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Sep 2019
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Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography
Cyanobacterium Synechocystis sp. PCC 6803 中蛋白混合物的标记亲和层析纯化方法   

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Abstract

Exploring the structure and function of protein complexes requires their isolation in the native state–a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism for the physiology of oxygenic phototrophs, and the biogenesis of membrane-bound photosynthetic complexes has traditionally been studied using this cyanobacterium. In a typical approach, the protein complexes are purified with a combination of His-affinity chromatography and a size-based fractionation method such as gradient ultracentrifugation and/or native electrophoresis. However, His-affinity purification harbors prominent contaminants and the levels of many proteins are too low for a feasible multi-step purification. Here, we have developed a purification method for the isolation of 3x FLAG-tagged proteins from the membrane and soluble fractions of Synechocystis. Soluble proteins or solubilized thylakoids are subjected to a single affinity purification step that utilizes the highly specific binding of FLAG-affinity resin. After an intensive wash, the captured proteins are released from the resin under native conditions using an excess of synthetic 3x FLAG peptide. The protocol allows fast isolation of low abundant protein complexes with a superb purity.

Keywords: Protein purification (蛋白质纯化), Membrane protein complexes (膜蛋白复合物), Synechocystis 6803 (集胞藻6803), Photosystems (光系统), FLAG-tag (FLAG标签), Affinity chromatography (亲和层析)

Background

Cyanobacteria have been used as the preferred model systems to study the biogenesis and function of photosynthetic protein complexes for decades. The photosynthetic apparatus in cyanobacteria is very similar to eukaryotic systems (algae and plants), but cyanobacteria have all the advantages of prokaryotic models, such as fast growth and small genomes that allow easy genetic manipulation and the use of standard tools of bacterial genetics. Particularly, Synechocystis sp. PCC 6803 (hereafter Synechocystis) is established as a favorite model organism in this field, mainly because it is transformable with high efficiency and integrates DNA by functional homologous recombination. Moreover, a glucose tolerant substrain of Synechocystis, which can proliferate using carbohydrate supplement, enables studying the function of photosynthetic genes by reverse genetics (Williams, 1988).

The biogenesis of photosystem II (PSII), a large membrane embedded pigment-protein complex responsible for photosynthetic water oxidation, has been studied intensively in Synechocystis (Nixon et al., 2010). The assembly of the PSII complex from individual subunits requires complicated machinery of many auxiliary (assembly) factors, which are typically involved in a certain assembly step (Komenda et al., 2012). A conventional approach for the analysis of PSII assembly complexes is using Synechocystis mutants lacking one or several PSII subunits. PSII biogenesis is then blocked at a particular step where the missing subunit would bind to an intermediate complex of the assembly process. In such mutants, the otherwise transient PSII pre-complexes can accumulate to a level that allows their detection and even purification, which has traditionally been achieved via His-tagged PSII (core) subunits (Dobáková et al., 2007 and 2009; Boehm et al., 2011 and 2012).

His-tag has several advantages: it is small enough to be easily introduced via a simple primer design, and it is relatively inexpensive to use. However, after His-affinity purification the preparations from Synechocystis often contain prominent contaminating proteins (Boehm et al., 2011 and 2012; Liu et al., 2011); therefore, additional purification steps are usually required to obtain the satisfactory quality for functional and structural studies. Additional purification steps may, however, compromise the yield and nativity of the complex of interest. Moreover, the levels of many protein complexes in the cell are simply too low for feasible multi-step purification protocols, PSII assembly intermediates are such an example. For these reasons, a fast, gentle, single-step purification method is highly desirable.

Affinity tags, based on biomolecular interactions offer superior specificity as compared to the general metal affinity of His-tag (Lichty et al., 2005). Large, proteinaceous tags such as small ubiquitin-like modifier (SUMO) or maltose binding protein (MBP) are well suited for the isolation of small soluble proteins due to their stable nature, which can even promote the solubility of the protein (Esposito and Chatterjee, 2006). However, for studying larger and potentially labile membrane-protein complexes, a less bulky tag with minimal interference on protein-protein interactions is preferable. Short peptide tags with high-affinity to proteins or antibodies seem to be the best suited for this purpose. One such tag is the short octapeptide called FLAG (DYKDDDDK; Hopp et al., 1988). Compared to His-, and several other tags, using FLAG-tag results in superior purity with little compromise in yield (Lichty et al., 2005). Moreover, because the elution of FLAG-tagged proteins is possible using a synthetic FLAG-peptide, the usage of disrupting chemicals, high ion concentrations or extreme pH can be avoided. Even though the price per mg purified protein of FLAG-affinity resin is approximately 50 times higher than that of Ni-NTA resin (Lichty et al., 2005); for the purification of labile and/or low-abundant complexes it can well worth the time and costs saved compared to optimizing multi-step purification procedures. Recently, FLAG-tag based approaches have been employed to purify pathogenic variants of the human Huntingtin protein from mammalian and insect cell lines (Harding et al., 2009), as well as the integral membrane Vo-part of the Pichia pastoris V-type ATPase (Li et al., 2017).

The FLAG-affinity purification protocol presented here is designed for the purification of proteins tagged with 3x FLAG epitope (DYKDDDDKDYKDDDDKDYKDDDDK). The protocol has been successfully used to resolve the roles of the PSII assembly factors Ycf39 (Knoppová et al., 2014), CyanoP (Knoppová et al., 2016), Pam68 (Bučinská et al., 2018) and RubA (Kiss et al., 2019); as well as pigment-protein complexes containing the chlorophyll-synthase (Chidgey et al., 2014), protoporphyrinogen oxidase (Skotnicová et al., 2018) and ferrochelatase enzymes (Pazderník et al., 2019). Although the method was originally developed for membrane-bound protein complexes, with minor modifications it can also be applied to soluble proteins.

Materials and Reagents

  1. Disposable semi-micro cuvettes (BRAND, catalog number: 759015 )
  2. 50 ml conical tubes (JET biofil, catalog number: CFT011500 )
  3. Round paint brush, ø 6 mm
  4. Pipette tips:
    10 µl (Neptune, catalog number: 2340 )
    200 µl (Eppendorf, catalog number: 00 30000870 )
    1,000 µl (Eppendorf, catalog number: 00 30000927 )
  5. 1.5 ml microtubes (Deltalabs, catalog number: 4092.3N )
  6. 7 ml screw cap vials for cell lysis (BioSpec Products, catalog number: 3205 )
  7. Plastic chromatography column (Bio-Rad, catalog number: 7311550 )
  8. Synechocystis cells expressing a 3x FLAG-tagged protein
  9. BG-11 liquid medium and agar plates (Rippka et al., 1979)
  10. 100-200 µm glass microbeads (PRECIOSA ORNELA, crystal microbeads B 134)
  11. cOmplete protease inhibitor cocktail tablets (Sigma-Aldrich, catalog number: 11836145001 )
  12. (Optional) Microconcentrators with suitable cut-off filter (e.g., Amicon Ultra-0.5, Sigma-Aldrich)
  13. Liquid nitrogen
  14. Crushed ice
  15. Styrofoam box or equivalent for keeping ice
  16. Reverse osmosis (RO) water
  17. Deionized water (Merck, catalog number: 1167545000 )
  18. Methyl alcohol (PENTA, catalog number: 21240-11000 )
  19. n-dodecyl-β-D-maltoside (DDM; BioChemica, catalog number: A0819,0005 )
  20. ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich, catalog number: A2220 )
  21. Proteus Clarification Mini Spin Column (Generon, catalog number: GEN-MSF500 )
  22. 3x FLAG peptide, 20 mg, > 85% purity (custom synthesis, Genscript, USA)
    Notes:
    1. An alternative is to purchase 3x FLAG peptide (Sigma-Aldrich, catalog number: F4799-4MG ), however, the custom synthesis is roughly 6 times cheaper.
    2. Prepare a 3 mg/ml solution in deionized water and store at -20 °C as 100-200 µl aliquots. Thawed peptide can be stored at 4 °C until use.
  23. 2-Morpholinoethanesulfonic acid (MES; AppliChem, catalog number: A0689,0250 )
  24. MgCl2·6H2O (PENTA, catalog number: 16330-31000 )
  25. Glycerol (PENTA, catalog number: 14550-11000 )
  26. CaCl2·2H2O (PENTA, catalog number: 16790-31000 )
  27. α-FLAG antibody (Merck, catalog number: F7425 )
  28. MES buffer (1 L) (see Recipes)
  29. 0.5 M MES-NaOH, pH 6.5 (250 ml) (see Recipes)
  30. 1 M MgCl2 (100 ml) (see Recipes)
  31. 1 M CaCl2 (100 ml) (see Recipes)

