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Mar 2020

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Expression and Purification of Arabidopsis Transmembrane Protein BCM1 in Saccharomyces cerevisiae
拟南芥跨膜蛋白BCM1在酿酒酵母中的表达与纯化   

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Abstract

Heterologous expression and purification of transmembrane proteins have remained a challenge for decades hampering detailed biochemical and structural characterization of key enzymes and their interacting regulators in multiple metabolic pathways. An in-depth study on the newly identified Arabidopsis thaliana integral membrane protein BALANCE OF CHLOROPHYLL METABOLISM 1 (BCM1) showed a stimulatory effect of the BCM1 on magnesium chelatase, the first enzyme of chlorophyll biosynthesis, through interaction with the GENOMES UNCOUPLED 4 (Wang et al., 2020). Here, we report a detailed and optimized method for heterologous expression and purification of His-tagged BCM1 in Saccharomyces cerevisiae. Following this method, we obtained native BCM1 used for in vitro enzymatic assay of magnesium chelatase (Wang et al., 2020). Currently, the crystallization studies of the BCM1 are underway. This protocol could be adapted to purify BCM1-like transmembrane proteins from eukaryotic organisms for enzymatic and structural studies.

Keywords: A transmembrane protein (跨膜蛋白), Expression and purification of BCM1 (BCM1表达与纯化), Saccharomyces cerevisiae (酿酒酵母), Arabidopsis thaliana (拟南芥)

Background

Identification of post-translational regulators which directly modulate the enzymatic activities of chlorophyll synthesis enzymes can greatly improve our understanding of molecular mechanisms, by which plants maintain highly efficient chlorophyll biosynthesis during leaf greening (Brzezowski et al., 2015). However, the detailed biochemical analyses of chlorophyll synthesis enzymes and their interacting proteins have been restricted by the availability of recombinant proteins in vitro. We recently identified a post-translational regulator BALANCE OF CHLOROPHYLL METABOLISM 1 (BCM1), which simultaneously stimulates chlorophyll biosynthesis and delays chlorophyll breakdown, thereby conferring chlorophyll homeostasis during leaf development (Wang et al., 2020). To examine BCM1’s effect on the enzymatic activity of magnesium chelatase (MgCh), the first enzyme of chlorophyll biosynthesis, we expressed and purified His-tagged BCM1 in S. cerevisiae. It has been shown that BCM1 is able to stimulate MgCh activity in vitro (Wang et al., 2020). Because BCM1 has six transmembrane domains, BCM1 will be used an example of multiple-pass protein herein. Thus, we provide an optimized method for expression and purification of BCM1 in S. cerevisiae. Although prokaryotic purification system hosted by Escherichia coli has been widely used to express hydrophilic proteins from prokaryotic and eukaryotic organisms, overexpression of BCM1 in Escherichia coli cells leads to accumulation of BCM1 aggregate and inclusion bodies instead of properly folded proteins at the membranes. In comparison with cell-free protein expression systems and other eukaryotic expression systems, such as mammalian and insect expression systems, the yeast protein expression method described here enables a large-scale purification of integral membrane proteins with high yield and low cost.

Materials and Reagents

  1. Sterile pipette tips
  2. Eppendorf microcentrifuge tubes 1.5 ml (Eppendorf, catalog number: 00 30121694 )
  3. Falcon, conical centrifuge tubes 50 ml (Corning, catalog number: 14-432-22 )
  4. 0.45 μm filter (VWR, catalog number: 28145-479 )
  5. 14 ml Open-top thinwall untra-clear centrifuge tube (Beckman Coulter, catalog number: 344060 )
  6. Dispensable plastic spin column (Thermo Fisher Scientific, catalog number: 10220544 )
  7. SnakeSkin Dialysis Tubing 10 kDa cut-off (Thermo Fisher Scientific, catalog number: 68100 )
  8. Saccharomyces cerevisiae L40ccua strain
  9. pDR296-His-BCM1 plasmid
  10. Salmon sperm (Thermo Fisher Scientific, catalog number: 15632011 )
  11. Demineralized water
  12. Bacto yeast extract (Cal Roth, catalog number: 2904 )
  13. Bacto peptone (Cal Roth, catalog number: 8952 )
  14. Glucose monohydrate (Cal Roth, catalog number: 6780 )
  15. Adenine sulfate (Sigma-Aldrich, catalog number: A3159 )
  16. Agar-Y (MP Biomedicals, catalog number: 4019012 )
  17. Yeast nitrogen base (YNB) with ammonium sulfate (MP Biomedicals, catalog number: 4027412 )
  18. Synthetic dropout/-tryptophan (SD/-Trp) (MP Biomedicals, catalog number: 4511012 )
  19. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  20. n-dodecyl-β-D-maltoside (β-DM) (Sigma-Aldrich, catalog number: D4641 )
  21. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA·Na2) (Sigma-Aldrich, catalog number: ED2SS )
  22. Tris(hydroxymethyl)aminomethane (Tris) (Cal Roth, catalog number: A411 )
  23. Hydrochloric acid (HCl) (VWR, catalog number: BDH7204
  24. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-212 )
  25. Lithium acetate (LiAc) (Cal Roth, catalog number: 5447 )
  26. Polyethylene glycol 4000 (PEG4000) (Sigma-Aldrich, catalog number: 8.0749 0)
  27. Liquid nitrogen
  28. cOmplete, mini, EDTA-free protease inhibitor tablets (Roche Diagnostics, catalog number: 11873580001 )
  29. Glass beads (425-600 μm) (Sigma-Aldrich, catalog number: G8772 )
  30. Sodium chloride (NaCl) (Cal Roth, catalog number: 9265 )
  31. Potassium chloride (KCl) (Cal Roth, catalog number: 6781 )
  32. Sodium phosphate monobasic dihydrate (NaH2PO4·2H2O) (Cal Roth, catalog number: T879 )
  33. Potassium dihydrogen phosphate (KH2PO4) (Cal Roth, catalog number: 3904 )
  34. Ni-NTA agarose resin (Thermo Fisher Scientific, catalog number: 88223 )
  35. Imidazole (Sigma-Aldrich, catalog number: I2399 )
  36. YPD medium (930 ml) (see Recipes)
  37. 40% (w/v) glucose (100 ml) (see Recipes)
  38. 0.2% (w/v) Adenine sulfate (see Recipes)
  39. YPDA medium (1 L) (see Recipes)
  40. SD/-Trp medium (1 L) (see Recipes)
  41. 80% (v/v) glycerol (100 ml) (see Recipes)
  42. 10x Tris-EDTA (TE) buffer (200 ml) (see Recipes)
  43. 1 M LiAc (200 ml) (see Recipes)
  44. 50% (w/v) PEG4000 (100 ml) (see Recipes)
  45. 10x phosphate-buffered saline (PBS) buffer (1 L) (see Recipes)
  46. 10% (w/v) β-DM (see Recipes)
  47. 1x TE buffer (see Recipes)
  48. TE/LiAc buffer (see Recipes)
  49. Polyethylene glycol (PEG)/LiAc buffer (see Recipes)
  50. Solubilization buffer (10 ml) containing 1% (w/v) β-DM (see Recipes)
  51. Wash buffer containing 0.0256% (w/v) β-DM and 20 mM imidazole (20 ml) (see Recipes)
  52. Elute buffer containing 0.0256% (w/v) β-DM (see Recipes)
  53. Dialysis buffer (1 L) containing 0.0256% (w/v) β-DM (see Recipes)

