector syringe with the liposome suspensions and inject it through the extruder and passing it between the syringes five times. The liposome suspension will be at the receiving syringe. At this step, the solution contains liposomes that contain carboxyfluorescein and are of ~200 nm in diameter. However, the solution also contains a high concentration of carboxyfluorescein outside the liposomes, which needs to be removed.
    11. Equilibrate the gravity sizing column with desired final buffer, of the same molarity as the buffer used for the liposome preparation.
    12. Remove all of the buffer placing the meniscus on the top of the resin.
    13. Gently add the liposome sample to the top of the resin, while causing minimal disruption of the packed resin.
    14. Allow the column to flow, and stop it as soon as the entire liposome sample meniscus enters the resin.
    15. Gently add buffer over the top of the beads to fill up the column. The dark orange/greenish colored liposome sample and the resin should remain intact (Figure 3A).
    16. Allow the column to run.
      Note: The liposomes, containing carboxyfluorescein, exceed the pore size of the resin and will elute in the void volume of the column. The free carboxyfluorescein molecules elute later because they enter the resin beads as they pass through the column (Figure 3B).
    17. Collect the elution fractions containing liposomes, which are dark orange in color (Figure 1B).
      Note: The free carboxyfluorescein is bright green and elutes after the liposomes. Expect about 0.5-1 ml of liposomes since liposomes were made by dissolving lipids in 500 µl of ether and carboxyfluorescein.
    18. To determine the integrity of the liposomes, dilute the liposome fraction 1,000 fold and measure the fluorescence intensity at 512 nm upon excitation at 492 nm. Addition of 0.1% Triton-X 100 to the liposomes should result in approximately a threefold increase in fluorescence intensity.


      Figure 3. Purifying liposomes containing carboxyfluorescein. A. Addition of sample to size-exclusion chromatography column. S: sample containing a mixture of liposomes containing carboxyfluorescein (L+CF), and free carboxyfluorescein outside liposomes (CF). B. Separation of liposome sample into two fractions. CF: free carboxyfluorescein that is bright green. L+CF: liposomes containing carboxyfluorescein that is orange in color.

  5. Determining the lipid concentration
    Note: If numerical studies are desired it is useful to determine the lipid content in the liposome preparation using the Stewart assay (Stewart, 1980). Lipid is lost during preparation especially during extrusion and gel filtration. The assay is based on the formation of a complex formed from the phospholipid and ammonium ferrothiocyanate, and sensitive for a lipid range of 0.01-0.1 mg.
    1. To generate a standard curve, dissolve known amounts of lipids covering a range between 0.01 and 0.1 mg of lipid in chloroform to 2 ml. Five to ten points between 0.01 and 0.1 mg of lipid is sufficient to make a standard curve.
    2. To determine the concentration of lipids in the prepared liposome sample, add an appropriate amount of chloroform to obtain a final volume of 2 ml.
      Note: Since 10 mg of lipid was used to prepare liposomes, ensure that the sample is diluted enough to be in the range of 0.01 and 0.1 mg.
    3. Add 2 ml of 0.1 M ammonium ferrothiocyanate (see Recipes) to each of the 2 ml volumes of chloroform containing known amounts of lipid and the test liposome sample.
    4. Vortex the sample three times for 30 sec each, and centrifuge at 300 x g. With centrifugation, two layers will form.
    5. Collect the lower layer with a Pasteur pipette.
    6. Measure the optical density at 488 nm, and make a graph of the optical density versus lipid amount to obtain a standard curve.
    7. The concentration of lipid in the liposome sample can be determined from the standard curve.

  6. Preparing the biomolecule with potentially membrane lytic properties
    1. Ensure that the biomolecule is dissolved in the same buffer as the liposomes.
      Notes:
      1. Commonly used buffers are buffer-KCl and buffer-NaCl. Remove detergent from the biomolecule or include detergent containing controls.
      2. When testing a biomolecule for the first time, it is critical to use protein of good quality. Impurities, poor handling, and storage can lead to degradation, or aggregation of the protein which may impact the activity and result in the inaccurate quantification of protein activity. The type of purification needed to obtain homogenous protein samples, such as gravity column based affinity chromatography or gel filtration is dependent on each protein. It is recommended to use protein that elutes from a gel filtration column as a single monodisperse species. The purity of the protein can be determined by SDS-PAGE where the target protein is ~90% of the total protein sample.
    2. Prepare a dilution series of the biomolecule. A range from low nanomolar to micromolar range can be recommended, since these are physiologically relevant concentrations for many proteins.