Equipment

  1. Temperature controlled growth facility equipped with an orbital shaker and a light source
  2. Laminar flow hood
  3. Metallic inoculation loop
  4. Gas burner
  5. 500 ml Erlenmeyer flasks
  6. Round culture flask (10 L)
  7. CimarecTM i Maxi magnetic stirrer (Thermo Fisher Scientific, catalog number: 50088143 ) and a magnetic stir bar
  8. Spectrophotometer (WPA S1200 Spectrawave)
  9. Centrifuges and rotors:
    Centrifuge (Sigma Laborzentrifugen GmbH, model: Sigma 8KS , rotor: 12505-H)
    Centrifuge (Sigma Laborzentrifugen GmbH, model: Sigma 3K30, rotors: 12155-H [+15 ml conical tube adapters, 13081] and 12158-H)
    Centrifuge (Eppendorf, model: 5418)
    Centrifuge (Eppendorf, model: 5415 R)
  10. Centrifuge bottles:
    500 ml (Nalgene, catalog number: 3141-0500 )
    80 ml (Sigma Laborzentrifugen GmbH, catalog number: 15080)
    27 ml (Sigma Laborzentrifugen GmbH, catalog number: 15032)
  11. Deep freezer
  12. Regular freezer
  13. Mini-Beadbeater-16 (BioSpec Products, catalog number: 607 ) equipped with a 7 ml vial adapter (BioSpec Products, catalog number: 607 TC8)
  14. Vortex V-1 Plus (Biosan, catalog number: BS-010203-AAG )
  15. 1 ml Hamilton syringe (Hamilton, catalog number: 81330 )
  16. Automatic pipettes:
    0.5-10 µl (Eppendorf, catalog number: 3120000020 )
    10-100 µl (Eppendorf, catalog number: 3120000046)
    100-1,000 µl (Eppendorf, catalog number: 3120000062)
  17. Multi-Bio RS-24 rotator (Biosan, catalog number: BS-010117-AAG )
  18. Cold hood or cold room, 10 °C
  19. A retort stand and a ring clamp
  20. 25 ml glass beakers

Procedure

  1. Cell culture and harvesting
    1. Working in a laminar flow hood, inoculate Synechocystis cells using a metallic inoculation loop into five, 500 ml Erlenmeyer flasks, each containing 200 ml BG-11 medium. Grow the cells at 28 °C and 40 µmol/m2/s photosynthetic photon flux density on a rotary shaker set to 150 rpm until the culture reaches mid/end of logarithmic growth phase.
      Note: When the optical density of the culture at 750 nm reaches 0.6-0.8, which corresponds to 9-12 x 107 Synechocystis cells/ml.
    2. Collect your cultures (OD750 ~0.8) into a 10 L flask and dilute it to 4 L final volume to gain OD750 ~0.2 density. Grow the culture until mid/end of logarithmic growth phase with magnetic stirrer (240 rpm) and air bubbling in the same conditions as above.
    3. Divide the cell culture into 500 ml centrifuge bottles and pellet the cells by centrifuging at 10,000 x g in a cooling centrifuge ( Sigma 8KS , rotor 12505-H) at 4 °C for 20 min.
    4. Suspend the cell pellets into a total of 30 ml of MES buffer.
    Notes:
    1. All media and equipment used for the preparation of the cell culture should be sterilized. Use the gas burner to sterilize the metal inoculation loop, and autoclave all glassware and growth media prior to use.
    2. Cyanobacterial cultures have negligible absorbance at 750 nm; therefore, the OD at this wavelength is primarily dependent on light scattering (turbidity). Different spectrophotometers differ greatly in their various optical properties, and hence the light scattering value by OD750 may vary. Therefore, it is important to establish the OD at which your culture is at the mid/end of logarithmic growth phase by measuring a growth curve a priori. Under our standard conditions, the doubling time of Synechocystis is approximately 12 h; hence, a culture with OD750 = 0.2 will reach desirable OD750 in about a day.
    3. In our experience, larger volumes of cyanobacterial cultures (i.e., 4 L) tend to grow slower, probably due to limitations in gas exchange. Therefore, an intensive bubbling of the culture by air is desirable.
    4. At Step A4 you may freeze the cells in liquid nitrogen and store them at -80 °C until use.

  2. Cell lysis and thylakoid isolation
    1. Pellet the cells in 80 ml centrifuge tubes at 10,000 x g (Sigma 3K30, rotor 12155-H) at 4 °C for 10 min.
    2. Discard the supernatant and resuspend the cells into 5 ml of MES buffer using a wet paint brush. Add 30 ml MES buffer to the centrifuge tube and pellet the cells as above.
    3. Resuspend the cells into 12 ml of MES buffer and add protease inhibitor: dissolve 1 tablet into 1 ml of deionized water to obtain a 50x stock solution and pipet accordingly to obtain 1x working concentration. You may store the rest of the inhibitor at -20 °C for later use.
    4. Add 3 ml of glass beads and 3 ml of cell suspension into four 7 ml screw cap vials (Figure 1A), close the lids and seal them with parafilm (Figure 1B). There should be approximately 1 cm of free space in the tube, you may add some buffer to reach the desired volume.
    5. Break the cells for 60 s by Mini-Beadbeater-16. Allow the tubes to cool on ice protected from light for 5 min and repeat the breaking cycle 5 times.
    6. After lysis, spin down the beads at 500 x g for 10 s at 4 °C, (Sigma 3K30, rotor 12155-H equipped with adapters for 15 ml conical tubes). Collect the supernatants into a 27 ml centrifuge tube. Wash the beads (Figure 1C) with one bed volume of MES buffer three-to-four times, until the supernatant and the beads appear light green (Figures 1D-1E).
    7. Pool all supernatants from Step B6 and pellet the thylakoids by centrifuging at 35,000 x g (Sigma 3K30, rotor 12158-H) at 4 °C for 20 min.
    8. At this point, if you are only interested in the isolation of membrane proteins, you may discard the supernatant. Otherwise, collect the supernatant into a new centrifuge tube (marked: SP, ‘soluble proteins’), and resuspend the pellet into 10 ml of MES buffer using a wet paint brush (mark the tube: TM, ‘thylakoid membranes’). Centrifuge the tubes as above.
    9. Collect the supernatant from the ‘SP’-tube into a 50 ml conical tube and store it on ice. Discard the supernatants from the ‘TM’-tube and resuspend the pellets from both tubes (‘SP’ and ‘TM’) into 1/3 of soluble fraction volume of MES buffer. If you are continuing only with the membrane fraction use a total of 10 ml MES buffer and combine the pellets into a 50 ml conical tube.
    10. Freeze a 300 µl aliquot of soluble proteins and a 100 µl aliquot of thylakoids in liquid nitrogen and store at -80 °C for further analysis by gel electrophoresis.
    Notes:
    1. After pelleting the cells, the supernatant may appear slightly bluish due to partial breaking of cells after freezing and thawing.
    2. From Step B5 onwards it is important to keep the sample cooled (4-10 °C) and protect it from light to prevent the excitation of chlorophyll molecules and the consequent formation of reactive oxygen species. We recommend working under dim green light.
    3. Thylakoids are easier to resuspend first into few ml volume, before adding the rest of the buffer.
    4. At Step B10 you may also freeze the rest of the soluble protein and thylakoid samples in liquid nitrogen and store them at -80 °C until subsequent solubilization and purification.