Equipment

  1. Glassware
    250 ml Erlenmeyer flask
    200 ml Erlenmeyer flask
    2 L Erlenmeyer flask
    1 L Erlenmeyer flask
    100 ml Erlenmeyer flask
    1 L transparent glass media bottle
    250 ml transparent glass media bottle
    100 ml transparent glass media bottle
  2. Rotor JA-25.50
  3. Rotor FA-48-45-11
  4. Milli-Q Reference Water Purification System (EMD Millipore, catalog number: Z00QSV0WW )
  5. Clean bench (Labconco, model: 3440009 )
  6. Water bath
  7. Temperature controlled shaker incubator 
  8. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 )
  9. Cold room
  10. Deep freezer (-20 °C)
  11. Ultra-low temperature freezer (-80 °C)
  12. Centrifuge (Eppendorf, model: 5430 R )
  13. High speed centrifuge (Beckman Coulter, model: Avanti J-26XP )
  14. Ultracentrifuge (Beckman Coulter, model: Optima L-80 XP )

Procedure

  1. Transformation of S. cerevisiae with pDR296-His-BCM1 plasmid
    1. The nucleotide sequences encoding mature BCM1 (amino acid 55-382) with depletion of N-terminal chloroplast transit peptide from Arabidopsis is cloned into the yeast expression vector pDR296. The construct contains an N-terminal histidine (His) tag to facilitate purification of BCM1 by means of immobilized nickel-affinity chromatography (Figure 1).


      Figure 1. Plasmid map of pDR296-BCM1. Unique restriction sites and important features in the plasmids are indicated. PMA1 promoter, transcription promoter for the S. cerevisiae plasma membrane ATPase 1 (PMA1) gene. ADH1 terminator, transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene. 2μ ori, yeast 2μ plasmid origin of replication. TRP1, tryptophan biosynthesis protein. AmpR, the gene encoding β-lactamase, which confer resistance to ampicillin.

    2. Streak out a glycerol stock of S. cerevisiae L40ccua on a YPDA agar plate and incubate the plate at 30 °C for two days.
    3. Inoculate 5 ml of YPDA medium with 1 colony of S. cerevisiae L40ccua in a 14 ml Falcon tube and grow at 30 °C and 250 rpm overnight.
    4. Transfer the overnight culture to 100 ml YPDA medium in a 250 ml Erlenmeyer flask and continue to grow at 30 °C and 250 rpm for 2-4 h until the optical density at 600 nm (OD600) of the culture reaches between 0.4-0.6.
    5. Harvest cells by centrifugation at 1,000 x g for 5 min at room temperature using rotor JA-25.50.
    6. Resuspend cells in demineralized water by vortexing for 5-10 s and harvest then cells by centrifugation at 1,000 x g for 5 min at room temperature using rotor JA-25.50.
    7. Resuspend cells in 1.5 ml TE/LiAc buffer (the cell titer is ~3 x 109 cells/ml). These are now yeast competent cells.
    8. Add 0.1-0.2 μg pDR296-His-BCM1 plasmids, 10 μl of denatured salmon sperm DNA, and 100 μl of yeast competent cells to a 1.5 ml Eppendorf tube and mix them well by pipetting for 2-3 times.
    9. Add 600 μl of freshly prepared PEG/LiAc buffer to the tube and mix them well by vortex for 5 s.
    10. Incubate the mixture at 30 °C for 30 min with shaking at 200 rpm.
    11. Add 70 μl of DMSO to the culture and mix well by gentle inversion.
    12. Heat the tube in a 42 °C water bath for 15 min, do not shake.
    13. Place the tube on ice for 1-2 min.
    14. Pellet cells by centrifugation at 12,000 x g for 5 s at room temperature using rotor FA-48-45-11.
    15. Resuspend cells in demineralized water and harvest then cells by centrifugation at 12,000 x g for 5 s at room temperature using rotor FA-48-45-11.
    16. Resuspend cells in 200 μl of 1x TE buffer and spread 100 μl of the transformation mixture on a SD/-Trp agar plate.
    17. Incubate the agar plate at 30 °C for 2-4 days until emergence of colonies. 
    18. Pick a single colony from the transformation plate using a sterile pipette tip and inoculate 5 ml of YPDA medium in a 14 ml Falcon tube at 30 °C and 250 rpm overnight.
    19. Add 250 μl of 80% (v/v) glycerol to 750 μl of the overnight culture in a 1.5 ml Eppendorf tube and mix well by vortexing for 5 s.
    20. Flash freeze the tube in liquid nitrogen and store the glycerol stock at -80 °C for future use.

