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Feb 2019

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In vitro and in vivo Assessment of Protein Acetylation Status in Mycobacteria
分枝杆菌中蛋白乙酰化状态的体外和体内检测   

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Abstract

Protein acetylation is one of the standard post-translational modifications found in proteins across all organisms, along with phosphorylation which regulates diverse cellular processes. Acetylation of proteins can be enzymatically catalyzed through acetyltransferases, acetyl CoA synthetases or non-enzymatically through acyl carrier metabolic intermediates. In this protocol, using response regulator proteins as targets we describe the experimental strategy for probing the occurrence of acetylation using purified recombinant proteins in an in vitro setup. Further using M. smegmatis strains overexpressing the wild type or mutant response regulator protein, we also describe how in vivo acetylation can be validated in Mycobacterial proteins. The described approach can be used for analyzing acetylation of any mycobacterial protein under both in vitro and in vivo conditions.

Keywords: TcrX (TcrX), Response regulator (反应调节子), Acetylation (乙酰化), KATms (KATms), M. tuberculosis (卡玛肺结核), Two-component signaling (双组份信号传导系统), Post-translational modification (翻译后调节)

Background

Lysine acetylation is a typical post-translational modification (PTMs) found to be present in proteins across all living organisms. It involves covalent attachment of an acetyl group from acetyl donor, e.g., acetyl phosphate, acetyl CoA, acetate, acetyladenylate, etc. on to the ε-NH2 group of the amino acid lysine in an acceptor protein. Protein acetylation has been extensively studied in context to the histone modification, and for many transcription factors and is associated with regulating chromatin remodeling to cell signaling, antibiotic resistance, environmental stress survival, and metabolism.

While many recent studies have revealed the presence of acetylated proteins in prokaryotes using global proteome analysis approaches, there have been limited attempts to study the impact of acetylation on the function of identified protein due to multistep validation needed for acetylated proteins. For Mycobacterium tuberculosis, which is a slow-growing bacterium, development of highly sensitive mass spectrometry approaches facilitated total acetylome analysis (Liu et al., 2014; Xie et al., 2015), which included many signaling proteins, thus warranting detailed mechanistic analysis of the impact of the identified modification. Recently, the effect of acetylation on the activity of two-component signaling protein DosR in the hypoxic response of Mycobacterium tuberculosis was reported (Bi et al., 2018; Yang et al., 2018). And more recently we demonstrated that acetylation of the response regulator TcrX alters the crosstalk known to be present in two-component signaling systems of M. tuberculosis as well as phosphatase activity of the sensor kinase and DNA binding activity of the response regulator (Singh et al., 2019). During the course of this study, we developed an optimized protocol for in vitro as well as in vivo acetylation analysis of various target proteins which is described here, using the response regulator TcrX as a template. This protocol can be used for testing acetylation status of any mycobacterial protein and involves two steps, first, where the presence of acetylation is probed in vitro in an enzymatically catalyzed reaction and second, the protein is probed for acetylation presence in vivo using M. smegmatis mc2155 as a surrogate host. Utilization of a protein carrying a Lys substitution mutation in vivo helps confirm that the identified Lys is indeed the target acetylation site.

Materials and Reagents

  1. Pipette tips
    1. 1 ml tip (Tarsons, catalog number: 521020)
    2. 2-200 µl (Tarsons, catalog number: 521010)
    3. 0.2-10 µl (Tarsons, catalog number: 521000)
  2. 1.5 ml microcentrifuge tubes (Tarsons, catalog number: 500010)
  3. 15 ml polypropylene centrifuge tubes (Tarsons, catalog number: 546021)
  4. 50 ml polypropylene centrifuge tubes (Tarsons, catalog number: 546041)
  5. 0.1 mm diameter zirconia/silica beads (Thomas Scientific, catalog number: 3411F09) 
  6. 2 ml polypropylene bead beating vials (Bio Spec Products Inc., catalog number: 10831)
  7. PVDF membrane (Bio-Rad, catalog numbers: 1620177 or 1620238)
  8. Whatman filter paper 1.5 MM (GE Healthcare, catalog number: 10426981)
  9. 0.45 µm filters (Sartorius Minisart syringe filter, catalog number:16555-K)
  10. 96-well Micro test plate (Tarsons, catalog number: 941196)
  11. Expression vector with affinity tag for recombinant protein overexpression and purification (pProEx-Ht vector contains 6x His-tag at the N-terminal has been used for overexpression and purification of TcrX response regulator protein (Agrawal et al., 2015) as well as KATms (acetyl transferase) was a kind gift from Prof. Sandhya Visweswariah, IISc (Nambi et al., 2010)
  12. Expression strain E. coli BL21 Arctic ExpressTM [B F ompT hsdS(rB mB) dcm+ Tetr gal λ(DE3) endA Hte [cpn10 cpn60 Gentr] (Ferrer et al., 2003) (Agilent Technologies, USA, catalog number: 230191) used for overexpression of TcrX and SP850cyc- strain for KATms as reported (Nambi et al., 2010)
  13. Mycobacterium smegmatis mc2155 (ATCC, catalog number: 700084) 
  14. pMV261 vector (Received as a kind gift from Prof. Dipankar Chatterji, IISc)
  15. pMV261 containing a gene coding for TcrX (Singh et al., 2019) or your gene of interest
  16. pMV261 containing a gene coding for TcrX K231R (Singh et al., 2019) or your gene of interest
  17. Middlebrook 7H9 medium (BD Biosciences, catalog number: 271310)
  18. Glycerol (Sisco Research Ltd., catalog number: 62417) 
  19. Tween 80 (Sigma-Aldrich, catalog number: P4780) 
  20. Tween 20 (Sigma-Aldrich, catalog number: P9416)
  21. Tyloxapol (Sigma-Aldrich, catalog number: T8761)
  22. Kanamycin (Goldbio, catalog number: K-120)
  23. Oleic acid (Sigma-Aldrich, catalog number: 75090)
  24. Albumin (Amersco, catalog number: 0332)
  25. Dextrose (Sisco Research Ltd., catalog number: 51758)
  26. Catalase (Sigma-Aldrich, catalog number: C9322)
  27. Coomassie Brilliant Blue G-250 (Sisco Research Ltd., catalog number: 64222)
  28. Methanol (Sisco Research Ltd., catalog number: 96446)
  29. Phosphoric Acid (Alfa Aesar, catalog number: A18067)
  30. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144SI-212) 
  31. Sodium chloride (NaCl) (Merck Millipore Corporation, catalog number: 1064040500) 
  32. Potassium chloride (KCl) (Merck Millipore Corporation, catalog number: 1049360500) 
  33. Di-sodium hydrogen phosphate (Merck Millipore Corporation, catalog number: 1065860500) 
  34. Potassium dihydrogen phosphate (KH2PO4) (Merck Millipore Corporation, catalog number: 1048730250) 
  35. Trizma base (Sigma-Aldrich, catalog number: T6066) 
  36. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250) 
  37. Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: P7626) 
  38. Benzamidine hydrochloride (Sigma-Aldrich, catalog number: B6506)
  39. 1% Nonidet-P40 (Sigma-Aldrich, CAS: 9016-45-9)
  40. Anti-acetyl antibody (Cell Signaling Technology Inc., catalog number: 9441, Dilution 1:7,500)
  41. HRPO-conjugated secondary antibody (Cell Signaling Technology Inc., catalog number: 7074, Dilution 1:5,000)
  42. Anti-His antibody (Cell Signaling Technology Inc., catalog number: 2365, Dilution 1:5,000)
  43. Anti-TcrX polyclonal antibody (Dilution 1:1,000) (generated in the laboratory [Singh et al., 2019])
  44. Western Lightning PLUS ECL containing enhanced luminol reagent and oxidizing reagent (PerkinElmer, catalog number: NEL100001EA)
  45. SDS (Affymetrix USB, catalog number: 18220)
  46. EDTA (Sisco Research Ltd., catalog number: 40648)
  47. Bromophenol blue (MERCK, catalog number: 1081220005)
  48. cAMP (Sigma-Aldrich, catalog number: A6885)
  49. Acetyl CoA (Sigma-Aldrich, catalog number: A2056)
  50. ADC solution (Albumin, Dextrose, Catalase) (see Recipes)
  51. 10x acetylation Buffer (see Recipes)
  52. cAMP, 5 mM stock (see Recipes) (Sigma-Aldrich, catalog number: A6885)
  53. Acetyl CoA, 5 mM stock (see Recipes) (Sigma-Aldrich, catalog number: A2056)
  54. 7H9-Glycerol-Tween 80 medium (see Recipes)
  55. Recovery Media (see Recipes)
  56. Phosphate buffered saline (see Recipes) 
  57. Lysis buffer (see Recipes)
  58. 5x Bradford Reagent (see Recipes)
  59. Storage buffer (see Recipes)
  60. SDS-PAGE running buffer (see Recipes)
  61. Transfer buffer (see Recipes)
  62. Tris Buffer Saline Tween 20 (TBS-T) (see Recipes)
  63. Stripping Buffer (see Recipes)
  64. 5x Protein sample buffer (see Recipes)