      Figure 1. Breaking of Synechocystis cells. A. Screwcap tubes are filled with 3 ml of glass beads and 3 ml of cyanobacterial suspension; B. The tubes are sealed with parafilm and cells lysed using a bead beater. C. After breaking cells, the beads are spun to the bottom and the supernatant is collected. D. The beads are washed with MES buffer; E. After each washing step, the supernatant is collected.

  3. Thylakoid solubilization
    1. Adjust the chlorophyll concentration of the thylakoids to 0.5 µg/µl with MES buffer.
    2. Add 1% n-dodecyl β-D-maltoside (DDM) to the thylakoid suspension and incubate the tube for 1 h at 10 °C in the dark (for example, you may cover the tube with aluminum foil) on a rotator mixer set to 10 rpm.
    3. Transfer the suspension into a 27 ml centrifuge tube and pellet insoluble material at 46,000 x g (Sigma 3K30, rotor 12158-H) at 4 °C for 30 min.
    4. Collect the supernatant into a 50 ml conical tube and discard the pellet.
    5. Freeze 100 µl of the solubilized thylakoids in liquid nitrogen and store at -80 °C for further analysis, particularly for the identification of a potential problem with protein solubility.

  4. FLAG-affinity purification
    1. Wash the purification column with 1 ml of RO-water followed by 1 ml of deionized water.
    2. Close the column, then add 1 ml of MES buffer and 600 µl of 1:1 Anti-FLAG-M2 agarose resin using a cut pipette tip (Video 1).

      Video 1. Column preparing

    3. Open the column and allow the buffer to flow through. Wash the resin with an additional 1 ml of MES buffer (pipet the buffer gently on top of the resin and allow it to flow through; Figure 2A; Video 2).

      Video 2. Gentle washing of column

    4. Load the protein suspension into the column and collect the flowthrough into a glass beaker (Figure 2B; Video 3).

      Video 3. Sample loading

    5. Wash the column with 1 ml of MES buffer. Discard the flowthrough.
    6. Load the flowthrough from Step D4 to the column for the second time and collect the flowthrough. Repeat the binding once more.
    7. Take a 0.5 ml sample from the final flowthrough, freeze it in liquid nitrogen and store at -80 °C for further analysis [Figure 3, flowthrough (FT)].
    8. Wash the column with 1 ml of MES buffer [Figure 2C; Figure 3, first wash (W1)].
    9. Close the column and add 1 ml of MES buffer: pipet the buffer with high enough speed to mix it thoroughly with the resin (Figure 2D; Video 4) (you may mix the buffer with the resin gently by pipetting up and down with a cut pipette tip, but some resin may stick to the tip and be lost). Open the column and allow the buffer to drain out [Figure 3, second wash (W2)].

      Video 4. Vigorous washing of the resin

    10. Wash the column with 1 ml of MES buffer without mixing the resin. Repeat this washing step 3 times. In total, the resin is washed by 20 column volumes of MES buffer (Figure 2E).


      Figure 2. Isolation of 3x FLAG-tagged proteins from Synechocystis using affinity chromatography. Individual purification steps for soluble proteins and detergent-solubilized membrane proteins are shown in upper panels and lower panels, respectively. A. Anti-FLAG resin is packed into a plastic chromatography column. B. Soluble protein fraction (upper panel) or detergent solubilized thylakoids (lower panel) are loaded to the column. C. Sample is allowed to flow through the column and the flowthrough is collected. Loading can be repeated several times. D. First wash (W1) is performed by gently pipetting buffer on top of the resin. E. Washing is continued as in panel D until the column is completely washed from excess pigment-proteins. FLAG resin is indicated with a black bar in panels A and E.

    11. Resuspend the resin into one bed volume of MES buffer and transfer it into a Proteus Clarification Mini Spin Column.
    12. Add 3x FLAG-peptide into a final concentration of 300 µg/ml. Seal the top of the tube with parafilm and cover the whole tube with aluminum foil. Mix in a rotator mixer set to 10 rpm for 30 min at 10 °C.
    13. Spin the column at 600 x g (Eppendorf 5415 R) at 4 °C for 3 min. Collect elution 1 into a 1.5 ml microtube and store on ice.
    14. Repeat the incubation with FLAG-peptide and centrifuge as above to yield elution 2.
    15. The obtained elutions can be pooled and the total elution volume (typically ~800 µl) can be immediately concentrated using microconcetrators (e.g., Amicon Ultra-0.5, Sigma-Aldrich) with a desired cut-off to achieve higher concentration for downstream analysis (protein gel electrophoresis, immunoblotting [see Figure 3, eluate (E)], protein mass spectrometry, single-particle analysis, size-exclusion chromatography, etc.).
    Notes:
    1. When working with solubilized thylakoids, use buffers supplemented with 0.04% DDM at all steps. Use buffers without detergent when working with soluble proteins.
    2. FLAG-resin should never be allowed to dry out during the purification.
    3. Thaw the FLAG resin on ice and mix thoroughly before use.
    4. The FLAG resin is stored in buffer as a 50% solution, which means that pipetting 600 µl of the well mixed solution will result in 300 µl resin bed volume.
    5. For low abundant proteins, the number of binding steps (Step D6) can be increased.
    6. The stringency of the purification can be altered by increasing the number of washing steps.


      Figure 3. Immunoblot analysis following the purification of geranylgeranyl reductase enzyme tagged with 3x FLAG-tag (FLAG-ChlP). The purification of FLAG-ChlP from Synechocystis was performed according to this protocol. Protein suspension that was loaded in Step D4 (L; solubilized thylakoid membranes of 0.5 µg chlorophyll), the first flowthrough in Step D4 (FT; equal volume to L), the first wash in Step D5 (W1; 1/25 volume of the wash fraction), the second wash in step 6 (W2; 1/25 volume of the wash fraction), and the eluate gained in Step D15 (E; 1/250 volume of total, non-concentrated eluate) were analyzed by SDS gel electrophoresis (A) and by a subsequent immunoblot (B). Prior to Western blotting on a PVDF membrane the gel was stained with SYPRO Orange (Sigma) to detect total proteins (A). Proteins blotted on the membrane were probed with the α-FLAG antibody (Merck) (A); the protein band, representing the isolated FLAG-ChlP, is indicated.

Notes

  1. The MES buffer system pH 6.5 containing Mg2+ and Ca2+ ions used in this procedure has been optimized to maximize the stability of PSII complexes (Dobáková et al., 2009). A different buffer composition may be required for the stability of different protein complexes.
  2. The protein concentration in soluble fraction can be assessed e.g., by BCA assay or other standard methods. However, for assessing the concentration of the membrane fraction, it is more convenient to relate to its chlorophyll content [the protein concentration (µg/µl) in Synechocystis thylakoids is roughly ten times higher than that of chlorophyll].
  3. Measurement of chlorophyll concentration: Pipet 10 µl of thylakoids into a 1.5 ml microtube and add 990 µl methanol using a Hamilton syringe. Vortex and incubate the tube in the dark at room temperature for 5 min. Pellet cell debris by centrifuging at 16,800 x g (Eppendorf 5418) at room temperature for 5 min. Measure the chlorophyll absorbance at 665.2 nm and sample turbidity at 750 nm, and calculate the chlorophyll a concentration (µg/µl) of the thylakoids: ((A665.2 - A750)/α) x 100 x 1 cm-1 (α = 79.95 L g-1 cm-1; Porra et al., 1989).
  4. Always perform a control purification with a wild-type strain to exclude contaminants from your analysis of results. For example, a small amount of Synechocystis trimeric photosystem I and tryptophanyl-tRNA synthetase are typical contaminants of our FLAG-tag purifications (Knoppová et al., 2014; Bučinská et al., 2018; Skotnicová et al., 2018).
  5. In case the purification fails or the protein yield is very low, make sure that your protein of interest is expressed and not degraded in vivo or during the preparation of cellular fractions (perform SDS-PAGE and immunodetection using commercially available anti-FLAG antibody (Merck). A representative SDS gel electrophoresis and subsequent immunoblot analysis of purification (Steps D7-D9) and elution (Step D15) are presented in Figure 3. In the case of membrane proteins, compare the unsolubilized and solubilized fractions to check protein solubility (a detergent with different properties than DDM may be required for the solubility or stability of the desired protein complex). In addition, compare your original SP and/or solubilized TM samples with the obtained flowthrough in Step D7 (Figure 3). The relative amount of your protein in flowthrough should significantly decrease when compared with the loaded material; otherwise it signifies a problem with binding to the resin. For the purification of proteins requiring metal cofactors an EDTA-free protease inhibitor should be used.