  2. Large scale expression of His-BCM1 in S. cerevisiae
    1. Streak out a glycerol stock of transformed S. cerevisiae L40ccua on a SD/-Trp agar plate and incubate the plate at 30 °C for two days.
    2. Inoculate 50 ml of SD/-Trp medium with 2-3 colonies of S. cerevisiae L40ccua from Step B1 in a 200 ml Erlenmeyer flask and grow at 30 °C and 250 rpm overnight.
    3. Transfer the overnight culture to 1 L of SD/-Trp medium in a 2 L Erlenmeyer flask and grow at 30 °C and 250 rpm for 3-4 h until the OD600 of the culture reaches between 0.4-0.6.
    4. Harvest cells by centrifugation at 1,000 x g for 5 min at 4 °C using rotor FA-48-45-11.
    5. Flash freeze the pellet in liquid nitrogen and store at -80 °C for future use.
    6. Analyze the expression of His-BCM1 in S. cerevisiae cells by immunoblotting (Figure 2).


      Figure 2. Immunoblotting analysis of His-BCM1 expression in S. cerevisiae. Lane 1: total lysate of untransformed S. cerevisiae cells. Lane 2: total lysate of transformed S. cerevisiae cells. Total yeast proteins were stained with Coomassie Brilliant Blue (CBB, upper panel) and probed with BCM1 antibody (lower panel), respectively. The immunoblotting analysis was conducted as described previously (Wang et al., 2020).

  3. Purification of His-BCM1 from total yeast membrane extracts
    1. Thaw the S. cerevisiae cell pellet obtained in Step B5 on ice.
    2. Resuspend the cells in chilled 20 ml of 1x phosphate-buffered saline (PBS) buffer supplemented with 2 tablets of protease inhibitor.
    3. Add 3 g chilled glass beads and vortex at maximum speed 5-7 times for 1 min, each time keeping the cells on ice for 1 min between vortexing.
    4. Remove unbroken yeast cells and glass beads by centrifugation at 1,000 x g for 5 min at 4 °C using rotor JA-25.50.
    5. Transfer supernatant to a clean 14-ml ultracentrifuge tube using a plastic pipette and pellet total yeast membranes by ultracentrifugation at 40,000 x g for 30 min at 4 °C using rotor SW 40-Ti.
    6. Resuspend the yeast membrane pellet in 10 ml of chilled solubilization buffer by gentle pipetting for 5-10 times.
    7. Incubate the suspension on ice at 100 rpm for 30 min.
    8. Pellet insoluble debris by centrifugation at 40,000 x g for 30 min at 4 °C using rotor SW 40-Ti and transfer the supernatant to a clean 50 ml tube.
    9. Add 1 ml of the Ni-NTA agarose resin (pre-equilibrated with solubilization buffer) in the supernatant and mix on a roller mixer at 4 °C for 1 h.
    10. Transfer the suspension to a 10 ml disposable plastic spin column and let the suspension run through by gravity.
    11. Wash the Ni-NTA agarose resin with 10 ml of wash buffer and let the wash buffer run through by gravity.
    12. Incubate Ni-NTA agarose resin with 2 ml of elution buffer on ice for 1-2 min and collect then flow through.
    13. Remove aggregates in the elute by centrifugation at 20,000 x g for 10 min at 4 °C using rotor FA-48-45-11.
    14. Dialyse the elute against 1 L of dialysis buffer at 4 °C for 1 h. Change the dialysis buffer and dialyse at 4 °C overnight.
    15. Transfer the elute to clean 1.5 ml Eppendorf tubes.
    16. Add 50 μl of glycerol to 1 ml of the elute and mix them well by gentle pipetting for 3-5 times.
    17. Divide the protein solution into 100 μl aliquots on ice.
    18. Flash freeze the aliquots in liquid nitrogen and store at -80 °C for future use.
    19. Analyze the purified His-BCM1 protein using 12% SDS-PAGE (Figure 3).


      Figure 3. SDS-PAGE analysis of purified His-BCM1. Lane M: protein marker; Lane 1: Purified His-BCM1 (approximately 36.5 kDa) indicated by the arrow; Lanes 2-4: 0.5, 1, and 2 μg bovine serum albumin standard.

Notes

  1. It is necessary to boil salmon sperm DNA at 100 °C for 5 min and chill immediately on ice before use.
  2. To induce the expression of pDR296-His-BCM1 in S. cerevisiae, we use the SD/-Trp medium instead of YPDA medium.