Equipment

  1. Pipettes
    a. 100-1,000 µl pipette (Rainin pipet-lite, catalog number: SL-1000)
    b. 20-200 µl pipette (Rainin pipet-lite, catalog number: SL-200)
    c. 2-20 µl pipette (Rainin pipet-lite, catalog number: SL-20)
    d. 0.1-2 µl pipette (Rainin pipet-lite, catalog number: SL-2)
  2. 2 mm electroporation cuvette (Bio-Rad Laboratories, catalog number: 165-2086)
  3. Mini-Protean gel electrophoresis system (Bio-Rad Laboratories, model: 165-8000)
  4. Mini Trans-Blot cell (Bio-Rad Laboratories, model: Trans-Blot® SD cell)
  5. Gel Doc Imaging System (Bio-Rad Laboratories, model: Universal Hood III)
  6. 37 °C incubator/shaker (N-BIOTEK, model: NB-205L)
  7. Refrigerated centrifuge (Thermo Fisher Scientific, model: Sorvall Legend X1R; Hettich Zentrifugen, model: Rotina 420R)
  8. Spectrophotometer (Eppendorf, BioPhotometer®, model: D30)
  9. Bead beater (Bio Spec Products, model: Mini-Bead beater)
  10. Microcentrifuge (Tomy Kogyo Co. Ltd., model: Kitman-T24)
  11. Vortexes (Shalom Instruments, model: SLM-VM-3000)
  12. Heating block (heats to 95 °C) (Biobee Lab)
  13. Ultra-low temperature freezer (Sanyo, model: MDF-U700VX)
  14. Electroporator (Eppendorf Eporator, catalog number: 4309000019)
  15. Multimode microplate reader (Tecan, model: Infinite M1000 PRO)
  16. Autoclave
  17. -80 °C freezer

Software

  1. ImageJ https://imagej.nih.gov/ij/download.html
  2. Image lab version 5.2.1 software from BIO-RAD (used in ChemiDocTM Gel Documentation system)

Procedure

  1. In vitro acetylation analysis
    The acetylation status of TcrX protein (as a representative) is assessed by probing the protein using an anti-acetyl antibody by Western blot analysis, which is discussed in a stepwise manner in this protocol. Response regulator TcrX (6x his-tagged) and mutant TcrX (K231R) proteins are overexpressed and purified from E. coli BL21 Arctic ExpressTM and KATms (acetyltransferase/MSMEG_5458) from SP850cyc-strain, as previously reported in Singh et al. (2019). The purified proteins are quantitated through Bradford’s protein estimation assay (Stoscheck, 1990; He, 2011a) and the proteins are stored in storage buffer (-20 °C; see Recipes) until use. The protocol below describes the experimental procedure subsequent to protein purification.
    Setting up in vitro acetylation reaction followed by anti-acetyl Western blotting:
    1. Mix the following ingredients in the following amounts:
      TcrX or TcrX K231R proteins, 3 µg
      10x acetylation buffer, 2 μl (see Recipes)
      cAMP, 0.4 μl (see Recipes for stock concentration)
      Acetyl CoA, 0.4 µl (see Recipes for stock concentration)
      KATms, 0.2 µg
      Add autoclaved MQ water to 20 µl 
    2. Incubate at 37 °C for 4 h (the incubation duration might vary between substrate proteins linked to extent of acetylation).
    3. Terminate reactions by diluting the reaction mixture with 5 µl of sample buffer (see Recipes). Heat the sample at 95 °C for 10 min.
    4. Resolve samples using 15% SDS-PAGE (He, 2011b) as per standard protocol at 100 V for 3-4 h.
    5. Transfer the protein to PVDF membrane by electroblotting using the Bio-Rad Mini Trans-Blot cell, as per the manufacturer’s protocol at 10 V for 1 h.
      Notes:
      1. Cut the PVDF membrane from the PVDF roll according to someone’s SDS-PAGE gel size and lanes loaded or use precut preassembled blotting membrane/filter paper sandwich from Bio-Rad.
      2. Transfer of proteins onto PVDF membrane takes 10 V for 1 h for this particular low molecular protein (~30 kDa) in a semidry transfer apparatus (Trans-Blot® SD cell) of Bio-Rad. It should be optimized according to someone’s protein of interest (high or low molecular weight) for voltage, current, timing and type of apparatus (Dry/Semi dry/Wet) to get an efficient transfer.
    6. Block the PVDF membrane with 5% BSA in 1x TBST (30 ml) for 90 min with gentle rocking at room temperature.
    7. Incubate with primary anti-acetyl lysine antibody diluted at 1:7,500 for overnight at 4 °C with gentle rocking. 
    8. Wash the membrane 3 times with 1x TBST (50 ml) for 10 min each with rapid rocking.
    9. Incubate with 1:5,000 diluted secondary antibody for 1 h at room temperature with gentle rocking.
    10. Wash the membrane again 3 times with 1x TBST (60-70 ml) for 10 min each with rapid rocking.
    11. Develop the blot using enhanced chemi-luminescence (ECL) as per the manufacturer’s protocol and image the blot in a Chemidoc gel documentation system (Figure 1).
    12. After developing of PVDF membrane wash the membrane with 1x TBST (50 ml) 3 times for 10 min each.
    13. Incubate the PVDF membrane at 55 °C with stripping buffer (30 ml; see Recipes) for 25-30 min (optimize accordingly).
    14. Pour off the stripping buffer and wash the PVDF membrane 6-7 times with 1x TBST (100 ml) with rapid rocking for 10 min each or β-mercaptoethanol is completely removed.
      Note: Do check for the typical smell of β-mercaptoethanol from PVDF membrane to ensure that it has been completely removed after washing with 1x TBST buffer. Repeat washing until it goes off completely.
    15. Block the PVDF membrane with 5% BSA in 1x TBST (30 ml) for 90 min at room temperature.
    16. Repeat the steps from Step A7 (use anti-his 6x his antibody at a dilution of 1:5,000) until Step A10 for developing the blot and acquire the image as specified in Step A11 (Figure 1). This step provides a reference image for the amount of protein loaded.