Recipes

  1. MES buffer (1 L)
    25 mM MES-NaOH, pH 6.5 (50 ml of 0.5 M stock)
    10 mM MgCl2 (10 ml of 1 M stock)
    10 mM CaCl2 (10 ml of 1 M stock)
    25% glycerol (250 ml)
    1. Adjust the volume to 1 L with deionized water in a measuring cylinder
    2. After membrane protein solubilization, use buffer supplemented with 0.04% DDM for all purification steps
  2. 0.5 M MES-NaOH, pH 6.5 (250 ml)
    24.41 g MES (2-Morpholinoethanesulfonic acid) (AppliChem)
    1. Dissolve into ~200 ml of deionized water and set pH to 6.5 with 10 M NaOH
    2. Adjust the volume to 250 ml in a measuring flask
  3. 1 M MgCl2 (100 ml)
    20.33 g MgCl2·6H2O
    Dissolve to deionized water and adjust the volume to 100 ml in a measuring flask
  4. 1 M CaCl2 (100 ml)
    14.70 g CaCl2·2H2O
    Dissolve to deionized water and adjust the volume to 100 ml in a measuring flask

Acknowledgments

This work was funded by the Czech Science Foundation (17‐08755S) and by the Czech Ministry of Education (project LO1416).

Competing interests

The authors declare no conflicts of interest.

References

  1. Boehm, M., Romero, E., Reisinger, V., Yu, J., Komenda, J., Eichacker, L. A., Dekker, J. P. and Nixon, P. J. (2011). Investigating the early stages of photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexes. J Biol Chem 286(17): 14812-14819.
  2. Boehm, M., Yu, J., Reisinger, V., Bečková, M., Eichacker, L. A., Schlodder, E., Komenda, J. and Nixon, P. J. (2012). Subunit composition of CP43-less photosystem II complexes of Synechocystis sp. PCC 6803: implications for the assembly and repair of photosystem II. Philos Trans R Soc Lond B Biol Sci 367(1608): 3444-3454.
  3. Bučinská, L., Kiss, É., Koník, P., Knoppová, J., Komenda, J. and Sobotka, R. (2018). The ribosome-bound protein Pam68 promotes insertion of chlorophyll into the CP47 subunit of photosystem II. Plant Physiol 176(4): 2931-2942.
  4. Chidgey, J. W., Linhartová, M., Komenda, J., Jackson, P. J., Dickman, M. J., Canniffe, D. P., Konik, P., Pilny, J., Hunter, C. N. and Sobotka, R. (2014). A cyanobacterial chlorophyll synthase-HliD complex associates with the Ycf39 protein and the YidC/Alb3 insertase. Plant Cell 26(3): 1267-1279.
  5. Dobáková, M., Sobotka, R., Tichý, M. and Komenda, J. (2009). Psb28 protein is involved in the biogenesis of the photosystem II inner antenna CP47 (PsbB) in the cyanobacterium Synechocystis sp. PCC 6803. Plant Physiol 149(2): 1076-1086.
  6. Dobáková, M., Tichý, M. and Komenda, J. (2007). Role of the PsbI protein in photosystem II assembly and repair in the cyanobacterium Synechocystis sp. PCC 6803. Plant Physiol 145(4): 1681-1691.
  7. Esposito, D. and Chatterjee, D. K. (2006). Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17(4): 353-358.
  8. Harding, R. J., Loppnau, P., Ackloo, S., Lemak, A., Hutchinson, A., Hunt, B., Holehouse, A. S., Ho, J. C., Fan, L., Toledo-Sherman, L., Seitova, A. and Arrowsmith, C. H. (2019). Design and characterization of mutant and wildtype huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems. J Biol Chem 294(17): 6986-7001.
  9. Hopp, T. P., Prickett, K. S., Price, V. L., Libby, R. T., March, C. J., Cerretti, D. P., Urdal, D. L. and Conlon, P. J. (1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Nature Biotechnology 6: 1204-1210.
  10. Kiss, É., Knoppová, J., Aznar, G. P., Pilný, J., Yu, J., Halada, P., Nixon, P. J., Sobotka, R. and Komenda, J. (2019). A Photosynthesis-Specific Rubredoxin-Like Protein Is Required for Efficient Association of the D1 and D2 Proteins during the Initial Steps of Photosystem II Assembly. Plant Cell 31(9): 2241-2258.
  11. Knoppová, J., Sobotka, R., Tichý, M., Yu, J., Konik, P., Halada, P., Nixon, P. J. and Komenda, J. (2014). Discovery of a chlorophyll binding protein complex involved in the early steps of photosystem II assembly in Synechocystis. Plant Cell 26(3): 1200-1212.
  12. Knoppová, J., Yu, J., Konik, P., Nixon, P. J. and Komenda, J. (2016). CyanoP is involved in the early steps of Photosystem II assembly in the cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 57(9): 1921-1931.
  13. Komenda, J., Sobotka, R. and Nixon, P. J. (2012). Assembling and maintaining the Photosystem II complex in chloroplasts and cyanobacteria. Curr Opin Plant Biol 15(3): 245-251.
  14. Li, S., Hong, T., Wang, K., Lu, Y. and Zhou, M. (2017). Dissociation and purification of the endogenous membrane-bound Vo complex from Pichia pastoris. Protein Expr Purif 138: 76-80.
  15. Lichty, J. J., Malecki, J. L., Agnew, H. D., Michelson-Horowitz, D. J. and Tan, S. (2005). Comparison of affinity tags for protein purification. Protein Expr Purif 41(1): 98-105.
  16. Liu, H., Roose, J. L., Cameron, J. C. and Pakrasi, H. B. (2011). A genetically tagged Psb27 protein allows purification of two consecutive photosystem II (PSII) assembly intermediates in Synechocystis 6803, a cyanobacterium. J Biol Chem 286(28): 24865-24871.
  17. Nixon, P. J., Michoux, F., Yu, J., Boehm, M. and Komenda, J. (2010). Recent advances in understanding the assembly and repair of photosystem II. Ann Bot 106(1): 1-16.
  18. Pazderník, M., Mareš, J., Pilný, J. and Sobotka, R. (2019). The antenna-like domain of the cyanobacterial ferrochelatase can bind chlorophyll and carotenoids in an energy-dissipative configuration. J Biol Chem 294(29): 11131-11143.
  19. Porra, R. J., Thompson, W. A. and Kriedemann, P. E. (1989). Determination og accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica et Biophysica Acta 975: 384-394.
  20. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Microbiology 111(1): 1-61.
  21. Skotnicová, P., Sobotka, R., Shepherd, M., Hajek, J., Hrouzek, P. and Tichy, M. (2018). The cyanobacterial protoporphyrinogen oxidase HemJ is a new b-type heme protein functionally coupled with coproporphyrinogen III oxidase. J Biol Chem 293(32): 12394-12404.
  22. Williams, J. G. K. (1988). Construction of specific mutations in photosystem II photosynthetic reaction center by genetic-engineering methods in Synechocystis 6803. Methods in Enzymology 167: 766-778.