Recipes

  1. Media and stock solution
    1. YPD medium (930 ml)
      1. Dissolve 10 g Bacto yeast extract and 20 g Bacto peptone in 930 ml of demineralized water in a 1 L transparent glass media bottle
      2. Add 20 g Agar-Y only for making solid agar plates
      3. Autoclave at 121 °C for 20 min on a liquid cycle
      4. Store for up to 6 months at room temperature 
    2. 40% (w/v) glucose (100 ml)
      1. Dissolve 40 g glycerol in 40 ml of demineralized water in a 50 °C water bath in a 100 ml Erlenmeyer flask
      2. Fill up to 100 ml with demineralized water
      3. Sterilize by filtering through a 0.45 μm filter
      4. Store for up to 1 month at 4 °C
    3. 0.2% (w/v) Adenine sulfate
      1. Dissolve 0.1 g Adenine sulfate in 50 ml of demineralized water in a 100 ml Erlenmeyer flask
      2. Sterilize by filtering through a 0.45 μm filter
      3. This solution is freshly prepared
    4. YPDA medium (1 L)
      1. Add 50 ml of 40% glucose and 20 ml of 0.2% (w/v) Adenine sulfate to 930 ml of YPD medium under aseptic conditions in a 1 L sterilized Erlenmeyer flask
      2. This medium is freshly prepared
    5. SD/-Trp medium (1 L)
      1. Dissolve 20 mg SD/-Trp and 6.7 g YNB in 950 ml of demineralized water in a 1 L transparent glass media bottle
      2. Adjust to pH 5.8 with NaOH
      3. Add 20 g Agar-Y only for making solid agar plates
      4. Autoclave at 121 °C for 20 min on a liquid cycle
      5. Add 50 ml of 40% (w/v) glucose 950 ml of the medium under aseptic conditions
      6. Store for up to 6 months at room temperature
    6. 80% (v/v) glycerol (100 ml)
      1. Add 80 ml of glycerol to 20 ml of demineralized water in a 100 ml transparent glass media bottle
      2. Autoclave at 121 °C for 20 min on a liquid cycle
      3. Store for up to 6 months at room temperature
    7. 10x TE buffer (200 ml)
      1. Dissolve 2.42 g Tris and 0.744 g EDTA·Na2 in 160 ml of demineralized water in a 250 ml transparent glass media bottle
      2. Adjust to pH 8.0 with hydrochloric acid
      3. Fill up to 200 ml with demineralized water
      4. Sterilize by filtering through a 0.45 μm filter
      5. Store for up to 6 months at room temperature 
    8. 1 M LiAc (200 ml)
      1. Dissolve 13.2 g LiAc in 160 ml of demineralized water in a 250 ml transparent glass media bottle
      2. Adjust to pH 7.5 with acetic acid
      3. Fill up to 200 ml with demineralized water
      4. Sterilize by filtering through a 0.45 μm filter
      5. Store for up to 6 months at room temperature
    9. 50% (w/v) PEG4000 (100 ml)
      1. Dissolve 50 g PEG4000 in 50 ml of demineralized water in a 50 °C water bath in a 100 ml transparent glass media bottle
      2. Fill up to 100 ml with demineralized water
      3. Sterilize by filtering through a 0.45 μm filter
      4. Store for up to 3 months at room temperature
    10. 10x PBS buffer (1 L)
      1. Dissolve 80 g NaCl, 2 g KCl, 15.6 g NaH2PO4·2H2O and 2.4 g KH2PO4 in 800 ml of demineralized water in a 1 L transparent glass media bottle
      2. Adjust to pH 7.4
      3. Fill up to 1 L with demineralized water
      4. Autoclave at 121 °C for 20 min on a liquid cycle
      5. Store for up to 6 months at room temperature
    11. 10% (w/v) β-DM
      1. Dissolve 0.1 g β-DM in 1 ml of demineralized water in a 1.5 ml Eppendorf tube
      2. Store for up to 6 months at -20 °C

  2. Buffer
    1. 1x TE buffer (100 ml)
      1. Add 10 ml of 10x TE buffer to 90 ml of demineralized water under aseptic conditions in a 100 ml transparent glass media bottle
      2. This buffer is freshly prepared
    2. TE/LiAc buffer (100 ml)
      1. Add 10 ml of 10x TE buffer and 10 ml of 1 M LiAc to 90 ml of demineralized water in a 100 ml transparent glass media bottle under aspartic conditions
      2. This buffer is freshly prepared
    3. PEG/LiAc buffer (10 ml)
      1. Add 1 ml of 10x TE buffer and 1 ml of 1 M LiAc to 8 ml of 50% (w/v) PEG4000 in a 14 ml Falcon tube under aspartic conditions
      2. This buffer is freshly prepared
    4. Solubilization buffer (10 ml) containing 1% (w/v) β-DM
      1. Add 5 ml of 10% (w/v) β-DM to 45 ml of 1x PBS buffer in a 100 ml transparent glass media bottle
      2. This buffer is freshly prepared
    5. Wash buffer containing 0.0256% (w/v) β-DM and 20 mM imidazole (20 ml)
      1. Dissolve 27.2 mg imidazole to 20 ml of 1x PBS buffer, supplemented with 51.2 μl of 10% (w/v) β-DM in a 50 ml transparent glass media bottle
      2. This buffer is freshly prepared
    6. Elution buffer containing 0.0256% (w/v) β-DM and 300 mM imidazole (20 ml)
      1. Dissolve 0.408 g imidazole to 20 ml of 1x PBS buffer, supplemented with 51.2 μl of 10% (w/v) β-DM in a 50 ml transparent glass media bottle
      2. This buffer is freshly prepared
    7. Dialysis buffer (1 L) containing 0.0256% (w/v) β-DM
      1. Dissolve 0.256 g β-DM in 1 L of 1x PBS buffer in a 1 L transparent glass media bottle
      2. This buffer is freshly prepared

Acknowledgments

This work was funded by the Alexander von Humboldt Foundation to P.W., and the Deutsche Forschungsgemeinschaft to P. W. (WA 4599/2-1), and to B.G. (FOR2092, GR 936/18-1 and SFB TR175, subproject C04). This protocol was adapted from a previously described method (Wang et al., 2020).

Competing interests

We declare no conflicting or competing interests.