  7. Liposome disruption assay (Figure 4)


    Figure 4. Overview of the liposome disruption assay. The cuvette with buffer has no fluorescence. Addition of liposomes results in a minor increase in fluorescence due to residual free carboxyfluorescein outside the liposomes. Addition of lytic protein or biomolecule disrupts liposomes resulting in the release of carboxyfluorescein and subsequent dequenching of fluorescence. Finally, addition of Triton-X disrupts all the liposomes in the cuvette.

    1. Set the spectrofluorimeter to record the fluorescence over time in order to determine the time dependence of liposome disruption. For carboxyfluorescein, program the fluorescence spectrophotometer to record the fluorescence emission at 512 nm upon excitation at 492 nm for a time range between 2 to 30 min.
      Note: It is important to empirically determine the time range appropriate for each biomolecule because they may have different rates of liposome disruption.
    2. Add 980 µl of buffer to a cuvette, and start the kinetic program to record the fluorescence emission over time.
    3. Add 10 µl of liposomes from a stock to ensure a final desired lipid concentration (usually between 250 nM and 2.5 µM).
      Note: Record the time that liposome sample is added. There should be a noticeable sudden increase in fluorescence mostly due to traces of free carboxyfluorescein outside liposomes remaining after previous purification steps.
    4. Add 10 µl of the biomolecule or protein being tested.
      Notes:
      1. There will be a gradual increase in fluorescence, due to leakage of carboxyfluorescein from liposomes, if the protein disrupts liposomes.
      2. Increase in fluorescence due to liposome disruption typically occurs within 5-30 min.
      3. It is necessary to include a no protein control in order to determine the passive leakage of dye from the liposomes that is independent of a membrane lytic protein.
    5. Finally, add 10 µl of 10% Triton-X to disrupt all liposomes in the system and obtain the 100% release value.

  8. Dextran-block experiment to determine if liposome disruption occurs via pore formation.
    1. Prepare 20 µM solutions of dextran molecules of different sizes dissolved in the buffer.
      Note: A typical liposome disruption assay has a total lipid concentration of 250 nM to 1 µM. Meaning that using the Dextran (20 µM) is in excess of the liposomes in the experimental system.
    2. Similar to the liposome disruption assay (described in Procedure G), determine the extent of liposome disruption in the absence of dextran in the buffer.
    3. Determine the extent of liposome disruption with buffers containing different molecular weight dextran molecules. It is recommended to test dextran molecules ranging from 5 kDa to 2,000 kDa since the size of the pore is unknown.

Data analysis

  1. Liposome disruption assay data analysis
    1. The following formula is used to determine the percent of liposome disruption with time:

      Disruptiontime = [(F512 ofliposome+protein - F512ofliposome)/(F512 ofliposome+triton - F512 ofliposome)] x 100%

      Where F512 is the fluorescence intensity at 512 nm upon excitation at 492 nm.
    2. For kinetic analyses, the time dependence of liposome disruption can be fitted to the following equation:

      % LiposomesDisruptedtime = A[1 - e - (time/tau)] + m x time

      where A is the maximum percentage of liposomes disrupted; Tau is the time constant for the exponential component and is the inverse of the rate constant (k); and m is the slope of the linear component.
      Note: Fitting the data to this equation will provide data that can be used to obtain a hill plot as previously described (Saito et al., 2000). Additionally, examples of representative data and analysis are previously reported in Saito et al., 2000 and Jimah et al., 2016.

  2. Dextran-block and pore-formation assay data analysis
    1. A one-way ANOVA analysis using GraphPad can be used to determine if dextran molecules of a particular size block liposome disruption compared to the no dextran control.
    2. This dextran-block experiment may reveal the size of the pore because radius of the pore will be similar to the radius of gyration of the dextran molecule that blocks liposome disruption.
      Note: Examples of representative data and analysis are previously reported in Saito et al., 2000 and Jimah et al., 2016.