      Figure 1. In vitro acetylation assessment. RR proteins TcrX and TcrXK231R were incubated with KATms (acetyltransferase, MSMEG_5458enzyme from M. smegmatis) as per the protocol. Acetylation site defective TcrX protein was generated after site identification by mass spectrometry, as reported in Singh et al. (2019). Top, an image of blot probed with anti-acetyl lysine antibody and bottom, anti-His antibody.

  2. In vivo acetylation analysis of TcrX protein
    In vivo assessment of acetylation of a specific protein requires it to be overexpressed in Mycobacterial cells. Towards this, the typical approach involves cloning and expression of the ORF under a strong mycobacterial promoter like hsp60 or msp12 in a mycobacterial shuttle vector. Introduce the plasmid in Mycobacterial strains such as M. smegmatis and then analyze the expression or modification status of the overexpressed protein. The following steps describe the entire workflow towards that.
    1. Preparation for electrocompetent Mycobacterium smegmatis mc2155
      Mycobacterium smegmatis mc2155 strain is made electrocompetent to introduce the recombinant pMV261 mycobacterial plasmids carrying a gene of interest. 
      1. Prepare 1% primary culture by inoculating 50 µl of Mycobacterium smegmatis mc2155 stock in 5 ml of Middlebrook 7H9 media containing 0.2% glycerol, 0.05% Tween-80 and 10% ADC.
      2. Incubate the culture at 37 °C in the shaker at 180 rpm until OD600 ~2 is obtained (approximately 2 days) 
      3. Inoculate 1 ml of the primary culture to 100 ml of 7H9 broth supplemented with 0.2% glycerol, 0.05% Tween-80 and 10% ADC and grow at 37 °C in the shaker at 180 rpm until the OD600 of 0.8-1.0 (~24-32 h).
      4. Pellet the cells at 4,500 x g, 4 °C for 15 min, followed by 3-4 washes in 20 ml of autoclaved MilliQ water.
      5. This is followed by 3 washes with 20 ml of sterile 10% glycerol. 
      6. Pellet the cells and resuspend in 2 ml of sterile 10% glycerol.
      7. Prepare aliquots of 200 μl each for electroporation in the 1.5 ml centrifugation tubes.
      8. Spot freeze the remaining aliquots of electrocompetent cells in 1.5 ml centrifugation tubes using pre-chilled 100% ethanol and store them at -80 °C until further use.
        Note: Keep a container filled with 100% ethanol at -80 °C for 6-8 h prior to start the preparation of electrocompetent cells. When the electrocompetent cells aliquots are ready in microcentrifuge tubes then dip the tubes in the container containing the prechilled ethanol before storage at -80 °C.
    2. Electroporation of plasmid DNA in M. smegmatis mc2155
      1. To an aliquot of electrocompetent cells add 1 µg of the recombinant plasmids and incubate on ice for 2 min.
      2. Prechill 2 mm electroporation cuvette on ice, add electrocompetent cells and the plasmid mixture into the cuvette and incubate on ice for 10 min. Wipe the outer walls of the cuvette and pulse at 2,500 V using an electroporator and immediately keep on ice.
        Note: At the end of electroporation, ~4-5 millisecond time can be observed on display of electroporator which good indication of correct current flow between the two parallel electrodes and efficient electroporation. If arcing takes place, timing is in seconds on display and set voltage differs from applied voltage on the cells during the electroporation. This is possibly due to high ionic strength of buffer, high salt or insufficient washing of cells which ultimately results in ineffective or no electroporation. 
      3. Add 200 µl recovery media (see Recipes) to the cuvette and transfer cells to a 15 ml conical tube. Add more media to bring the volume to 3 ml and shake the tube for 4 h at 37 °C at 180 rpm in shaker incubator for recovery of cells.
        Note: Keep the recovery media (see Recipes) in excess (20 ml) ready before electroporation. 
      4. Harvest the cells at 4,500 x g for 10 min at room temperature. Discard the supernatant and resuspend pellet in 100 µl of recovery media.
      5. Plate cells on 7H11 media (agar base) supplemented with 0.2% glycerol, 0.05% Tween 80, 10% ADC containing appropriate antibiotics (here 25 µg/ml kanamycin).
      6. Incubate the plate at 37 °C for 2 days to obtain transformants.
    3. Lysate preparation and in-vivo expression and acetylation status analysis 
      1. For starter culture, inoculate single colony each of the recombinant strains in 5 ml of 7H9 media containing 0.2% glycerol, 0.05% Tween 80 and 10% ADC along with 25 µg/ml kanamycin and incubate it at 37 °C at 180 rpm in a shaker for 2 days. 
      2. Subculture 0.5-1 ml of the primary culture in 50 ml of 7H9 broth supplemented with 0.2% glycerol, 0.05% tyloxapol and 10% ADC along with 25 µg/ml kanamycin and grow at 37 °C in the shaker at 180 rpm.
        Note: When a large volume of secondary culture (e.g., 1/2 liter) is needed, primarily to get good amount of protein, primary culture should also be of a larger volume (10-50 ml). 
      3. Once OD600 reaches ~2-3 (approximately 2-3 days), pellet the cells at 4 °C at 4,500 x g, wash 3 times each with 10 ml of sterile PBS and resuspend pellet in 500 µl of Tris lysis buffer (pH ~7.5).
      4. Transfer the suspension in 2 ml bead beating vial containing 0.1 mm diameter zirconia/silica beads (~250 µl volume of buffer) and bead beat for 30 s, 8-10 times with intermittent incubation on ice (optimize according to the instructions from the bead homogenizer manufacturer).
      5. Centrifuge the lysate for 10 min at 4 °C at 10,000 x g and collect ~400 µl of supernatant. Care should be taken to avoid collecting the unlysed cells and debris.
      6. Quantify the lysate by Bradford’s protein estimation assay.
      7. Resolve 100 µg of protein lysate by 15% SDS-PAGE as per standard protocol.
      8. Transfer the resolved protein to PVDF membrane by electroblotting as per the manufacturer’s protocol and proceed for Western blotting with anti-acetyl lysine antibody as described in the section above, followed by stripping and probing with protein-specific antibody (here anti-TcrX).
      9. Figure 2 depicts a representative developed blot reporting the overexpression of candidate response regulator protein TcrX and its acetylation status through specific antibodies. The blot also shows an absence of acetylation when a lysine mutant protein is used, confirming the site which undergoes acetylation in vivo.