简介

[摘要] 探索蛋白质复合物的结构和功能需要在天然状态下对其进行分离- 当研究不稳定和/或低丰度复合物时,这项任务变得具有挑战性。制备膜-蛋白质复合物的困难尤其出名。蓝藻集胞藻 PCC 6803是一种用于氧合营养养分生理的广泛使用的模式生物,传统上已经使用这种蓝细菌研究了膜结合的光合复合物的生物发生。在典型方法中,蛋白质复合物是通过His-affinity色谱法和基于大小的分级分离方法结合纯化的 例如梯度超速离心和/或天然电泳。但是,His亲和纯化带有明显的污染物,许多蛋白质的含量太低,无法进行可行的多步纯化。在这里,我们已经开发出一种纯化方法,用于从膜突囊藻的膜和可溶性级分中分离出3x FLAG标签的蛋白。可溶性蛋白或可溶类囊体经过单一亲和纯化步骤,该步骤利用FLAG亲和树脂的高度特异性结合。彻底洗涤后,使用过量的合成3x FLAG肽在自然条件下将捕获的蛋白质从树脂中释放出来。该方案可以快速分离出纯度极高的低丰度蛋白质复合物。

[背景 ] 蓝藻已被用作优选的模型系统以研究光合蛋白复合物的生物合成和功能的几十年。蓝细菌中的光合作用装置与真核系统(藻类和植物)非常相似,但是蓝细菌具有原核模型的所有优点,例如快速生长和小的基因组,可以轻松地进行基因操作以及使用细菌遗传学的标准工具。特别是,集胞藻。PCC 6803(此后称为集胞囊藻)被确立为该领域最喜欢的模式生物,主要是因为它可以高效转化并通过功能同源重组整合DNA。此外,葡萄糖耐受亚株的集胞藻属,其可使用增殖补糖,使学习光合基因通过反向遗传学(威廉姆斯,1988)的功能。

光系统II(PSII)的生物发生,是一种大型膜嵌入的色素-蛋白质复合物,负责光合作用的水氧化,已在集胞藻中进行了深入研究(Nixon et al。,2010)。从单个亚基组装PSII复合物需要具有许多辅助(组装)因素的复杂机械,这些因素通常涉及某个组装步骤(Komenda 等人,2012)。用于分析PSII装配体复合物的常规方法是使用缺少一个或几个PSII亚基的集胞藻突变体。然后,PSII生物发生在一个特定步骤被阻止,在该步骤中,缺失的亚基将与组装过程的中间复合物结合。在此类突变体中,原本短暂的PSII预复合物可以积累到可以对其进行检测甚至纯化的水平,传统上这是通过His标记的PSII(核心)亚基实现的(Dobáková 等,2007和2009; Boehm 等)等人,201 1 和2012年)。

His-tag具有几个优点:它足够小,可以通过简单的引物设计轻松引入,并且使用起来相对便宜。然而,在His亲和纯化后,来自集胞藻的制品通常含有显着的污染蛋白(Boehm 等,201 1 和2012; Liu 等,2011);等等。因此,通常需要额外的纯化步骤以获得所述令人满意的对功能和结构研究的质量。然而,额外的纯化步骤可能会损害目标复合物的产率和天然度。此外,对于可行的多步纯化方案,细胞中许多蛋白质复合物的水平太低了,PSII组装中间体就是一个例子。由于这些原因,非常需要一种快速,温和的一步纯化方法。

与His-tag的一般金属亲和力相比,基于生物分子相互作用的亲和力标签具有更高的特异性(Lichty 等,2005)。大的蛋白质标签,例如小的泛素样修饰剂(SUMO)或麦芽糖结合蛋白(MBP),由于其稳定的性质而非常适合分离小的可溶性蛋白质,甚至可以提高蛋白质的溶解度(Esposito和Chatterjee ,2006)。然而,为了研究更大且可能不稳定的膜-蛋白质复合物,优选的是体积较小的标签,其对蛋白质-蛋白质相互作用的干扰最小。与蛋白质或抗体高度亲和的短肽标签似乎最适合此目的。一种这样的标签是称为FLAG的短八肽(DYKDDDDK;Hopp 等,1988)。与His-标签和其他几个标签相比,使用FLAG-标签可产生出众的纯度,而收率却几乎没有下降(Lichty 等,2005)。此外,由于可以使用合成的FLAG肽洗脱带有FLAG标签的蛋白质,因此可以避免使用破坏性化学物质,高离子浓度或极端pH值。即使FLAG亲和树脂的每毫克纯化蛋白的价格比Ni-NTA树脂的价格高约50倍(Lichty 等,2005)。与优化多步纯化程序相比,用于纯化不稳定和/或低丰度复合物的方法值得节省时间和成本。最近,已经采用基于FLAG标签的方法从哺乳动物和昆虫细胞系中纯化人类Huntingtin 蛋白的致病变异(Harding 等,2009),以及巴斯德毕赤酵母V型的完整膜Vo-部分。ATPase(Li 等,2017)。

本文介绍的FL AG亲和纯化方案旨在纯化带有3个FLAG表位(DYKDDDDKDYDYKDDDDKDYKDDDDK)的蛋白质。该协议已成功用于解决PSII装配因子Ycf39(Knoppová 等,2014),CyanoP (Knoppová 等,2016),Pam68(Bučinská 等,2018)和RubA (Kiss 等)的作用。 。,2019); 以及含有叶绿素合酶(Chidgey 等,2014),原卟啉原原氧化酶(Skotnicová 等,2018)和铁螯合酶的色素-蛋白质复合物(Pazderník 等,2019)。尽管该方法最初是为结合膜的蛋白质复合物开发的,但经过很小的修改,它也可以应用于可溶性蛋白质。

关键字:蛋白质纯化, 膜蛋白复合物, 集胞藻6803, 光系统, FLAG标签, 亲和层析

材料和试剂


 


一次性半微量比色杯(品牌,目录号:759015)
50 ml锥形管(JET biofil ,目录号:CFT011500)
圆形油漆刷,ø6毫米
移液器提示:
10 µl(海王星,目录号:2340)


200微升(Eppendorf,货号:0030000870)


1,000 µl(Eppendorf,目录号:0030000927)


1.5 ml 微管(Deltalabs ,目录号:4092.3N)
7 ml螺旋盖小瓶,用于细胞裂解(BioSpec 产品,目录号:3205)
塑料色谱柱(Bio-Rad,目录号:7311550)
表达3x FLAG标签蛋白的囊泡细胞
BG-11液体培养基和琼脂平板(Rippka et al。,1979)
100-200 µm玻璃微珠(PRECIOSA ORNELA,晶体微珠B 134)
完整PROT 便于抑制剂混合物片剂(Sigma-Aldrich公司,目录号:11836145001)
(可选)具有合适截止过滤器的微浓缩器(例如,Amicon Ultra -0.5,Sigma-Aldrich)
液氮
碎冰
保丽龙盒或同等品,用于保存冰块
反渗透(RO)水
去离子水(默克(Merck),货号:1167545000)
甲醇(PENTA,目录号:21240-11000)
正十二烷基-β-D- 麦芽糖苷(DDM; BioChemica ,目录号:A0819,0005)
抗FLAG M2亲和凝胶(Sigma-Aldrich,目录号:A2220)
Proteus澄清微型旋转柱(Generon ,目录号:GEN-MSF500)
3x FLAG肽,20 mg,纯度> 85%(定制合成,Genscript ,美国)
ñ OTES:


另一种选择是购买3x FLAG肽(Sigma-Aldrich,目录号:F4799-4MG),但是定制合成的价格大约便宜6倍。
在去离子水中制备3 mg / ml的溶液,并以100-200 µl的等分试样形式存储在-20 °C下。Ť hawed肽可以被存储在4 ℃直至使用。
2-巯基乙烷磺酸(MES; 应用liChem,目录号:A0689,0250)
MgCl 2 ·6H 2 O(PENTA,目录号:16330-31000)
甘油(PENTA,目录号:14550-11000)
CaCl 2 ·2H 2 O(PENTA,目录号:16790-31000)
α-FLAG抗体(Merck,目录号:F7425)
MES缓冲液(1 L)(请参阅配方)
0.5 M MES-NaOH,pH 6.5(250 ml)(请参阅食谱)
1 M MgCl 2 (100毫升)(请参阅食谱)
1 M CaCl 2 (100 ml)(请参阅食谱)
 