Notes

  1. Good protein purification, handling, and storage are critical because membrane active proteins are frequently unstable and subject to aggregation, in addition the presence of proteases could degrade the protein resulting in a loss of activity. It is useful to determine the activity of freshly purified protein and determine how it compares to freeze-thawed samples.
  2. Since proteins that disrupt membranes most likely have a solution form and membrane bound form, it might be necessary to activate the protein with detergent, or another molecule known to induce a conformational change in the protein. The amount of detergent or molecule should be enough to activate the protein without disrupting the liposomes and this must be tested with control samples.
  3. Ensure that the liposomes are intact before testing the ability of protein to disrupt liposomes. Liposomes can be stored for approximately two days depending on the lipid composition. The fluorescence intensity of viable liposomes containing 20 mM carboxyfluorescein should increase three fold upon addition of 0.1% Triton-X.
  4. The same buffers should be used in all steps to ensure there are no differences in osmolarity between the protein and liposome buffers. Differences in osmolarity may result in lysis of liposomes.
  5. The Stewart assay may be less accurate because the absorbance reading of the chloroform phase, containing the lipid and ammonium ferrothiocyanate, may be skewed by the leakage or presence of carboxyfluorescein in the chloroform phase. An alternative 1H NMR based method for determining the phospholipid content of liposomes is described by Hein et al., 2016.
  6. Exercise safety precautions when handling and storing solvents such as chloroform and ether.

Recipes

  1. Buffer-KCl
    10 mM HEPES pH 7.4
    150 mM KCl
  2. Buffer-NaCl
    10 mM HEPES pH 7.4
    150 mM NaCl
  3. 0.1 M ammonium ferrothiocyanate
    Dissolve 27.03 g ferric chloride hexahydrate and 30.4 g ammonium thiocyanate in 1 L distilled water

Acknowledgments

This work was supported by the Burroughs Wellcome Fund (to NHT) and National Institutes of Health (R56 AI080792 to NHT). This protocol is adapted from Saito et al., 2000 and Jimah et al., 2016.

References

  1. Hein, R., Uzundal, C. B. and Henning, A. (2016). Simple and rapid quantification of phospholipids for supramolecular membrane transport assays. Org Biomol Chem 4(7): 2182-5.
  2. Jimah, J. R., Salinas, N. D., Sala-Rabanal, M., Jones, N. G., Sibley, L. D., Nichols, C. G., Schlesinger, P. H. and Tolia, N. H. (2016). Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption. Elife 5.
  3. Jimah, J. R., Schlesinger, H. P. and Tolia, H. N. (2017). Membrane lipid screen to identify molecular targets of biomolecules. Bio Protoc 7(15): e2427.
  4. Saito, M., Korsmeyer, S. J. and Schlesinger, P. H. (2000). BAX-dependent transport of cytochrome c reconstituted in pure liposomes. Nat Cell Biol 2(8): 553-555.
  5. Stewart, J. C. (1980). Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal Biochem 104(1): 10-14.

简介

蛋白质可以具有三维结构或氨基酸特征,其表明在细胞膜内靶向和破坏脂质的作用。通常有必要进行实验研究,如果这些蛋白质和生物分子能够破坏膜,以确定性地表征这些生物分子的功能。在这里,我们描述了一种体外实验来评估蛋白质和生物分子的膜裂解性质。用荧光猝灭浓度含有羧基荧光素的大单层囊泡(脂质体)用感兴趣的生物分子进行处理。由于染料从脂质体泄漏而导致的荧光增加,随后在缓冲液中的稀释表明生物分子足以破坏脂质体和膜。另外,由于脂质体破裂可能通过孔形成或通过类似于洗涤剂的脂类的一般溶解而发生,因此我们提供了区分这两种机制的方法。可以通过检查与适合孔的葡聚糖分子的羧基荧光素释放的封锁来鉴定和评估孔形成。这里描述的方法用于确定疟疾疫苗候选人CelTOS和促凋亡Bax通过孔形成破坏脂质体(Saito等人,2000; Jimah等人,2016) )。由于生物分子的膜脂质结合先于膜破裂,我们推荐使用伴侣方案:Jimah等人,2017。
【背景】该方案提出了评估蛋白质和其他生物分子的膜裂解性质的程序。该方案旨在清楚地描述研究生物分子的膜破坏性质所需的各种实验步骤。最后,该方案描述了膜破坏的定量测量,可用于提供对脂质体破坏的动力学和机制的了解。该方案成功地用于研究疟疾疫苗候选人CelTOS,其提供了对CelTOS的第一次体外功能测定的清楚描述。该方案的应用揭示,对于不饱和脂肪酸和多孔元素的CelTOS(透明性蛋白质)破坏了含有磷脂酸的膜。这一见解表明,CelTOS由入侵宿主细胞内的疟疾寄生虫分泌,以破坏宿主细胞膜并使寄生虫出现(Jimah等人,2016)。最后,该测定可以容易地应用于研究小分子,抗体或肽对CelTOS介导的脂质体破坏的抑制。当为CelTOS设计时,该协议易于泛化,适用于任何其他感兴趣的生物分子。