        Figure 2. In vivo acetylation analysis. M. smegmatis mc2155 strains carrying either vector pMV261 alone or expressing wild type TcrX or its mutant protein were cultured in Middlebrook 7H9 medium (7H9). A total of 100 µg of total protein was analyzed by Western blotting using anti-acetyl lysine (top) and anti-TcrX (bottom) antibodies. Arrow represents the TcrX protein.

Notes

  1. Care should be taken while preparing M. smegmatis cell lysates. The lysis step involving the bead-beating have to be optimized based on the homogenizer which might vary in intensity and beating frequency. 
  2. Dilution of anti-lysine antibody should be optimized as it might vary between batch and batch.

Recipes

  1. ADC solution (Albumin Dextrose Catalase) (100 ml)
    Albumin
    5 g
    Dextrose
    2 g
    Catalase
    3 mg
    Dissolve albumin, dextrose and catalase in ~50 ml MilliQ water (~50 ml) and then make up the volume to 100 ml. Filter sterilize with 0.45-micron filters.
  2. 10x acetylation buffer (1 ml)
    Tris (pH ~7.4)
    250 mM
    NaCl
    1,000 mM
  3. cAMP, 5 mM stock (1 ml)
    Dissolve 1.84 mg of powder in ~0.5 ml MilliQ water till it completely dissolves and then make up the volume to 1 ml
  4. Acetyl CoA, 5 mM stock (1 ml)
    Dissolve 4.046 mg of Acetyl CoA powder in ~0.5 ml MilliQ water till it completely dissolves and then make up the volume to 1 ml
  5. 7H9-Glycerol-Tween 80 medium (90 ml)
    7H9 powder
    0.47 g
    Glycerol (50%)
    400 µl
    Tween 80 (20%)
    250 µl
    1. Dissolve 0.47 g of 7H9 powder in ~50 ml MilliQ water
    2. Add glycerol and Tween 80 as mentioned above and make up the total volume to 90 ml with MilliQ water
    3. Sterilize by autoclaving and store at RT
    Note: Since glycerol and Tween 80 are viscous, they are diluted to above-mentioned stock percentages from a 100% stock in water, sterilized by autoclaving and stored at RT. 
  6. Recovery Media (7H9-Glycerol-Tween 80-ADC) (20 ml)
    7H9
    17.87 ml
    ADC
    2 ml
    Glycerol (50%)
    80 µl
    Tween 80 (20%)
    50 µl
  7. Phosphate Buffer Saline (1,000 ml)
    NaCl
    8 g
    KCl
    0.2 g
    Na2HPO4
    1.44 g
    KH2PO4
    0.24 g
    Sterilize by autoclaving and store at RT
  8. Lysis Buffer (5 ml)
    Tris-Cl (pH ~7.4)
    20 mM
    NaCl
    100 mM
    Glycerol
    10%
    PMSF
    1 mM
    Benzamidine Hydrochloride
    1 mM
  9. 5x Bradford Reagent (100 ml)
    Coomassie Brilliant Blue G-250
    50 mg
    Methanol (100%)
    23.5 ml
    Phosphoric Acid (85%)
    50 ml
    Make up the volume to 100 ml by adding MilliQ water
  10. Storage buffer (500 ml)
    Tris-HCl, pH 8.0
    50 mM
    Glycerol
    50%
    NaCl
    50 mM
    DTT
    1 mM
  11. SDS-PAGE Running Buffer (1,000 ml)
    Tris
    3 g
    Glycine
    14.4 g
    SDS
    1 g
    Make up the volume by adding MilliQ water
    Note: SDS-PAGE running buffer will be of pH ~8.2-8.5.
  12. Transfer Buffer (1 L)
    Tris
    3 g
    Glycine
    14.4 g
    Methanol
    200 ml
    Make up the volume to 1 L by adding MilliQ water
    Note: Transfer buffer also will be of pH ~8.2-8.5.
  13. Tris Buffer Saline Tween 20 (TBS-T) (1,000 ml)
    Tris (pH ~7.5)
    10 mM
    NaCl
    153.8 mM
    Tween 20
    0.1%
  14. Stripping Buffer (20 ml)
    Tris-Cl (pH 6.8)
    62.5 mM
    SDS
    2%
    β-mercaptoethanol
    0.7% (140 μl/20 ml)
  15. 5x Protein Sample buffer (10 ml)
    Tris (pH-6.8)
    0.25 M
    Glycerol
    40% (4 ml/10 ml)
    SDS
    10%
    Bromophenol blue
    0.25%
    β-mercaptoethanol
    5% (0.5 ml/10 ml)

Acknowledgments

This work was supported by Department of Biotechnology, India (Grant Nos. BT/PR17357/MED/29/1019/2016). The study was also supported in part by the DBT partnership program to Indian Institute of Science (DBT/BF/PRIns/2011-12) and Infosys Foundation; Equipment support by DST–Funds for Infrastructure in Science and Technology program (SR/FST/LSII-036/2016).

Competing interests

Authors declare no competing interests.

References

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  2. Bi, J., Gou, Z., Zhou, F., Chen, Y., Gan, J., Liu, J., Wang, H. and Zhang, X. (2018). Acetylation of lysine 182 inhibits the ability of Mycobacterium tuberculosis DosR to bind DNA and regulate gene expression during hypoxia. Emerg Microbes Infect 7(1): 108. 
  3. Ferrer, M., Chernikova, T. N., Yakimov, M. M., Golyshin, P. N. and Timmis, K. N. (2003). Chaperonins govern growth of Escherichia coli at low temperatures. Nat Biotechnol 21(11): 1266-1267.
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  6. Liu, F., Yang, M., Wang, X., Yang, S., Gu, J., Zhou, J., Zhang, X. E., Deng, J. and Ge, F. (2014). Acetylome analysis reveals diverse functions of lysine acetylation in Mycobacterium tuberculosis. Mol Cell Proteomics 13(12): 3352-3366.
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  8. Singh, K. K., Bhardwaj, N., Sankhe, G. D., Udaykumar, N., Singh, R., Malhotra, V. and Saini, D. K. (2019). Acetylation of response regulator proteins, TcrX and MtrA in M. tuberculosis tunes their phosphotransfer ability and modulates two-component signaling crosstalk. J Mol Biol 431(4): 777-793.
  9. Stoscheck, C. M. (1990). Quantitation of protein. Methods Enzymol 182: 50-68.
  10. Xie, L., Wang, X., Zeng, J., Zhou, M., Duan, X., Li, Q., Zhang, Z., Luo, H., Pang, L., Li, W., Liao, G., Yu, X., Li, Y., Huang, H. and Xie, J. (2015). Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis. Int J Biochem Cell Biol 59: 193-202.
  11. Yang, H., Sha, W., Liu, Z., Tang, T., Liu, H., Qin, L., Cui, Z., Chen, J., Liu, F., Zheng, R., Huang, X., Wang, J., Feng, Y. and Ge, B. (2018). Lysine acetylation of DosR regulates the hypoxia response of Mycobacterium tuberculosis. Emerg Microbes Infect 7(1): 34.