设备


 


温控生长设备,配备定轨振荡器和光源
层流罩
金属接种环
煤气灶
500 ml锥形瓶
圆形培养瓶(10升)
Cimarec TM i Maxi磁力搅拌器( Thermo Fisher Scientific,目录号:50088143)和磁力搅拌棒
分光光度计(WPA S1200 Spectrawave )
离心机和转子:
离心机(Sigma Laborzentrifugen GmbH ,型号:Sigma 8KS,转子:12505-H)


离心机(Sigma Laborzentrifugen GmbH ,型号:Sigma 3K30,转子:12155-H [ +15 ml锥形管适配器,13081 ] 和12158-H )


离心机(Eppendorf,型号:5418)


离心机(Eppendorf,型号:5415 R)


离心瓶:
500 ml(Nalgene,货号:3141-0500)


80 ml(Sigma Laborzentrifugen GmbH,目录号:15080)


27 ml(Sigma Laborzentrifugen GmbH,目录号:15032)


低温冷冻箱
普通冰柜
配备7毫升样品瓶适配器的Mini-Beadbeater-16(BioSpec Products,目录号:607)(BioSpec Products,目录号:607TC8)
Vortex V-1 Plus(Biosan ,目录号:BS-010203-AAG)
1 ml汉密尔顿注射器(Hamilton,目录号:81330)
自动移液器:
0.5-10微升(Eppendorf,目录号:3120000020)


10-100微升(Eppendorf,货号:3120000046)


100-1 ,000微升(的Eppendorf,目录号:3120000062)


Multi-Bio RS-24旋转器(Biosan ,目录号:BS-010117-AAG)
冷藏室或冷藏室,10 °C
脱水缸架和环形夹
25毫升玻璃烧杯
 


程序


 


细胞培养和收获
在层流罩的工作,接种集胞藻使用金属接种环成五个细胞,500ml锥形烧瓶,每个含有200ml BG-11培养基。在28 岁时培养细胞 在设定为150 rpm的旋转振荡器上,以°C和40 µmol / m 2 / s的光合作用光子通量密度设定,直到培养物达到对数生长期的中/末期。
注:W 母鸡的培养物在750度纳米达到0.6-0.8的光密度,这对应于9-12×10 7 集胞藻细胞/ ml。


将您的培养物(OD 750〜0.8 )收集到10 L的烧瓶中,并稀释至4 L最终体积以获得OD 750〜0.2 密度。生长培养物,直到有磁性搅拌器(转速240rpm)和空气鼓泡对数生长期的中期/结束在所述相同的条件如上述。
将细胞培养物分成500 ml离心瓶,并通过在4 °C 的冷却离心机(Sigma 8KS,转子12505-H)中以10,000 x g 离心20分钟来沉淀细胞。
将细胞沉淀悬浮在总共30 ml的MES缓冲液中。
笔记:


用于制备细胞培养物的所有培养基和设备均应灭菌。使用燃气燃烧器对金属接种环进行消毒,并在使用前对所有玻璃器皿和生长培养基进行高压灭菌。
蓝细菌培养物在750 nm处的吸光度可忽略不计;因此,在此波长下的OD主要取决于光散射(浊度)。不同的分光光度计的各种光学特性差异很大,因此OD 750 的光散射值可能会变化。因此,重要的是通过先验测量生长曲线来确定培养物处于对数生长期中/末期的OD 。在我们的标准条件下,集胞藻的倍增时间约为12小时;因此,OD 750 = 0.2 的培养物将在大约一天内达到所需的OD 750 。
根据我们的经验,更大的蓝藻文化(即卷,4 L)一般增长较慢,可能是由于气体交换的限制。因此,期望通过空气强烈鼓泡培养物。
在小号TEP 甲4你可能会冻结的细胞在液氮中,并存储它们在- 80 ℃直至使用。
 


细胞裂解和类囊体分离
在10,000 x g (Sigma 3K30,转子12155-H )在4 °C的80 ml离心管中将细胞沉淀10分钟。
丢弃上清液,并用湿油漆刷将细胞重悬于5 ml MES缓冲液中。向离心管中添加30 ml MES缓冲液,然后如上所述沉淀细胞。
将细胞重悬于12 ml MES缓冲液中,并添加蛋白酶抑制剂:将1片药片溶解于1 ml去离子水中以获得50x储备液,并吸取相应的溶液以达到1x工作浓度。你可以存储在在-20抑制剂的其余℃下用于以后使用。
将3 ml玻璃珠和3 ml细胞悬液添加到四个7 ml螺帽小瓶中(图1A),合上盖子并用封口膜密封(图1B)。管中应该有大约1厘米的自由空间,您可以添加一些缓冲液以达到所需的体积。
用Mini-Beadbeater-16将电池破碎60秒。让试管在避光的冰上冷却5分钟,并重复5次破碎循环。
裂解后,将磁珠在4 °C 下以500 x g的转速旋转10 s (Sigma 3K30,装有用于15 ml锥形管的适配器的转子12155-H)。将上清液收集到27 ml离心管中。用一倍床体积的MES缓冲液洗涤磁珠(图1C)三到四次,直到上清液和磁珠呈浅绿色(图1D-1E)。
合并来自S tep B 6的所有上清液,并通过在4 °C下以35,000 x g (Sigma 3K30,转子12158-H)离心20分钟来沉淀类囊体。
此时,如果您仅对膜蛋白的分离感兴趣,则可以丢弃上清液。否则,将上清液收集到新的离心管中(标记为:SP,“可溶性蛋白”),并使用湿油漆刷将沉淀重悬于10 ml MES缓冲液中(标记为管:TM,类囊体膜)。如上离心管。
将上清液从“ SP”管中收集到50 ml锥形管中,并保存在冰上。丢弃“ TM”管中的上清液,然后将两个管(“ SP”和“ TM”)中的沉淀重悬到MES缓冲液的1/3可溶性级分中。如果仅继续使用膜部分,请使用总计10 ml的MES缓冲液,然后将沉淀物合并到50 ml的锥形管中。
在液氮中冷冻300 µl等分的可溶性蛋白和100 µl类囊体的等分试样,并储存在-80 °C下以通过凝胶电泳进一步分析。
笔记:


沉淀细胞后,由于冷冻和解冻后细胞的部分破裂,上清液可能会略带蓝色。
从小号TEP 乙5 起它是进口蚂蚁保持在样品冷却(4-10 ℃) ,保护它的光,以防止所述激发叶绿素分子和反应性氧物质的随之形成。我们建议在昏暗的绿色灯光下工作。
类囊体在添加其余缓冲液之前更容易先重悬至数ml体积。
在小号TEP 乙10也可能会冻结可溶性蛋白和类囊体样品的其余部分在液氮中,并将它们存储在-80 ℃直至随后的溶解和纯化。
 


D:\ Reformatting \ 2020-3-2 \ 1903040--1374 816046 \ Figs jpg \图1 .jpg


图1.裂囊藻细胞的破裂。A. 螺帽管中装有3 ml玻璃珠和3 ml蓝细菌悬浮液;B. Ť 他管密封用石蜡膜和细胞使用珠打浆机裂解。C.破坏细胞后,将珠子旋转到底部,并收集上清液。D.用MES缓冲液洗涤珠;E. 在每个洗涤步骤之后,收集上清液。


 