关键字:膜, 脂质体, 脂质, 破碎, 裂解, 孔, 脂质体渗漏, 羧基荧光素, 右旋糖酐

材料和试剂

  1. 离心管,50ml(Genesee Scientific,目录号:21-106)
  2. 微量离心管,1.7 ml(Midsci,目录号:AVSS1700RA)
  3. 石蜡膜(Bemis,目录号:PM996)
  4. 玻璃巴斯德移液器(Fisher Scientific,目录号:13-678-20C)
  5. 重力定径柱(Bio-Rad Laboratories,目录号:7371032)
  6. 玻璃管平原(Fisher Scientific,目录号:14-961-27)
  7. 环架(Fisher Scientific,目录号:S13747)
    制造商:GSC Go Science Crazy,目录号:4SSC16。
  8. 夹具(Fisher Scientific,目录号:02-217-005)
  9. Whatman过滤器(GE Healthcare,目录号:230300)
  10. 200 nm聚碳酸酯轨道蚀刻过滤器(GE Healthcare,目录号:800281)
  11. 羧酰荧光素(Sigma-Aldrich,目录号:C0662)
  12. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
  13. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:P5958)
  14. Sephadex G ® G-25(Sigma-Aldrich,目录号:G25300)
  15. 磷脂(溶于氯仿,由制造商提供)。常用的磷脂有:
    1,2-二油酰-sn-甘油-3-磷酸胆碱(DOPC)(Avanti Polar Lipids,目录号:850375℃)
    1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱(POPC)(Avanti Polar Lipids,目录号:850457C)
    1-棕榈酰-2-油酰-sn-甘油-3-磷酸(POPA)(Avanti Polar Lipids,目录号:840857C)
    1-棕榈酰-2-油酰-sn-甘油-3-磷酸-L-丝氨酸(POPS)(Avanti Polar Lipids,目录号:840034C)
  16. 氮气罐(Airgas,目录号:NI HP300)
  17. Triton-X 100(Sigma-Aldrich,目录号:X100)
  18. 不同分子量和回转半径的葡聚糖分子:
    5 kDa(Sigma-Aldrich,目录号:31417)
    9kDa(Sigma-Aldrich,目录号:D9260)
    39kDa(Sigma-Aldrich,目录号:D4133)
    注意:本产品已停产。
    66.9kDa(Sigma-Aldrich,目录号:D1537)
    注意:本产品已停产。
    148 kDa(Sigma-Aldrich,目录号:D4876)
    500 kDa(Sigma-Aldrich,目录号:D5251)
    注意:本产品已停产。
    1,500-2800 kDa(Sigma-Aldrich,目录号:D5376)
  19. HEPES(Sigma-Aldrich,目录号:H3375)
  20. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  21. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  22. 乙醚(Fisher Scientific,目录号:E138-500)
  23. 六水合氯化铁(III)(Sigma-Aldrich,目录号:236489)
  24. 硫氰酸铵(Sigma-Aldrich,目录号:221988)
  25. Buffer-KCl(参见食谱)
  26. 缓冲液 - NaCl(参见食谱)
  27. 0.1M硫氰酸铵(见配方)

设备

  1. 玻璃烧杯(Fisher Scientific,目录号:FB1012000)
  2. pH计(Fisher Scientific,型号:Accumet TM AE150,目录号:13-636-AE153)
  3. 搅拌棒
  4. BenchRocker TM 变量2D摇杆(Benchmark Scientific,目录号:BR2000)
  5. 水超声波仪(实验室用品,型号:G112SP1T)
  6. 气压调节器(林德,目录号:UPE-3-75-580)
  7. 冷冻干燥系统(Labconco,目录号:7750021)
  8. Hi-vac烧瓶(Labconco,目录号:7544400)
  9. Vortexer(Fisher Scientific,目录号:02-215-414)
  10. 旋转烧瓶蒸发器:Rotovap(IKA,型号:RV 8 FLEX,目录号:0010002178)
  11. 圆底烧瓶,用于旋转蒸发器(IKA,目录号:0003743200)
  12. 迷你挤出机(Avanti Polar Lipids,目录号:610023)
  13. 离心机(Beckman Coulter,型号:J6-MI)
  14. Cary Eclipse荧光分光光度计(Varian Inc./Agilent,Santa Clara,CA,USA)
  15. UV / Vis / NIR分光光度计(能够测量Stewart测定的488nm处的光密度)(Beckman Coulter,型号:DU-640)