简介

蛋白质乙酰化是在所有生物体中的蛋白质中发现的标准翻译后修饰之一,以及调节不同细胞过程的磷酸化。 蛋白质的乙酰化可以通过乙酰转移酶,乙酰辅酶A合成酶或非酶促通过酰基载体代谢中间体酶促催化。 在该方案中,使用响应调节蛋白作为靶标,我们描述了在体外装置中使用纯化的重组蛋白质探测乙酰化发生的实验策略。 进一步使用 M. 耻垢分子菌株过表达野生型或突变体反应调节蛋白,我们还描述了体内乙酰化如何在分枝杆菌蛋白中得到验证。 所描述的方法可用于分析体外和体内条件下的任何分枝杆菌蛋白质的乙酰化。
【背景】赖氨酸乙酰化是发现存在于所有生物体中的蛋白质中的典型翻译后修饰(PTM)。它涉及乙酰基供体乙酰基的共价连接,例如,,乙酰基磷酸酯,乙酰基CoA,乙酸酯,乙酰基腺苷酸,等。在受体蛋白质中的氨基酸赖氨酸的ε-NH 2 组。蛋白质乙酰化已经在组蛋白修饰和许多转录因子的背景下进行了广泛研究,并且与调节染色质重塑细胞信号传导,抗生素抗性,环境应激存活和代谢有关。

尽管最近的许多研究已经揭示了使用全局蛋白质组分析方法在原核生物中存在乙酰化蛋白质,但由于乙酰化蛋白质所需的多步验证,研究乙酰化对所鉴定蛋白质功能的影响的尝试有限。对于结核分枝杆菌,这是一种生长缓慢的细菌,开发高灵敏度的质谱方法促进了总乙酰分析(Liu et al。,2014; Xie et al。,2015),其中包括许多信号蛋白,因此需要对所识别的修饰的影响进行详细的机械分析。最近,报道了乙酰化对结核分枝杆菌缺氧反应中双组分信号蛋白DosR活性的影响(Bi et al。,2018; Yang 等人,2018)。最近我们证明了响应调节器TcrX的乙酰化改变了已知存在于 M的双组分信号系统中的串扰。结核病以及传感器激酶的磷酸酶活性和响应调节因子的DNA结合活性(Singh et al。,2019)。在本研究过程中,我们使用响应调节器TcrX开发了一种优化的体外协议以及体内乙酰化分析各种靶蛋白的方法。作为模板。该方案可用于测试任何分枝杆菌蛋白的乙酰化状态,并涉及两个步骤,首先,在酶催化反应中体外探测乙酰化的存在,其次,探测蛋白质的乙酰化。使用 M存在体内。 smegmatis mc2155作为代理主机。在体内利用携带Lys取代突变的蛋白质有助于确认鉴定的Lys确实是靶乙酰化位点。

关键字:TcrX, 反应调节子, 乙酰化, KATms, 卡玛肺结核, 双组份信号传导系统, 翻译后调节

材料和试剂

  1. 移液器吸头
    1. 1毫升尖头(Tarsons,目录号:521020)
    2. 2-200μl(Tarsons,目录号:521010)
    3. 0.2-10μl(Tarsons,目录号:521000)
  2. 1.5毫升微量离心管(Tarsons,目录号:500010)
  3. 15毫升聚丙烯离心管(Tarsons,目录号:546021)
  4. 50毫升聚丙烯离心管(Tarsons,目录号:546041)
  5. 0.1毫米直径的氧化锆/硅胶珠(Thomas Scientific,目录号:3411F09) 
  6. 2毫升聚丙烯珠粒打浆瓶(Bio Spec Products Inc.,目录号:10831)
  7. PVDF膜(Bio-Rad,目录号:1620177或1620238)
  8. Whatman滤纸1.5 MM(GE Healthcare,目录号:10426981)
  9. 0.45μm过滤器(Sartorius Minisart注射式过滤器,目录号:16555-K)
  10. 96孔微量测试板(Tarsons,目录号:941196)
  11. 具有用于重组蛋白过表达和纯化的亲和标签的表达载体(pProEx-Ht载体在N末端含有6x His标签,已用于过表达和纯化TcrX反应调节蛋白(Agrawal 等。 ,2015)以及KATms(乙酰转移酶)是来自IIS的Sandhya Visweswariah教授的礼物(Nambi et al。,2010)
  12. 表达菌株 E.大肠杆菌 BL21 Arctic Express TM [BF - omp T hsd S(r B - mB - ) dcm + Tet r gal λ(DE3) end A Hte [ cpn 10 cpn 60 Gent r ](费雷尔) et al。,2003)(安捷伦科技,美国,目录号:230191)用于过表达KATms的TcrX和SP850cyc-菌株(Nambi et al。, 2010)
  13. 耻垢分枝杆菌 mc 2 155(ATCC,目录号:700084) 
  14. pMV261载体(作为来自Dipankar Chatterji教授,IISc的礼物收到)
  15. pMV261含有编码TcrX的基因(Singh et al。,2019)或您感兴趣的基因
  16. pMV261含有编码TcrX K231R的基因(Singh et al。,2019)或您感兴趣的基因
  17. Middlebrook 7H9培养基(BD Biosciences,目录号:271310)
  18. 甘油(Sisco Research Ltd.,目录号:62417) 
  19. 吐温80(西格玛奥德里奇,产品目录号:P4780) 
  20. 吐温20(西格玛奥德里奇,目录号:P9416)
  21. Tyloxapol(Sigma-Aldrich,目录号:T8761)
  22. 卡那霉素(Goldbio,目录号:K-120)
  23. 油酸(Sigma-Aldrich,目录号:75090)
  24. 白蛋白(Amersco,目录号:0332)
  25. 葡萄糖(Sisco Research Ltd.,目录号:51758)
  26. 过氧化氢酶(Sigma-Aldrich,目录号:C9322)
  27. 考马斯亮蓝G-250(Sisco Research Ltd.,目录号:64222)
  28. 甲醇(Sisco Research Ltd.,目录号:96446)
  29. 磷酸(Alfa Aesar,目录号:A18067)
  30. 盐酸(HCl)(Fisher Scientific,目录号:A144SI-212) 
  31. 氯化钠(NaCl)(Merck Millipore Corporation,目录号:1064040500) 
  32. 氯化钾(KCl)(Merck Millipore Corporation,目录号:1049360500) 
  33. 磷酸氢二钠(Merck Millipore Corporation,目录号:1065860500) 
  34. 磷酸二氢钾(KH 2 PO 4 )(Merck Millipore Corporation,目录号:1048730250) 
  35. Trizma基地(西格玛奥德里奇,目录号:T6066) 
  36. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250) 
  37. 苯基甲基磺酰氟(Sigma-Aldrich,目录号:P7626) 
  38. 盐酸苯甲脒(Sigma-Aldrich,目录号:B6506)
  39. 1%Nonidet-P40(Sigma-Aldrich,CAS:9016-45-9)
  40. 抗乙酰抗体(Cell Signaling Technology Inc.,目录号:9441,Dilution 1:7,500)
  41. HRPO缀合的二抗(Cell Signaling Technology Inc.,目录号:7074,稀释1:5,000)
  42. 抗His抗体(Cell Signaling Technology Inc.,目录号:2365,Dilution 1:5,000)
  43. 抗TcrX多克隆抗体(稀释1:1,000)(在实验室中产生[Singh et al。,2019])
  44. Western Lightning PLUS ECL含有增强型鲁米诺试剂和氧化剂(PerkinElmer,目录号:NEL100001EA)
  45. SDS(Affymetrix USB,目录号:18220)
  46. EDTA(Sisco Research Ltd.,目录号:40648)
  47. 溴酚蓝(MERCK,目录号:1081220005)
  48. cAMP(西格玛奥德里奇,目录号:A6885)
  49. 乙酰辅酶A(Sigma-Aldrich,目录号:A2056)
  50. ADC溶液(白蛋白,葡萄糖,过氧化氢酶)(见食谱)
  51. 10x乙酰化缓冲液(见食谱)
  52. cAMP,5 mM原液(参见食谱)(Sigma-Aldrich,目录号:A6885)
  53. 乙酰辅酶A,5 mM原液(参见食谱)(Sigma-Aldrich,目录号:A2056)
  54. 7H9-甘油 - 吐温80培养基(见食谱)
  55. 恢复媒体(见食谱)
  56. 磷酸盐缓冲盐水(见食谱) 
  57. 裂解缓冲液(见食谱)
  58. 5x Bradford Reagent(参见食谱)
  59. 存储缓冲区(参见食谱)
  60. SDS-PAGE运行缓冲液(见食谱)
  61. 转移缓冲区(见食谱)
  62. Tris缓冲盐水吐温20(TBS-T)(见食谱)
  63. 剥离缓冲液(参见食谱)
  64. 5x蛋白质样品缓冲液(见食谱)