类囊体溶解
用MES缓冲液将类囊体的叶绿素浓度调节至0.5 µg / µl。
加入1%的正十二烷基β-D- 麦芽糖苷(DDM)到类囊体悬浮液和我ncubate在管1个小时10 ° 下在所述暗(例如,可能会用铝箔覆盖的管)在旋转器上混合器设置为10 rpm。
将悬浮液转移到27 ml离心管中,在4 °C下以46,000 x g (Sigma 3K30,转子12158-H)沉淀不溶物30分钟。
将上清液收集到50 ml锥形管中,弃去沉淀。
在液氮中冷冻100 µl溶解的类囊体,并储存在-80 °C进行进一步分析,尤其是用于识别蛋白质溶解性的潜在问题。
 


FLAG亲和纯化
先用1 ml的RO-水,再用1 ml的去离子水洗涤纯化柱。
关闭该柱,然后一个DD1毫升MES缓冲液和600微升的1:1的反- FLAG - M2琼脂糖使用切割移液管尖端树脂(视频1)。




D:\ Reformatting \ 2020-3-2 \ 1903040--1374 816046 \ video1.jpg


视频1. 专栏准备


 


打开色谱柱,让缓冲液流过。洗涤树脂与另外的1毫升MES缓冲液(轻轻吸管缓冲器上的树脂的顶部,并允许它以通过流动;图2A;视频2)。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ 2020-5-20 \ 3616--1903040--1374 816046 \ video 2.jpg


视频2 。轻柔洗涤的列


 


将蛋白质悬浮液加载到色谱柱中,并将流通液收集到玻璃烧杯中(图2B;视频3)。
 


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录像3 。样品上样


 


用1 ml MES 缓冲液洗涤色谱柱。丢弃流通液。
第二次将来自S tep D 4 的流出物上样至色谱柱,并收集流出物。再次重复绑定。
从最终流通液中取出0.5 ml样品,将其在液氮中冷冻,并储存在-80 °C进行进一步分析[ 图3,流通液(FT)] 。
用1 ml MES缓冲液洗涤色谱柱[ 图2C; 图3,先洗(W1)] 。
关闭柱子并添加1 ml MES缓冲液:以足够高的速度吸移缓冲液,以使其与树脂充分混合(图2D;视频4)(您可以通过上下移液,轻轻地将缓冲液与树脂混合移液器吸头,但有些树脂可能会粘在吸头上而丢失)。打开色谱柱,让缓冲液流干[图3,第二次清洗(W2)] 。
 


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视频4 。大力清洗树脂


 


用1 ml MES缓冲液洗涤色谱柱,而不要混合树脂。重复此洗涤步骤3次。总共用20倍柱体积的MES缓冲液洗涤树脂(图2E)。
 


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图2. 使用亲和色谱法从集胞藻中分离3x FLAG标签的蛋白。上图和下图分别显示了可溶性蛋白和去污剂溶解的膜蛋白的单独纯化步骤。A.将抗FLAG树脂填充到塑料色谱柱中。B.将可溶性蛋白质级分(上图)或去污剂溶解的类囊体(下图)上样至色谱柱。C.使样品流过色谱柱,并收集流过液。加载可以重复多次。D.第一步清洗(W1)是通过将缓冲液轻轻移液到树脂顶部进行的。E.洗涤继续在面板d 直到所述柱被完全从过量颜料蛋白洗涤。FLAG树脂在面板A和E中用黑条表示。


 


将树脂重悬于一床体积的MES缓冲液中,然后将其转移到Proteus Clarification Mini Spin Column中。
加入3x FLAG肽至终浓度300 µg / ml。用封口膜密封管的顶部,并用铝箔覆盖整个管。在转子混合器组混合以10rpm下为30 分钟,在10 ℃。
在4 °C下以600 x g (Eppendorf 5415 R )旋转色谱柱3分钟。将洗脱液1收集到1.5 ml微量管中,并保存在冰上。
重复与FLAG肽一起孵育并按上述离心方法进行洗脱2。
将所得到的洗脱可被合并,并在总洗脱体积(通常为〜800微升)可以使用立即集中microconcetrators (例如,的Amicon 超0.5,Sigma-Aldrich公司)与所需的截止,以实现用于下游分析较高浓度(蛋白质凝胶电泳,免疫印迹[见图3,洗脱液(E)] ,蛋白质质谱,单颗粒分析,尺寸排阻色谱法等)。
笔记:


当使用溶解的类囊体时,请在所有步骤中使用补充有0.04%DDM的缓冲液。处理可溶性蛋白质时,请使用不含去污剂的缓冲液。
在纯化过程中,切勿让FLAG树脂变干。
使用前,将FLAG树脂在冰上解冻并彻底混合。
FLAG树脂以50%的溶液形式存储在缓冲液中,这意味着吸取600 µl充分混合的溶液将产生300 µl的树脂床体积。
对于丰度低的蛋白质,可以增加结合步骤(S tep D 6)。
纯化的严格性可以通过增加洗涤步骤的数量来改变。
 


D:\ Reformatting \ 2020-3-2 \ 1903040--1374 816046 \ Figs jpg \图3.jpg


图3.纯化带有3x FLAG-tag(FLAG- ChlP )的geranylgeranyl还原酶的酶后的免疫印迹分析。根据该方案,从集胞藻中纯化FLAG-ChIP 。这是在装载蛋白悬浮液小号TEP d 4(L; 0.5微克叶绿素的溶解的类囊体膜),第一通流在小号TEP d 4(FT ;等体积到L),在所述第一洗涤小号TEP d 5(W1; 1/25体积的洗涤级分),步骤6中的第二次洗涤(W2; 1/25体积的洗涤级分)和在S tep D 15中获得的洗脱液(E; 1/250体积的总洗脱液,非浓缩洗脱液通过SDS凝胶电泳(A)和随后的免疫印迹(B)进行分析。在PVDF膜上进行Western印迹之前,将凝胶用SYPRO Orange(Sigma)染色以检测总蛋白(A)。用α-FLAG 抗体(Merck )(A)探测印迹在膜上的蛋白; 显示了代表分离的FLAG-ChIP 的蛋白带。


 


笔记


 


缓冲含Mg体系的pH 6.5的MES 2+ 和Ca 2+ 在此过程中使用的离子已被优化以最大化PSII复合物的稳定性(杜伯á KOVA 等人,2009)。不同蛋白质复合物的稳定性可能需要不同的缓冲液组成。
在可溶性级分中的蛋白质浓度可以评估例如。,通过BCA分析或其他标准方法。但是,为了评估膜级分的浓度,将其叶绿素含量与[叶囊藻类囊体中的蛋白质浓度(μg/μl)大约比叶绿素高出十倍] 更为相关。
叶绿素浓度的测量:吸取10 µl类囊体移入1.5 ml微管中,并使用汉密尔顿注射器添加990 µl甲醇。涡流和孵化在管的黑暗中在室温下搅拌5分钟。通过在室温下以16,800 x g (Eppendorf 5418)离心5分钟来沉淀细胞碎片。测量665.2 nm处的叶绿素吸光度和750 nm处的样品浊度,并计算类囊体的叶绿素a 浓度(µg / µl):(((A 665.2 - A 750 )/α)x 100 x 1 cm -1 (α= 79.95 L g -1 cm -1 ;Porra 等,1989)。
始终使用野生型菌株进行对照纯化,以从结果分析中排除污染物。例如,少量的集胞藻三聚体光系统I和色氨酸-tRNA合成酶是我们FLAG标签纯化的典型污染物(Knoppová 等人,2014; Bučinská 等人,2018; Skotnicová 等人,2018)。
如果纯化失败或蛋白质产量非常低,请确保您感兴趣的蛋白质在体内或制备细胞级分过程中表达且未降解(使用商业化的抗FLAG抗体进行SDS- PAGE和免疫检测(Merck )。代表性的SDS凝胶电泳以及随后的纯化(S teps D 7- D 9 )和洗脱(S tep D 15)的免疫印迹分析如图3所示。对于膜蛋白,将未溶解和可溶解的级分与检查蛋白质的溶解度(所需蛋白质复合物的溶解度或稳定性可能需要使用性能与DDM不同的去污剂)。此外,将原始的SP和/或溶解的TM样品与在S tep D 7中获得的流通量进行比较(图3)。与负载的材料相比,流过的蛋白质的相对数量应显着降低;否则表明与树脂的结合存在问题。为了纯化需要金属辅因子的蛋白质,应使用不含EDTA的蛋白酶抑制剂。
 