References

  1. Brzezowski, P., Richter, A. S. and Grimm, B. (2015). Regulation and function of tetrapyrrole biosynthesis in plants and algae. Biochim Biophys Acta 1847(9): 968-985.
  2. Wang, P., Richter, A. S., Kleeberg, J. R. W., Geimer, S. and Grimm, B. (2020). Post-translational coordination of chlorophyll biosynthesis and breakdown by BCMs maintains chlorophyll homeostasis during leaf development. Nat Commun 11(1): 1254.

简介

[摘要 ] 异源表达和公顷跨膜蛋白的纯化VE 仍然几十年来阻碍了关键酶详述生物化学和结构表征一个挑战小号和它们的相互作用调节在多个代谢途径。上新鉴定进行了深入的研究拟南芥拟南芥叶绿素代谢1(BCM1)的整合膜蛋白BALANCE显示一个通过与相互作用对镁螯合,叶绿素生物合成的第一个酶,所述BCM1的刺激效应基因组中脱开4 (王等等人,2020)。这里 ,我们报告了酿酒酵母中His-tagged BCM1异源表达和纯化的详细和优化方法。˚F ollowing这种方法,我们获得用于本机BCM1 体外酶测定的镁螯合(王等人,2020) 。目前,BCM1的结晶研究正在进行中。这个协议可以适于纯化BCM 1一样从用于酶和结构研究真核生物的跨膜蛋白。

[背景 ] 鉴定翻译后单组的lators其指导LY 调制enzym 一个叶绿素合成的酶的抽动活动可以大大提高我们理解的分子机制,通过该植物保持高效叶绿素叶期间LL合成绿化(Brzezowski 等人,2015年)。然而,叶绿素合成酶及其相互作用蛋白的详细生化分析受到体外重组蛋白可用性的限制。我们最近发现一个叶绿素代谢1(BCM1)的翻译后调节平衡,同时刺激小号叶绿素合成和延迟叶绿素分解,日ERE 被授予叶发育过程中的叶绿素稳态(王等人,2020年)。为了检查BCM1对镁螯合酶(MgCh )(叶绿素生物合成的第一种酶)的酶活性的影响,我们在酿酒酵母中表达和纯化了带His标签的BCM1 。已经显示出BCM1 能够在体外刺激MgCh 活性(Wang 等人,2020)。由于BCM1具有六个跨膜结构域,因此BCM 1 将在此处用作多次通过蛋白的一个例子。因此,我们提供了一个优化的方法表达和纯化BCM 在1 S. CE revisiae 。虽然原核净化系统托管由大肠杆菌已被广泛地用于表达亲水蛋白质从原核和真核生物,过表达的BCM1 在大肠杆菌细胞中导致小号到的积累BCM 1骨料和包涵体而不是在膜正确折叠的蛋白质。与无细胞蛋白表达系统和其他真核表达系统(例如哺乳动物和昆虫表达系统)相比,此处描述的酵母蛋白表达方法能够以高收率和低成本大规模纯化整合膜蛋白。

关键字:跨膜蛋白, BCM1表达与纯化, 酿酒酵母, 拟南芥

材料和试剂
1. 小号terile的枪头      
2. 1.5毫升Eppendorf微量离心管(Eppendorf,目录号:0030121694)      
3. 隼,Ç onical Ç entrifuge 吨ubes50毫升(康宁,目录号:14-432-22)      
4. 0.45 微米过滤器(VWR ,目录号:28145-479)      
5. 14 ml开顶薄壁超透明离心管(Beckman Coulter ,目录号:344060)      
6. D型可分配的塑料旋转柱(Thermo Fisher Scientific,目录号:10220544)      
7. 小号nakeSkin 透析袋10 kDa的截止(赛默飞世尔科技,产品目录号:68100)      
8. 酿酒酵母L40cc ua 菌株      
9. pDR296-His-BCM1 质粒      
10. 鲑鱼精子(Thermo Fisher Scientific,目录号:15632011)   
11. 软化水   
12. Bacto y east 提取物(Cal Roth,目录号:2904)   
13. Bacto 蛋白ept(Cal Roth,目录号:8952 )   
14. ģ lucose一水合物(卡尔罗斯,目录号:6780 )   
15. 甲丹尼恩硫酸盐(Sigma-Aldrich公司,目录号:A3159)   
16. Agar-Y(MP Biomedicals,目录号:4019012)   
17. Y 东氮碱(YNB)和硫酸铵(MP Biomedicals,目录号:4027412)   
18. 合成漏失/ -色氨酸(SD / - 色氨酸)(MP Biomedicals公司,目录号:4511012)   
19. 甘油(Sigma-Aldrich,目录号:G5516 )   
20. 正十二烷基-β-D- 麦芽糖苷(β-DM)(Sigma-Aldrich,目录号:D4641)   
21. 乙二胺四乙酸二钠盐二水合物(EDTA    · Na 2 )(Sigma-Aldrich,目录号:ED 2SS)
22. 三(羟甲基)氨基甲烷(T ris)(Cal Roth,目录号:A411)   
23. 盐酸(VWR),目录号:BDH7204   
24. 氢氧化钠(Fisher Scientific,目录号:BP359-212)   
25. 醋酸锂(LiAc )(Cal Roth,目录号:5447)   
26. 聚乙二醇4000(P EG4000)(Sigma-Aldrich,目录号:8.07490)   
27. 大号iquid氮   
28. 不含E DTA的cOmplete 微型蛋白酶抑制剂片剂(Roche Diagnostics,目录号:11873580001)   
29. ģ 小姑娘珠(425-600 微米)(Sigma-Aldrich公司,目录号:G8772)   
30. 氯化钠(Ñ ACL)(卡尔罗斯,目录号:9265)   
31. 钾氯化物(ķ 氯)(卡尔罗斯,目录号:6781)   
32. 一水磷酸二氢钠(Na H 2 PO 4 · 2H 2 O)(Cal Roth,目录号:T879)   
33. 磷酸二氢钾(K H 2 PO 4 )(Cal Roth,目录号:3904 )   
34. Ni-NTA琼脂糖树脂(Thermo Fisher Scientific,目录号:88223 )   
35. 咪唑(Sigma-Aldrich,目录号:I 2399)   
36. YP D培养基(930毫升)(请参阅食谱)   
37. 40%(w / v)葡萄糖(100 ml)(请参阅食谱)   
38. 0.2%(w / v)硫酸腺嘌呤(请参阅食谱)   
39. YP D A 培养基(1升)(请参阅食谱)   
40. SD / -Trp 培养基(1升)(请参阅食谱)   
41. 80%(v / v)甘油(100毫升)(请参阅食谱)   
42. 10 x Tris-EDTA (TE)缓冲液(200毫升)(参见食谱)   
43. 1 M LiAc (200毫升)(请参阅食谱)   
44.50 %(w / v)PEG4000(100毫升)(请参阅食谱)   
45. 10 x 磷酸盐缓冲盐水(PBS )缓冲液(1 L)(请参阅食谱)   
46. 10%(w / v)β-DM(请参阅食谱)   
47. 1 x TE 缓冲区(请参阅食谱)   
48. TE / LiAc 缓冲液(请参阅配方)   
49. 聚乙二醇(PEG)/ LiAc 缓冲液(请参阅食谱)   
50. 含有1%(w / v)β-DM的增溶缓冲液(10毫升)(请参阅食谱)   
51. 洗涤缓冲液,其中含有0.02 56%(w / v)β-DM和20 mM咪唑(20 ml)(请参阅食谱)   
52. Ë 琵琶缓冲含0.0256%(W / V)β-DM(见配方)   