设备

  1. 移液器
    一个。 100-1,000μl移液器(Rainin pipet-lite,目录号:SL-1000)
    湾20-200μl移液器(Rainin pipet-lite,目录号:SL-200)
    C。 2-20μl移液器(Rainin pipet-lite,目录号:SL-20)
    d。 0.1-2μl移液器(Rainin pipet-lite,目录号:SL-2)
  2. 2 mm电穿孔比色皿(Bio-Rad Laboratories,目录号:165-2086)
  3. Mini-Protean凝胶电泳系统(Bio-Rad Laboratories,型号:165-8000)
  4. Mini Trans-Blot细胞(Bio-Rad Laboratories,型号:Trans-Blot ® SD细胞)
  5. Gel Doc成像系统(Bio-Rad Laboratories,型号:Universal Hood III)
  6. 37°C培养箱/振动筛(N-BIOTEK,型号:NB-205L)
  7. 冷冻离心机(Thermo Fisher Scientific,型号:Sorvall Legend X1R; Hettich Zentrifugen,型号:Rotina 420R)
  8. 分光光度计(Eppendorf,BioPhotometer ®,型号:D30)
  9. Bead beater(Bio Spec Products,型号:Mini-Bead beater)
  10. 微量离心机(Tomy Kogyo Co. Ltd.,型号:Kitman-T24)
  11. Vortexes(Shalom Instruments,型号:SLM-VM-3000)
  12. 加热块(加热至95°C)(Biobee Lab)
  13. 超低温冰箱(三洋,型号:MDF-U700VX)
  14. Electroporator(Eppendorf Eporator,目录号:4309000019)
  15. 多模式酶标仪(Tecan,型号:Infinite M1000 PRO)
  16. 高压灭菌器
  17. -80°C冰柜

软件

  1. ImageJ https://imagej.nih.gov/ij/download.html
  2. 来自BIO-RAD的图像实验室版本5.2.1软件(用于ChemiDoc TM 凝胶文档系统)

程序

  1. 体外乙酰化分析
    通过蛋白质印迹分析使用抗乙酰基抗体探测蛋白质来评估TcrX蛋白质(作为代表)的乙酰化状态,其在该方案中以逐步方式讨论。响应调节因子TcrX(6x his标记)和突变TcrX(K231R)蛋白从 E过表达并纯化。如先前在Singh 等人中报道的,来自SP850cyc-菌株的大肠杆菌BL21 Arctic Express TM 和KATms(乙酰转移酶/ MSMEG_5458)(2019)。通过Bradford的蛋白质估计测定法(Stoscheck,1990; He,2011a)对纯化的蛋白质进行定量,并将蛋白质储存在储存缓冲液(-20℃;参见食谱)中直至使用。以下方案描述了蛋白质纯化后的实验程序。
    设置 体外 乙酰化反应,然后进行抗乙酰基蛋白质印迹:
    1. 以下列量混合以下成分:
      TcrX或TcrX K231R蛋白,3μg
      10x乙酰化缓冲液,2μl(见食谱)
      cAMP,0.4μl(参见库存浓度配方)
      乙酰辅酶A,0.4μl(见库存浓度配方)
      KATms,0.2μg
      将高压灭菌的MQ水加入20μl 
    2. 在37°C孵育4小时(孵育持续时间可能因与乙酰化程度相关的底物蛋白质而异)。
    3. 通过用5μl样品缓冲液稀释反应混合物来终止反应(参见配方)。将样品在95°C加热10分钟。
    4. 使用15%SDS-PAGE(He,2011b)按照标准方案在100V下溶解样品3-4小时。
    5. 使用Bio-Rad Mini Trans-Blot细胞通过电印迹将蛋白质转移至PVDF膜,按照制造商的方案在10 V下保持1小时。
      注意:
      1. 根据某人的SDS-PAGE凝胶尺寸切割PVDF膜并加载泳道或使用Bio-Rad的预切割预组装印迹膜/滤纸夹层。
      2. 在Bio-Rad的半干转移装置(Trans-Blot ® SD细胞)中,将蛋白质转移到PVDF膜上需要10 V,持续1 h,以获得特定的低分子蛋白质(~30 kDa) 。应根据某人感兴趣的蛋白质(高或低分子量)对电压,电流,时间和设备类型(干/半干/湿)进行优化,以获得有效的转移。
    6. 用1%TBST(30ml)中的5%BSA封闭PVDF膜90分钟,同时在室温下轻轻摇动。
    7. 与1:7,500稀释的初级抗乙酰赖氨酸抗体孵育,在4°C温和摇动过夜。 
    8. 用1x TBST(50ml)洗涤膜3次,每次10分钟,快速摇动。
    9. 在室温下与1:5,000稀释的二抗孵育1小时,轻轻摇动。
    10. 用1x TBST(60-70ml)再次洗涤膜3次,每次10分钟,快速摇动。
    11. 根据制造商的方案使用增强的化学发光(ECL)开发印迹,并在Chemidoc凝胶文献系统中对印迹成像(图1)。
    12. 在显影PVDF膜后,用1x TBST(50ml)洗涤膜3次,每次10分钟。
    13. 将PVDF膜在55℃下与汽提缓冲液(30ml;参见配方)孵育25-30分钟(相应地优化)。
    14. 倒出汽提缓冲液,用1x TBST(100 ml)冲洗PVDF膜6-7次,每次快速摇动10分钟,或完全除去β-巯基乙醇。
      注意:从PVDF膜检查β-巯基乙醇的典型气味,确保在用1x TBST缓冲液洗涤后已完全除去。重复洗涤直至完全脱落。
    15. 用1%TBST(30ml)中的5%BSA在室温下封闭PVDF膜90分钟。
    16. 重复步骤A7中的步骤(以1:5,000的稀释度使用抗his 6x抗体)直至步骤A10,以产生印迹并获得步骤A11(图1)中指定的图像。该步骤提供了加载的蛋白质量的参考图像。