菜谱


 


MES缓冲液(1 L)
25 mM MES-NaOH,pH 6.5(50 ml的0.5 M库存)             


              10 mM MgCl 2 (10 ml的1 M库存)


10 mM CaCl 2 (10 ml的1 M库存)


25%克lycerol(250毫升)


在量筒中用去离子水将体积调节至1 L
膜蛋白溶解后,所有纯化步骤均应使用添加了0.04%DDM的缓冲液
0.5 M MES-NaOH,pH 6.5(250毫升)
24.41 g MES(2-Morpholinoethanesulfonic酸)(AppliChem )


溶解于约200 ml去离子水中,并用10 M NaOH将pH设置为6.5
在量瓶中将体积调节至250 ml
1 M氯化镁2 (100毫升)
20.33 g氯化镁2 ·6H 2 O


溶解到去离子水和调整的体积至100ml在测量FLAS ķ


1 M CaCl 2 (100毫升)
14.70克CaCl 2 ·2H 2 O


溶于去离子水,并在量瓶中将体积调节至100 ml


             


致谢


 


这项工作是由捷克科学基金会(17资助- 由捷克教育部08755S)教育(项目LO1416)。


 


利益争夺


 


在一个uthors宣称没有利益冲突。


 


参考文献


 


Boehm,M.,Romero,E.,Reisinger,V.,Yu,J.,Komenda,J.,Eichacker,LA,Dekker,JP和Nixon,PJ(2011)。调查Synechocystis sp。中光系统II组装的早期阶段。PCC 6803:隔离CP47和CP43复合物。生物化学杂志286(17):14812-14819。
波姆,M.,于,J.,赖辛格,V.,成为č KOV á ,M.,Eichacker,LA,Schlodder ,E.,柯门达,J。和尼克松,PJ(2012)。少CP43的Synechocystis sp。的光系统II复合物的亚基组成。PCC 6803:对光系统II的组装和维修的影响。Philos Trans R Soc Lond B Biol Sci 367(1608):3444-3454。
卜č insk á ,L.,吻É 。,昆í ķ ,P.,Knoppov á ,J.,柯门达,J。和索博特卡,R。(2018)。所述ř ibosome- b ound p rotein Pam68 p romotes 我nsertion的Ç hlorophyll到CP47 小号的ubunit p hotosystem II。植物生理学176(4):2931-2942。
Chidgey,JW,Linhartov á ,M.,柯门达,J.,杰克逊,PJ,迪克曼,MJ,Canniffe ,DP,Konik ,P.,Pilny ,J.,猎人,CN和索博特卡,R。(2014)。蓝藻叶绿素合酶-HliD复合体与Ycf39蛋白和YidC / Alb3插入酶缔合。植物细胞26(3):1267-1279。
DOB á KOV á ,M.,索博特卡,R.,TICH ý ,M。和柯门达,J。(2009)。Psb28蛋白与蓝藻集胞藻属中的光系统II内部天线CP47(PsbB)的生物发生有关。PCC6803。植物生理学149(2):1076-1086。
DOB á KOV á ,M.,TICH ý ,M。和柯门达,J。(2007)。PsbI蛋白在光系统II装配和修复蓝藻Synechocystis sp。中的作用。PCC6803。植物生理学145(4):1681-1691。
Esposito,D。和Chatterjee,DK(2006年)。通过使用融合标签增强可溶性蛋白表达。Curr Opin Biotechnol 17(4):353-358。
哈丁,RJ,Loppnau ,P.,Ackloo ,S.,饭,A.,哈钦森,A.,亨特,B.,Holehouse ,AS,何,JC,电风扇,L.,托莱多-谢尔曼,L.,Seitova ,A.和Arrowsmith,CH(2019)。从可扩展的真核表达系统工具包中产生的突变和野生型亨廷顿蛋白的设计和表征。生物化学杂志294(17):6986-7001。
霍普(Hopp),TP,普里基特(Prickett),KS,Price,VL,利比(Libby),RT,三月,CJ,塞雷蒂(Cerrretti),DP,乌达(Urdal),DL和康隆(Conlon),PJ(1988)。用于重组蛋白鉴定和纯化的短多肽标记序列。Nature Biotechnology 6:1204-1210。
吻,É 。,Knoppov á ,J.,阿斯纳尔,GP,Piln ý ,J.,于,J.,Halada ,P.,尼克松,PJ,索博特卡,R。和柯门达,J。(2019)。在光系统II组装的初始步骤中,D1和D2蛋白的有效缔合需要光合作用特定的Rubredoxin-like蛋白。植物细胞31(9):2241-2258。
Knoppov á ,J.,索博特卡,R.,TICH ý ,M.,于,J.,Konik ,P.,Halada ,P.,尼克松,PJ和柯门达,J。(2014)。涉及光系统II组件的早期步骤的叶绿素结合蛋白复合物的发现集胞藻属。植物细胞26(3):1200-1212。
Knoppov á ,J.,于,J.,Konik ,P.,尼克松,PJ和柯门达,J。(2016)。CyanoP参与了蓝藻集胞藻属中光系统II组装的早期步骤。PCC6803。植物细胞生理学57(9):1921-1931。
Komenda,J.,Sobotka,R.和Nixon,PJ(2012)。在叶绿体和蓝细菌中组装和维护Photosystem II复合物。Curr Opin 植物生物学15(3):245-251。
Li,S.,Hong,T.,Wang,K.,Lu,Y. and Zhou,M.(2017年)。解离和纯化巴斯德毕赤酵母内膜结合的Vo复合物。Protein Expr Purif 138:76-80。
Lichty ,JJ,Malecki ,JL,Agnew,HD,Michelson-Horowitz,DJ和Tan,S。(2005)。亲和标签用于蛋白质纯化的比较。Protein Expr Purif 41(1):98-105。
Liu,H.,Roose ,JL,Cameron,JC和Pakrasi ,HB(2011)。基因标记的Psb27蛋白允许纯化蓝藻集胞藻6803中的两个连续的光系统II(PSII)组装中间体。生物化学杂志286(28):24865-24871。
Nixon,PJ,Michoux ,F.,Yu,J.,Boehm,M.和Komenda,J.(2010)。在了解光系统II的组装和维修方面的最新进展。Ann Bot 106(1):1-16。
Pazdern í ķ ,M.,母马š ,J.,Piln ý ,J。和索博特卡,R。(2019)。蓝藻亚铁螯合酶的天线样结构域可以能量耗散的方式结合叶绿素和类胡萝卜素。生物化学杂志294(29):11131-11143。
Porra ,RJ,Thompson,WA和Kriedemann ,PE(1989)。判定OG 准确消光系数和联立方程用于测定叶绿素一个和b 具有四个不同的溶剂提取:用原子吸收光谱的叶绿素浓度标准的验证。 Biochimica et Biophysica Acta 975:384-394。
Rippka ,R.,Deruelles ,J.,Waterbury,JB,Herdman ,M。和Stanier,RY(1979)。蓝细菌纯属培养的一般任务,菌株历史和特性。微生物学111(1):1-61。
Skotnicová ,P.,Sobotka,R.,Shepherd,M.,Hajek,J.,Hrouzek ,P.和Tichy ,M.(2018)。蓝细菌原卟啉原氧化酶HemJ是功能上与原卟啉原III氧化酶偶联的新型b型血红素蛋白。生物化学杂志293(32):12394-12404。
威廉姆斯,JGK(1988)。通过遗传工程方法在集胞藻6803中构建光系统II光合反应中心中的特定突变。酶学方法167:766-778。
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引用:Koskela, M. M., Skotnicová, P., Kiss, É. and Sobotka, R. (2020). Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography. Bio-protocol 10(10): e3616. DOI: 10.21769/BioProtoc.3616.
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