53. d ialysis缓冲器含有0.0256%(1 L)(W / V)β-DM(见配方)   


设备
玻璃器皿
250 ml锥形瓶
2 0 0 ml锥形瓶
2 L 锥形瓶
1 L 锥形瓶
100 ml锥形瓶
1 L透明玻璃瓶
25 0 ml透明玻璃瓶
100 ml透明玻璃瓶
转子JA-25.50
转子FA-48-45-11
Milli-Q参考净水系统(EMD Millipore,目录号:Z00QSV0WW)
无尘工作台(Labconco ,型号:3440009)
水浴
温控振荡器培养箱
涡旋混合器(科学工业公司,型号:Vortex-Genie 2)
C 老房
Dee p冷冻机(-20°C)
超低温冰箱(-80°C)
离心机(Eppe ndorf,型号:5430 R)
高速离心机(Beckman Coulter公司,型号:阿凡提J-26XP)
Ul 微量离心机(贝克曼库尔特公司,型号:Optima L-80 XP )
 
程序
变换的S. CE revisiae 与pDR296-HIS-BCM1 质粒
Ť 他的核苷酸序列编码的成熟BCM1 (具有耗尽氨基酸55-382)ñ 末端叶绿体转运肽从拟南芥我š 克隆到酵母表达载体pDR29 6 。该构建体包含N端组氨酸(His)标签,以利于通过固定的镍亲和色谱法纯化BCM1(图1)。

图1 。pDR296-BCM1的质粒图谱。指出了质粒中独特的限制性位点和重要特征。PMA1启动子,转录促销之三为酿酒酵母质膜ATP酶1(PMA1)基因。ADH1终止子,酿酒酵母酒精脱氢酶1(ADH1)基因的转录终止子。2 μ ORI ,复制的酵母2μ质粒起点。TRP1,色氨酸生物合成蛋白。AmpR ,编码β-内酰胺酶的基因,赋予对氨苄青霉素的抗性。

条纹出甘油STOC 的k个S. 酵母L40cc UA 上一个YPDA琼脂平板并孵育在30℃下将板两天。
接种5ml YPDA介质与1个菌落的酿酒酵母L40cc UA 在一个14毫升˚F 爱尔康管并生长在30℃和250rpm下过夜。
将过夜培养物转移到250 ml锥形瓶中的100 ml YPDA培养基中,并继续在30°C和250 rpm下生长2-4 h,直到培养物在600 nm(OD 600 )处的光密度达到0.4-0.6 。
使用转子JA-25.50在室温下以1,000 xg离心5分钟以收获细胞。
ř esuspend细胞在软化水中由v ø rtexing 5-10 s,在1000,通过离心收获细胞,然后XG 使用转子JA-25.50在室温下5分钟。
在1.5 ml TE / LiAc 缓冲液中重悬细胞(细胞滴度为〜3 x 10 9 细胞/ ml )。这些都是现在的酵母感受态细胞。
添加0.1-0.2 μ 克pDR296-HIS-BCM1 质粒,10 微升变性的鲑精DNA中,100 微升酵母感受态细胞到1.5ml Eppendorf管中,并通过将它们充分混合移液2-3次。
向试管中加入600μl 新鲜制备的PEG / LiAc 缓冲液,并通过涡旋充分混合5秒钟。
我ncubate 的在30℃下混合物30分钟以200rpm振荡。
向培养物中添加70μlDMSO ,并轻轻颠倒混合均匀。
H 将试管在42°C水浴中吃15分钟,不要摇晃。
将试管放在冰上1-2分钟。
通过使用转子FA-48-45-11 在室温下以12,000 xg离心 5 s 来沉淀细胞。
ř esuspend细胞在软化水中,并在12000通过离心收获细胞,然后XG 在室温下5秒使用转子FA-48-45-11。
ř esuspend细胞200 微升1的X TE 缓冲液和扩散100 微升上的SD /转化混合物的- 色氨酸琼脂平板上。
将琼脂平板在30°C下孵育2-4天,直到出现菌落。
挑单个栏从变换板使用无菌移液管尖端和接种5ml YPDA介质的ONY 在一个14毫升˚F 在30爱尔康管℃下和250rpm下过夜。
甲DD 250 微升的80%(V / V)甘油,以750 微升在1.5ml Eppendorf管中过夜培养,并通过搅拌均匀涡旋荷兰国际集团为5秒。
闪光冻结管在液氮中,小号撕的甘油原液在-80℃下以供将来使用。
酿酒酵母中His-BCM1的大规模表达
条纹化转化S 的甘油储备。酵母L40cc UA上的SD / - 色氨酸琼脂板上并在30℃下孵育板两天。
接种5 0 ml的SD的/ - 色氨酸与2-介质3 结肠IES ö ˚F 小号。在2 0 0 ml锥形瓶中,从步骤B1 中提取啤酒L40cc ua,并在30°C和250 rpm下生长过夜。
Ť 转让(BOT)的过夜培养至1L的SD / - 色氨酸介质在2 大号Erlenmeyer烧瓶中并在30℃下生长,并以250rpm 3- 4 小时,直到OD 600 0.4-0.6之间的培养物达到的。
通过使用转子FA-48-45-11 在4°C下以1,000 xg离心5分钟收获细胞。
闪光冷冻在液氮中并储存沉淀在-80℃下以供将来使用。
通过免疫印迹分析His -BCM1在酿酒酵母细胞中的表达(图2 )。
 