软件

  1. Microsoft Excel
  2. GraphPad棱镜

程序

  1. 制备20mM羧基荧光素
    1. 称量适量的羧基荧光素,使所需体积的20mM羧基荧光素。
      注意:40ml足以制备80个脂质体制剂。每个制剂都足够用于500个实验测定。
    2. 将〜70%的目标体积的缓冲液加入到羧基荧光素粉末中 注意:常用的缓冲液是缓冲液KCl和缓冲液 - NaCl(参见食谱部分)。所有实验中也应使用相同的缓冲液,包括所有脂质体和蛋白质制备步骤。
    3. 用搅拌棒搅拌溶液时,一次用1N KOH或NaOH一滴一滴 注意:确保不要超过pH值8,因为增加pH导致羧基荧光素沉淀,并且pH也影响荧光光谱。
    4. 沉淀溶解后,溶液变暗,将最终pH调节至7.4 注意:浓缩的黑色溶液是荧光淬灭的。
    5. 加入适量的缓冲液,得到最终浓度为20 mM的羧基荧光素
  2. 大小排阻色谱柱的制备
    1. 用50ml锥形管将35g缓冲液悬浮1g Sephadex G-25树脂。
      注意:常用的缓冲液是缓冲液KCl和缓冲液 - NaCl。
    2. 在室温下以30rpm轻轻摇动管12-16小时
    3. 向重力柱中加入约2ml的缓冲液,等份的树脂浆料经常足以避免在填充柱中分层。
      注意:当缓冲液沉降到柱中时,缓冲液对树脂产生阻力,防止树脂形成通道。
    4. 在加入等分试样期间,色谱柱缓慢而持续流动。
      注意:在包装柱子时避免引入气泡,并在树脂顶部保持缓冲液,以防止其干燥或形成通道。
    5. 将约30ml重力柱填充至顶部约95%。
      注意:顶部放置空间以添加样品(脂质体)。通过施胶柱的透明玻璃杆,能够看到深橙色脂质体的顶部和底部弯月面很重要。
    6. 如果没有干燥,树脂不破裂,并且树脂中没有气泡被捕获,该柱可以无限次地重复使用。
      注意:每次使用后应清洗柱子,并在使用前用1柱体积的缓冲液平衡3次。

  3. 干燥磷脂
    1. 通过在超声波发生器中超声处理30秒,测试玻璃管的完整性。
    2. 将10毫克脂质(由制造商提供的脂质溶解在氯仿中)放入玻璃瓶中
    3. 将小瓶固定在支架上,同时将其浸入装满水的烧杯中(图1)

      图1.干燥磷脂

    4. 通过将氮气流流过磷脂溶液直到有一层薄薄的脂质,使所有氯仿蒸发掉。
    5. 用Parafilm覆盖试管,并在Parafilm中戳一个小洞。
    6. 将管置于高温烧瓶中,真空干燥3小时。
    7. 干燥脂质后,用氮气填充小瓶,并用Parafilm盖住。
      注意:干性脂质通常在-20°C下储存1-3个月。

  4. 制备含有羧基荧光素的大单层囊泡(脂质体)
    1. 向含有干燥脂质的玻璃小瓶中加入500μl乙醚和500μl20mM羧基荧光素溶液。
    2. 用Parafilm盖住小瓶。
      注意:提供多余的玻璃膜,因为Parafilm随着挥发性气体从小瓶中逸出而膨胀。
    3. 涡旋5秒,将脂质溶解在乙醚和羧基荧光素溶液中。
      注意:一层乙醚将位于绿色荧光素的绿色层之上。
    4. 在水中超声波处理3次,每次30秒。
      注意:涡流样品在每个超声处理步骤之间15秒。为了获得最佳的超声处理,小瓶应该位于在水声发射器中形成的谐波驻波的中心。
    5. 使用巴斯德吸管将水 - 磷脂 - 磷脂悬浮液转移到圆底烧瓶中
    6. 将圆底烧瓶安装到旋转烧瓶蒸发器中以除去乙醚。
      注意:使用水泵真空启动真空度低以避免过多的冒泡并减少碰撞。随着乙醚的去除,逐渐增加真空约一分钟。
    7. 释放真空并从旋转轴中取出圆底烧瓶。嗅觉以确定所有的醚是否已蒸发。通常,在最高真空度下,样品停止鼓泡3分钟后,所有的醚都蒸发掉。
      注意:
      1. 嗅探时使用'wafting技术'。
      2. 在此步骤中,含有羧基荧光素的脂质体通常在剩余的缓冲液中形成为悬浮液。然而,通过挤压获得均匀尺寸的脂质体至关重要。
    8. 按照制造商的说明,将挤出机(Avanti mini-extrusion)与100nm直径的截止过滤器组装(图2)。