      图1. 体外乙酰化评估。 RR蛋白TcrX和TcrXK231R与KATms(乙酰转移酶,来自 M. smegmatis 的MSMEG_5458酶)一起培养。协议。如Singh 等人报道的那样,在通过质谱法鉴定位点后产生乙酰化位点缺陷的TcrX蛋白(2019)。顶部,用抗乙酰赖氨酸抗体和底部抗His抗体探测印迹的图像。

  2. TcrX蛋白的体内乙酰化分析
    特定蛋白质乙酰化的体内评估需要它在分枝杆菌细胞中过表达。为此,典型的方法涉及在分枝杆菌穿梭载体中的强分枝杆菌启动子如 hsp60 或 msp12 下克隆和表达ORF。在分枝杆菌菌株如 M中引入质粒。耻垢分析然后分析过表达蛋白的表达或修饰状态。以下步骤描述了整个工作流程。
    1. 制备电感受态耻垢分枝杆菌 mc 2 155
      使耻垢分枝杆菌 mc 2 155菌株具有电感受性,以引入携带目的基因的重组pMV261分枝杆菌质粒。 
      1. 通过在含有0.2%甘油,0.05%吐温-80和10%的5ml Middlebrook 7H9培养基中接种50μl耻垢分枝杆菌 mc 2 155原液制备1%原代培养物ADC。
      2. 将培养物在37℃下在摇床中以180rpm孵育直至OD 600 ~2(约2天) 
      3. 将1ml原代培养物接种到100ml补充有0.2%甘油,0.05%Tween-80和10%ADC的7H9肉汤中,并在37℃下在摇床中以180rpm生长直至OD 600 0.8-1.0(~24-32小时)。
      4. 将细胞在4,500 x g ,4℃下沉淀15分钟,然后在20ml高压灭菌的MilliQ水中洗涤3-4次。
      5. 然后用20ml无菌10%甘油洗涤3次。 
      6. 将细胞沉淀并重悬于2ml无菌10%甘油中。
      7. 在1.5ml离心管中制备各200μl的等分试样用于电穿孔。
      8. 使用预先冷却的100%乙醇将剩余的电感受态细胞等分试样放入1.5 ml离心管中,并将其储存在-80°C直至进一步使用。
        注意:在开始制备电感受态细胞之前,将装有100%乙醇的容器在-80°C保持6-8小时。当电感受态细胞等分试样准备好在微量离心管中时,然后将管浸入含有预冷乙醇的容器中,然后在-80℃下储存。
    2. M中质粒DNA的电穿孔。耻垢分子 mc 2 155
      1. 向等份的电感受态细胞中加入1μg重组质粒并在冰上孵育2分钟。
      2. 在冰上预冷2mm电穿孔杯,将电感受态细胞和质粒混合物加入比色皿中并在冰上孵育10分钟。擦拭比色杯的外壁,用电穿孔仪以2,500 V脉冲,立即保持在冰上。
        注意:在电穿孔结束时,在电穿孔仪的显示上可以观察到约4-5毫秒的时间,这表明两个平行电极之间的电流正确并且有效的电穿孔。如果发生电弧放电,则显示时间以秒为单位,并且设定电压与电穿孔期间电池上施加的电压不同。这可能是由于缓冲液的高离子强度,高盐或细胞洗涤不足,最终导致无效或无电穿孔。 
      3. 将200μl回收培养基(参见食谱)添加到比色杯中,并将细胞转移到15ml锥形管中。添加更多培养基,使体积达到3 ml,在37°C,振荡器培养箱中以180 rpm的速度摇动试管4小时,以恢复细胞。
        注意:在电穿孔前准备好超过(20 ml)的恢复培养基(参见食谱)。 
      4. 在室温下以4,500 x g 收获细胞10分钟。弃去上清液,将沉淀重悬于100μl回收培养基中。
      5. 在7H11培养基(琼脂基质)上的平板细胞补充有0.2%甘油,0.05%吐温80,10%含有适当抗生素的ADC(此处为25μg/ ml卡那霉素)。
      6. 将板在37℃孵育2天以获得转化体。
    3. 裂解液制备和体内表达和乙酰化状态分析 
      1. 对于起子培养,将每个重组菌株的单个菌落接种于含有0.2%甘油,0.05%吐温80和10%ADC以及25μg/ ml卡那霉素的5ml 7H9培养基中,并在37℃,180rpm下培养。摇床2天。 
      2. 在50ml补充有0.2%甘油,0.05%泰洛沙泊和10%ADC以及25μg/ ml卡那霉素的7H9肉汤中继代培养0.5-1ml原代培养物,并在37℃下在摇床中以180rpm生长。 /> 注意:当需要大量的二次培养( 例如 ,1/2升)时,主要是为了获得大量的蛋白质,原代培养也应该是更大的体积(10-50毫升)。 
      3. 一旦OD 600 达到~2-3(约2-3天),将细胞在4℃下以4,500 xg 沉淀,每次用10ml无菌洗涤3次PBS并在500μlTris裂解缓冲液(pH~7.5)中重悬沉淀。
      4. 将悬浮液转移到含有0.1 mm直径氧化锆/二氧化硅珠(~250μl体积缓冲液)的2 ml珠粒打浆小瓶中,并将珠子敲打30秒,在冰上间歇孵育8-10次(根据珠子的说明进行优化)均质器制造商)。
      5. 将裂解物在4℃下以10,000 x g离心10分钟并收集~400μl上清液。应注意避免收集未溶解的细胞和碎片。
      6. 通过Bradford的蛋白质估计测定法对裂解物进行定量。
      7. 按照标准方案,通过15%SDS-PAGE分离100μg蛋白质裂解物。
      8. 按照制造商的方案通过电印迹将解析的蛋白质转移到PVDF膜上,并如上面部分所述用抗乙酰赖氨酸抗体进行Western印迹,然后用蛋白质特异性抗体(这里是抗-TcrX)进行剥离和探测。
      9. 图2描绘了代表性开发的印迹,其报告了候选反应调节蛋白TcrX的过表达及其通过特异性抗体的乙酰化状态。当使用赖氨酸突变蛋白时,印迹也显示没有乙酰化,证实了体内乙酰化的位点。