图2 。免疫印迹婷分析的His-B的在CM1表达小号。酿酒酵母。泳道1:吨otal 裂解物未转化的小号。啤酒酵母细胞。泳道2:吨转化的裂解物otal 小号。啤酒酵母细胞。将总酵母蛋白用考马斯亮蓝(CBB,上图)染色,并用BCM 1抗体(下图)进行探测。如先前所述进行免疫印迹分析(Wang 等,2020)。

从总酵母膜提取物中纯化His-BCM1
在冰上解冻在步骤B5中获得的酿酒酵母细胞沉淀。
将细胞重悬于冰冷的20 ml 1 x 磷酸盐缓冲盐水(PBS)缓冲液中,并补充2片蛋白酶抑制剂。
加入3 g 冷冻玻璃珠,并以最大速度涡旋5-7次,每次1分钟,每次涡旋之间将细胞在冰上保持1分钟。
使用转子JA-25.50在4°C下以1,000 xg离心5分钟,以除去未破碎的酵母细胞和玻璃珠。
Ť 转让(BOT)的上清液,使用塑料吸管和一个干净的14毫升的超速离心管沉淀通过超速离心总酵母膜以40,000 ×g下在4℃下30分钟℃下使用转子SW 40的Ti。
通过轻轻吸打5-10次,将酵母膜沉淀物重悬于10 ml冷冻增溶缓冲液中。
我将悬浮液在冰上以100 rpm的速度孵育30分钟。
P 以40,000 ellet不溶性碎片通过离心XG 为30分钟,在4℃下使用转子SW 40-Ti和上清液转移到一个干净的50ml管中。
在上清液中加入dd 1 ml的Ni-NTA琼脂糖树脂(用增溶缓冲液预先平衡),并在4°C的辊式混合器上混合1 h。
将悬浮液转移至10 ml一次性塑料旋转柱中,并使悬浮液在重力作用下通过。
W¯¯ 灰的Ni-NTA琼脂糖以10ml洗涤缓冲液的树脂和让洗涤缓冲运行通过重力。
我ncubate Ni-NTA琼脂糖树脂与在冰上2毫升洗脱缓冲液的1-2分钟和收集然后流过。
使用转子FA-48-45-11 在4°C下以20,000 xg离心10分钟,以除去洗脱液中的聚集体。
d ialyse 的洗脱对1升透析缓冲液在4℃下1个小时。改变透析缓冲液和透析过夜,在4℃ 。
将洗脱液转移至干净的1.5 ml Eppendorf管中。
加入50 微升的甘油至1ml的洗脱液,并将它们混合瓦特ELL通过温和移液3-5次。
在冰上将蛋白质溶液分成100μl 等分试样。
闪冻在液氮中,存储中的等分试样在-80℃下以供将来使用。
使用12%SDS-PAGE 分析纯化的His -BCM1蛋白(图3 )。

图3 。纯化的His-BCM1的SDS-PAGE分析。泳道中号:蛋白质标记; 泳道1:纯化的His-BCM1 (约36.5 kDa的)指示由所述箭头; 泳道2-4:0.5、1和2μg 牛血清白蛋白标准品。

笔记
有必要煮沸的鲑鱼精子DNA,在100℃ 进行5分钟,并在使用前立即冰上冷却。
Ť Ó诱导的表达在pDR296-HIS-BCM1 小号。酿酒厂,我们使用SD / -Trp 培养基代替YPDA培养基。
 