      图2. Avanti迷你挤出机(Avanti Polar Lipids)的组装
      注意:
      1. “迷你挤出机装配说明”的图像。 Avanti极地脂质。网页。 2017年6月19日 https ://avantilipids.com/divisions/equipment/mini-extruder-assembly-instructions/
      2. 除了“过滤器支架”(Sigma-Aldrich)和“聚碳酸酯膜”(Sigma-Aldrich)之外,所有上述组件都包含在Avanti微型挤出机(Avanti Polar Lipids)中。

    9. 将缓冲液拉入一个注射器,并将其放置在挤出腔上的空针筒上。轻轻地将缓冲液穿过挤出室和膜进入空针筒。
      注意:在挤出机上注入缓冲液,润湿并制备脂质体。将接收的注射器清空,并将其放回挤出机。
    10. 将注射器注射器与脂质体悬浮液填充,并将其注射通过挤出机并将其在注射器之间传递五次。脂质体悬浮液将在接收注射器处。在该步骤中,该溶液含有含有羧基荧光素并且直径为〜200nm的脂质体。然而,该溶液还含有高浓度的脂质体外的羧基荧光素,需要除去。
    11. 将重力筛分柱与所需的最终缓冲液平衡,与用于脂质体制备的缓冲液相同的摩尔浓度。
    12. 将所有缓冲液放置在树脂顶部的半月板上。
    13. 将脂质体样品轻轻添加到树脂的顶部,同时使填充树脂的破坏最小
    14. 允许色谱柱流动,一旦整个脂质体样品弯液面进入树脂,就立即停止
    15. 在珠顶上轻轻加入缓冲液以填满色谱柱。深橙色/绿色的脂质体样品和树脂应保持完整(图3A)。
    16. 允许列运行。
      注意:含有羧基荧光素的脂质体超过树脂的孔径,并将在柱的空隙体积中洗脱。游离的羧基荧光素分子随着它们通过柱进入树脂珠而洗脱(图3B)。
    17. 收集含有脂质体的洗脱级分,其为深橙色(图1B)。
      注意:游离羧基荧光素是明亮的绿色,在脂质体后洗脱。预期约0.5-1ml的脂质体,因为通过将脂质溶解在500μl的乙醚和羧基荧光素中制成脂质体。
    18. 为了确定脂质体的完整性,将脂质体部分稀释1000倍,并在492nm激发时测量512nm处的荧光强度。向脂质体中加入0.1%Triton-X 100可导致荧光强度增加约三倍。