        图2. 体内乙酰化分析。 M.耻垢分子 mc 2 在Middlebrook 7H9培养基(7H9)中培养155个单独携带载体pMV261或表达野生型TcrX或其突变蛋白的菌株。使用抗乙酰赖氨酸(上图)和抗TcrX(下图)抗体通过蛋白质印迹分析总共100μg的总蛋白质。 Arrow代表TcrX蛋白。

笔记

  1. 在准备 M时应该小心。耻垢分子细胞裂解物。涉及珠粒打浆的裂解步骤必须基于均化器进行优化,均化器的强度和打浆频率可能不同。 
  2. 应优化抗赖氨酸抗体的稀释,因为它可能在批次和批次之间变化。

食谱

  1. ADC溶液(白蛋白葡萄糖过氧化氢酶)(100毫升)
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>白蛋白
    5克
    葡萄糖
    2克
    过氧化氢酶
    3毫克 将白蛋白,右旋糖和过氧化氢酶溶解在约50ml MilliQ水(~50ml)中,然后将体积补足至100ml。用0.45微米过滤器过滤灭菌。
  2. 10x乙酰化缓冲液(1 ml) class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>Tris(pH~7.4)
    250 mM
    NaCl
    1,000 mM
  3. cAMP,5 mM原液(1 ml)
    将1.84mg粉末溶于~0.5ml MilliQ水中直至其完全溶解,然后将体积补足至1ml
  4. 乙酰CoA,5 mM原液(1 ml)
    将4.046毫克乙酰辅酶A粉末溶于~0.5毫升MilliQ水中直至完全溶解,然后将体积补足至1毫升
  5. 7H9-甘油 - 吐温80培养基(90ml)
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>7H9粉末
    0.47克
    甘油(50%)
    400μl
    吐温80(20%)
    250μl
    1. 将0.47克7H9粉末溶于~50毫升MilliQ水中
    2. 如上所述加入甘油和吐温80,用MilliQ水将总体积补足至90ml
    3. 通过高压灭菌消毒并在室内储存
    注意:由于甘油和吐温80是粘性的,因此将它们从100%的水中稀释至上述存量百分比,通过高压灭菌消毒并在室温下储存。 
  6. 回收介质(7H9-Glycerol-Tween 80-ADC)(20 ml) class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>7H9
    17.87毫升
    ADC
    2毫升
    甘油(50%)
    80μl
    吐温80(20%)
    50μl
  7. 磷酸盐缓冲盐水(1,000毫升)
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>NaCl
    8克
    KCl
    0.2克
    Na 2 HPO 4
    1.44克
    KH 2 PO 4
    0.24克
    通过高压灭菌消毒并在室温下储存
  8. 裂解缓冲液(5毫升) class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>Tris-Cl(pH~7.4)
    20 mM
    NaCl
    100 mM
    甘油
    10%
    PMSF
    1 mM
    苯扎米啶盐酸盐
    1 mM
  9. 5x Bradford Reagent(100 ml)
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>考马斯亮蓝G-250
    50毫克
    甲醇(100%)
    23.5毫升
    磷酸(85%)
    50毫升
    加入MilliQ水,使体积达到100毫升
  10. 储存缓冲液(500毫升) class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>Tris-HCl,pH 8.0
    50 mM
    甘油
    50%
    NaCl
    50 mM
    DTT
    1 mM
  11. SDS-PAGE运行缓冲液(1,000 ml)
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>Tris
    3克
    甘氨酸
    14.4克
    SDS
    1克
    通过添加MilliQ水来弥补体积
    注意:SDS-PAGE运行缓冲液的pH值为~8.2-8.5。
  12. 转移缓冲液(1升)
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>Tris
    3克
    甘氨酸
    14.4克
    甲醇
    200毫升
    通过添加MilliQ水将体积补足至1L
    注意:转移缓冲液的pH值也应为8.2-8.5。
  13. Tris缓冲盐水吐温20(TBS-T)(1,000 ml) class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>Tris(pH~7.5)
    10 mM
    NaCl
    153.8 mM
    吐温20
    0.1%
  14. 剥离缓冲液(20 ml) class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>Tris-Cl(pH 6.8)
    62.5 mM
    SDS
    2%
    β-巯基乙醇
    0.7%(140μl/ 20 ml)
  15. 5x蛋白质样品缓冲液(10 ml)
    class =“ke-zeroborder”bordercolor =“#000000”style =“width:300px;” border =“0”cellspacing =“0”cellpadding =“2”>Tris(pH-6.8)
    0.25 M
    甘油
    40%(4毫升/ 10毫升)
    SDS
    10%
    溴酚蓝
    0.25%
    β-巯基乙醇
    5%(0.5毫升/ 10毫升)

致谢

这项工作得到了印度生物技术部的支持(Grant No. BT / PR17357 / MED / 29/1019/2016)。该研究还得到了印度科学院DBT合作项目(DBT / BF / PRIns / 2011-12)和Infosys基金会的部分支持; DST-基础科学和技术基础设施项目的设备支持(SR / FST / LSII-036/2016)。

利益争夺

作者宣称没有竞争利益。

参考

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  2. Bi,J.,Gou,Z.,Zhou,F.,Chen,Y.,Gan,J.,Liu,J.,Wang,H。和Zhang,X。(2018)。 赖氨酸182的乙酰化抑制结核分枝杆菌 DosR结合DNA的能力并且在缺氧期间调节基因表达。 Emerg Microbes Infect 7(1):108。 
  3. Ferrer,M.,Chernikova,T。N.,Yakimov,M。M.,Golyshin,P。N.和Timmis,K。N.(2003)。 伴侣蛋白在低温下控制大肠杆菌的生长。 Nat Biotechnol 21(11):1266-1267。
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  6. Liu,F.,Yang,M.,Wang,X.,Yang,S.,Gu,J.,Zhou,J.,Zhang,X.E.,Deng,J。和Ge,F。(2014)。 乙酰化分析显示结核分枝杆菌中赖氨酸乙酰化的多种功能。 Mol Cell Proteomics 13(12):3352-3366。
  7. Nambi,S.,Basu,N。和Visweswariah,S。S.(2010)。 分枝杆菌中cAMP调节的蛋白赖氨酸乙酰化酶。 J Biol Chem 285(32):24313-24323。
  8. Singh,K.K.,Bhardwaj,N.,Sankhe,G.D.,Udaykumar,N.,Singh,R.,Malhotra,V。和Saini,D。K.(2019)。 M中响应调节蛋白,TcrX和MtrA的乙酰化。结核病调节其磷酸转移能力并调节双组分信号传导串扰。 J Mol Biol 431(4):777-793。
  9. Stoscheck,C.M。(1990)。 蛋白质定量。 Methods Enzymol 182:50- 68。
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引用:Singh, K. K., Singh, D. P., Singh, R. and Saini, D. (2019). In vitro and in vivo Assessment of Protein Acetylation Status in Mycobacteria. Bio-protocol 9(13): e3291. DOI: 10.21769/BioProtoc.3291.
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