菜谱
媒体和库存解决方案
YP D培养基(930毫升)
溶解1种0克细菌用酵母提取物和20g 细菌用蛋白胨在93 0米升的软化水在1L透明玻璃介质瓶
甲DD20克琼脂-Y仅用于制造固体GAR 板
甲在121℃utoclave 20分钟上的液体循环
商店的长达6个月,在室温下
40%(w / v)葡萄糖(100毫升)
溶解4 0克克lycerol在40毫升的软化水中50℃的水浴中的100ml的Erlenmeyer 烧瓶
用去离子水最多填充100毫升
通过经由0.45过滤消毒微米过滤器
商店为长达1个月,在4℃
0.2%(w / v)硫酸腺嘌呤
解散0 。1 克腺嘌呤苏升命运5 0毫升软化水的ë ralized水在100毫升的锥形烧瓶
通过经由0.45过滤消毒微米过滤器
这是新鲜配制的溶液
YP D A 培养基(1升)
在1 L 无菌锥形瓶中,在无菌条件下,向930 ml的YPD 培养基中添加50 ml的40%葡萄糖和20 ml的0.2%(w / v)硫酸腺嘌呤。
该培养基是新鲜制备的
SD / -Trp 培养基(1升)
d issolve 20毫克SD / - 色氨酸和6.7克YNB在9 5 0 ml的软化水在1L透明玻璃介质瓶
用NaOH调节至pH 5.8
甲DD20克琼脂-Y仅用于制造固体GAR 板
甲在121℃utoclave 20分钟上的液体循环
在无菌条件下添加50 ml 40%(w / v)葡萄糖9 5 0 ml 培养基
商店的长达6个月,在室温下
80%(v / v)甘油(100毫升)
80米添加升甘油到20ml的软化水中的100毫升的透明玻璃介质瓶
甲在121℃utoclave 20分钟上的液体循环
商店的长达6个月,在室温下
10 x TE 缓冲液(200毫升)
将2402毫升Tris和0.744克EDTA · Na 2 溶解在25 毫升0ml透明玻璃瓶中的160毫升去离子水中
用盐酸调节至pH 8.0
用去离子水加满200毫升
通过经由0.45过滤消毒微米过滤器
商店的长达6个月,在室温下
1 M LiAc (200毫升)
在25 0 ml透明玻璃瓶中将13.2 g LiAc 溶于160 ml去离子水中
调节至pH 7.5与ACET 我Ç 酸
用去离子水加满200毫升
通过经由0.45过滤消毒微米过滤器
商店的长达6个月,在室温下
50%(w / v)PEG4000(100毫升)
在100 ml透明玻璃介质瓶中的50°C水浴中,将50 g PEG4000溶解在50 ml的去离子水中
用去离子水最多填充100毫升
通过经由0.45过滤消毒微米过滤器
商店的长达3个月,在室温下
10x PBS 缓冲液(1 L)
溶解80克的NaCl,2g的氯化钾,15.6克的NaH 2 PO 4 · 2 ħ 2 O和2 4克ķ ħ 2 PO 4 在800 毫升软化水中在1升的透明玻璃介质瓶
调整至pH 7.4
填补了1 大号用软化水
甲在121℃utoclave 20分钟上的液体循环
商店的长达6个月,在室温下
10%(w / v)β-DM
d issolve 0.1 Gβ-DM 1 ml的去离子水中在1.5米升微量离心管
商店的长达6个月- 20 ℃,
 
缓冲
1 x TE 缓冲液(100毫升)
在无菌条件下,在100 ml透明玻璃培养基瓶中,将10 ml的10 x TE 缓冲液添加到90 ml的去离子水中
该缓冲区是新准备的
TE / LiAc 缓冲液(100毫升)
在天冬氨酸条件下,在100 ml透明玻璃介质瓶中,将10 ml的10 x TE 缓冲液和10 ml的1 M LiAc 加到90 ml的去离子水中。
该缓冲区是新准备的
PEG / LiAc 缓冲液(10毫升)
在天冬氨酸条件下在14 ml F alcon管中将1 ml 10 x TE 缓冲液和1 ml 1 M LiAc 加到8 ml 50%(w / v)PEG4000 中
该缓冲区是新准备的
含有1%(w / v)β-DM的溶解缓冲液(10 ml)
在100 ml透明玻璃培养基瓶中的45 ml 1 x PBS缓冲液中加入5 ml 10%(w / v)β-DM
该缓冲区是新准备的
含有0.0256%(w / v)β-DM和20 mM咪唑(20 ml)的洗涤缓冲液
溶解27.2毫克咪唑至20ml 1 X PBS缓冲液,补充有51.2 微升的10%(W / V)β-DM在50ml透明玻璃介质瓶
该缓冲区是新准备的
含有0.0256%(w / v)β-DM和300 mM咪唑(20 ml)的洗脱缓冲液
溶解0.408 克咪唑至20ml 1 X PBS缓冲液,补充有51.2 微升的10%(W / V)β-DM在50ml透明玻璃介质瓶
该缓冲区是新准备的
含有0.0256%(w / v)β-DM的透析缓冲液(1 L)
将0.256 gβ-DM溶解于1 L透明玻璃培养基瓶中的1 L 1 x PBS缓冲液中
该缓冲区是新准备的
 
致谢
这项工作是由亚历山大·冯·洪堡基金会(Alexander von Humboldt Foundation)向PW资助的,德国Deutsche Forschungsgemeinschaft 向PW(WA 4599 / 2-1)和BG(FOR2092,GR 936 / 18-1和SFB TR175,子项目C04)资助的。该协议改编自先前描述的方法(Wang 等,2020)。

利益争夺

我们声明没有利益冲突或利益冲突。

参考文献
Brzezowski,P.,Richter,AS和Grimm,B.(2015)。植物和藻类中四吡咯生物合成的调控和功能。Biochim Biophys Acta 1847(9):968-985。
Wang,P.,Richter,AS,Kleeberg,JRW,Geimer,S.和Grimm,B.(2020年)。叶绿素生物合成的翻译后协调和BCM的分解在叶片发育过程中维持叶绿素稳态。Nat Commun 11(1):1254。
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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, P. and Grimm, B. (2020). Expression and Purification of Arabidopsis Transmembrane Protein BCM1 in Saccharomyces cerevisiae. Bio-protocol 10(18): e3758. DOI: 10.21769/BioProtoc.3758.
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