      图3.纯化含有羧基荧光素的脂质体。 A.将样品加入大小排阻色谱柱。 S:含有含有羧基荧光素(L + CF)的脂质体和脂质体外的游离羧基荧光素(CF)的混合物的样品。 B.将脂质体样品分成两部分。 CF:亮绿色的游离羧基荧光素。 L + CF:含有橙色色素的羧基荧光素的脂质体
  5. 确定脂质浓度
    注意:如果需要数值研究,使用Stewart测定法(Stewart,1980)确定脂质体制剂中的脂质含量是有用的。在制备过程中脂质会在挤出和凝胶过滤过程中丧失。该测定法基于由磷脂和硫氰酸铵形成的络合物的形成,并且对于0.01-0.1mg的脂质范围是敏感的。
    1. 为了产生标准曲线,将已知量的脂质在0.01至0.1mg脂质的氯仿中溶解至2ml。 0.01至0.1 mg脂质之间的五至十分就足以制定标准曲线
    2. 为了确定所制备的脂质体样品中脂质的浓度,加入适量的氯仿以获得最终体积为2ml。
      注意:由于使用10mg的脂质来制备脂质体,确保样品被稀释至0.01至0.1mg范围内。
    3. 加入2ml 0.1M硫氰酸铵(参见食谱)至含有已知量脂质的2ml体积的氯仿和测试脂质体样品。
    4. 将样品涡旋3次,每次30秒,并以300×g离心。随着离心,将形成两层
    5. 用巴斯德吸管收集下层。
    6. 测量488 nm处的光密度,并制作光密度与脂质量的曲线图,得到标准曲线。
    7. 脂质体样品中脂质的浓度可以从标准曲线确定
  6. 准备具有潜在的膜裂解性质的生物分子
    1. 确保生物分子溶解在与脂质体相同的缓冲液中。
      注意:
      1. 通常使用的缓冲液是缓冲液KCl和缓冲液 - NaCl。从生物分子中去除洗涤剂或包含含有洗涤剂的对照。
      2. 首次测试生物分子时,使用质量好的蛋白质至关重要。杂质,不良处理和储存可导致蛋白质的降解或聚集,这可能影响活性,并导致蛋白质活性的不准确定量。获得均质蛋白质样品(如基于重力柱的亲和层析或凝胶过滤)所需的纯化类型取决于每种蛋白质。建议使用从凝胶过滤柱洗脱的蛋白质作为单一的单分散物质。蛋白质的纯度可以通过SDS-PAGE测定,其中目标蛋白质是总蛋白质样品的约90%。
    2. 准备稀释系列的生物分子。可以推荐从低纳摩尔到微摩尔范围的范围,因为它们是许多蛋白质的生理相关浓度。

  7. 脂质体破碎测定(图4)


    图4.脂质体破碎测定的概述。 具有缓冲液的比色皿没有荧光。由于脂质体外的残留游离羧基荧光素,脂质体的添加导致荧光的微小增加。溶解蛋白或生物分子的添加破坏脂质体,导致羧基荧光素的释放和随后的荧光脱色。最后,添加Triton-X会破坏比色皿中的所有脂质体。

    1. 设置分光荧光计以记录随时间的荧光,以确定脂质体破裂的时间依赖性。对于羧基荧光素,编程荧光分光光度计,以在492nm激发2至30分钟之间的时间范围内记录512nm的荧光发射。
      注意:经验性地确定适合每个生物分子的时间范围是重要的,因为它们可能具有不同的脂质体中断速率。
    2. 将980μl缓冲液加入比色杯中,开始动力学程序记录荧光发射随时间的变化
    3. 从原液中加入10μl脂质体以确保最终所需的脂质浓度(通常在250 nM和2.5μM之间)。
      注意:记录添加脂质体样品的时间。主要是由于在先前的纯化步骤之后留下的脂质体之外的游离羧基荧光素的痕迹,荧光显着突然增加。
    4. 加入10μl待测试的生物分子或蛋白质。
      注意:
      1. 如果蛋白质破坏脂质体,则会由于来自脂质体的羧基荧光素的渗漏而逐渐增加荧光。
      2. 由于脂质体破坏引起的荧光增加通常发生在5-30分钟内。
      3. 为了确定与膜溶解蛋白无关的脂质体中染料的被动泄漏,必须包括无蛋白质控制。
    5. 最后,加入10μl10%Triton-X以破坏系统中的所有脂质体,并获得100%释放值。

  8. 确定脂质体破裂是否通过孔形成发生的葡聚糖区块实验。
    1. 准备20μM溶解在缓冲液中的不同大小的葡聚糖分子溶液 注意:典型的脂质体破坏测定的总脂质浓度为250nM至1μM。意思是使用葡聚糖(20μM)在实验系统中超过脂质体。
    2. 类似于脂质体破碎测定(在方法G中描述),确定缓冲液中不存在葡聚糖时的脂质体破坏的程度。
    3. 用含有不同分子量葡聚糖分子的缓冲液确定脂质体破裂的程度。建议测量5kDa至2,000kDa的葡聚糖分子,因为孔的大小是未知的。

数据分析

  1. 脂质体破碎测定数据分析
    1. 以下公式用于确定脂质体中断时间的百分比:

      分裂时间 = [(脂质体+蛋白质F512> F2双链脂质体“)/(512 ofliposome )]×100%

      其中 F 512 是在492nm激发时512nm处的荧光强度。
    2. 对于动力学分析,脂质体破坏的时间依赖性可以适应于以下等式:

      %脂质体破裂时间 = A [1-e - (时间/τ)] + m x时间

      其中 A 是脂质体破坏的最大百分比; Tau 是指数分量的时间常数,是速率常数(k)的倒数;而 m 是线性分量的斜率。
      注意:将数据拟合到这个方程式将提供可用于获得山前图的数据,如前所述(Saito等,2000)。此外,Saito等人,2000年和Jimah等人,2016年以前报告了代表性数据和分析的例子。

  2. 葡聚糖块和孔形成实验数据分析
    1. 与不使用葡聚糖对照相比,使用GraphPad的单因素方差分析可用于确定特定大小块状脂质体的葡聚糖分子是否破裂。
    2. 该葡聚糖块实验可以揭示孔的大小,因为孔的半径将类似于阻断脂质体中断的葡聚糖分子的回转半径。
      注意:以前在Saito等人,2000年和Jimah等人,2016中报道了代表性数据和分析的例子。

笔记

  1. 良好的蛋白质纯化,处理和储存是至关重要的,因为膜活性蛋白质通常是不稳定的并且可能会聚集,此外蛋白酶的存在可能降解蛋白质导致活性丧失。确定新鲜纯化蛋白质的活性是有用的,并确定它如何与冻融样品进行比较。
  2. 由于破坏膜的蛋白质最可能具有溶液形式和膜结合形式,所以可能需要用洗涤剂或已知诱导蛋白质构象变化的另一种分子来激活蛋白质。洗涤剂或分子的量应足以激活蛋白质而不破坏脂质体,并且必须用对照样品进行测试。
  3. 在测试蛋白质破坏脂质体的能力之前,确保脂质体是完整的。根据脂质组成,可将脂质体储存约两天。含有20mM羧基荧光素的可行脂质体的荧光强度在添加0.1%Triton-X时应增加3倍。
  4. 应在所有步骤中使用相同的缓冲液,以确保蛋白质和脂质体缓冲液之间的渗透压无差异。渗透压的差异可能导致脂质体裂解。
  5. 斯图尔特测定可能不太准确,因为含有硫酸亚铁和硫氰酸铵的氯仿相的吸光度读数可能会被氯仿相中的渗漏或羧基荧光素的存在所倾斜。用于确定脂质体的磷脂含量的另一种基于1 H NMR的方法由Hein等人,2016描述。
  6. 处理和储存溶剂如氯仿和乙醚时,请执行安全预防措施。

食谱

  1. Buffer-KCl
    10 mM HEPES pH 7.4
    150 mM KCl
  2. Buffer-NaCl
    10 mM HEPES pH 7.4
    150 mM NaCl
  3. 0.1M硫氰酸铵,
    将27.03g氯化铁六水合物和30.4g硫氰酸铵在1L蒸馏水中溶解

致谢

这项工作得到了Burroughs Wellcome基金(NHT)的支持。该协议由Saito等人,2000和Jimah等人,2016改编。

参考

  1. Hein,R.,Uzundal,CB and Henning,A。(2016)。  用于超分子膜转运测定的磷脂的简单且快速的定量。 4g(7):2182-5。 >
  2. Jim,JR,Salinas,ND,Sala-Rabanal,M.,Jones,NG,Sibley,LD,Nichols,CG,Schlesinger,PH and Tolia,NH(2016)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/27906127”target =“_ blank”>疟疾寄生虫CelTOS靶向细胞膜的内部小叶,以致孔依赖性破坏。 Elife 5.
  3. Jimah,JR,Schlesinger,HP和Tolia,HN(2017)。膜脂质屏幕,以确定生物分子的分子靶标。生物原型 7(15):e2427。
  4. Saito,M.,Korsmeyer,SJ和Schlesinger,PH(2000)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/10934477”target = “_blank”>在纯脂质体中重构的细胞色素c的BAX依赖性转运。细胞生物学 2(8):553-555。
  5. Stewart,JC(1980)。  比色测定磷脂与硫氰酸铵。分析生化 104(1):10-14。
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Copyright Jimah et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jimah, J. R., Schlesinger, P. H. and Tolia, N. H. (2017). Liposome Disruption Assay to Examine Lytic Properties of Biomolecules. Bio-protocol 7(15): e2433. DOI: 10.21769/BioProtoc.2433.
  2. Jimah, J. R., Salinas, N. D., Sala-Rabanal, M., Jones, N. G., Sibley, L. D., Nichols, C. G., Schlesinger, P. H. and Tolia, N. H. (2016). Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption. Elife 5.
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