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Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity
蓝细菌生物钟蛋白KaiC的表达和纯化及其自磷酸化酶活性测定   

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Scientific Reports
May 2016

Abstract

Circadian rhythms are biological processes displaying an endogenous oscillation with a period of ~24 h. They allow organisms to anticipate and get prepared for the environmental changes caused mainly by the rotation of Earth. Circadian rhythms are driven by circadian clocks that consist of proteins, DNA, and/or RNA. Circadian clocks of cyanobacteria are the simplest and one of the best studied models. They contain the three clock proteins KaiA, KaiB, and KaiC which can be used for in vitro reconstitution experiments and determination of the auto-phosphatase activity of KaiC as described in this protocol.

Keywords: KaiA (KaiA), KaiC (KaiC), Circadian clock (生物钟), Phosphorylation (磷酸化), Oscillator (振荡器)

Background

The rotation of planet Earth causes the ~24 h day-night oscillation. To fit in and then efficiently take advantage of this rhythmic change of the environment, most if not all organisms have an endogenous activity rhythm of ~24 h, which is called circadian rhythm. Circadian rhythms provide evolutionary advantages to those organisms. The long-term disruption of circadian rhythms is extremely harmful (Ma et al., 2013). In humans, many diseases, including cancer, hypertension, and sleep disorders, are closely related with a disrupted circadian rhythm (Shi et al., 2013; Roenneberg and Merrow, 2016).

Circadian rhythms are controlled by endogenous rhythm generators called circadian clocks. A functional circadian clock has three functionalities: accepting the environmental information, turning the environmental cues into oscillating signals, and relaying these signals to down-stream modulators (Pattanayak and Rust, 2014). Cyanobacteria are the simplest organisms having a well-studied circadian clock, in which the oscillation generator is controlled by three proteins: KaiA, KaiB, and KaiC (Mackey et al., 2011; Johnson et al., 2011; Chen et al., 2013; Egli and Johnson, 2013). KaiC is a multi-functional protein, which has auto-kinase, auto-phosphatase, and ATPase activity (Egli, 2015). The auto-kinase activity results in the phosphorylation of the two key residues T432 and S431 in KaiC, whereas the auto-phosphatase activity results in their de-phosphorylation (Rust, 2012). When incubated alone, KaiC shows mainly phosphatase activity (Nishiwaki and Kondo, 2012). KaiA can stimulate the auto-kinase activity of KaiC, and KaiB antagonizes KaiA’s function, which makes the phosphorylation state of KaiC oscillate in a ~24 h rhythm (Dong et al., 2016).

In 2005, Nakajima et al. successfully reconstituted the KaiABC oscillator in vitro by mixing the purified proteins, KaiA, KaiB, and KaiC, in a buffer containing ATP and Mg2+ (Nakajima et al., 2005). The simple procedure made the KaiABC system a highly attractive model for studying the molecular mechanism of circadian clocks. In this protocol, a major part of the reconstitution system, the in vitro determination of the auto-phosphatase activity of KaiC is described, in which the phosphorylation states of KaiC are analyzed by SDS-PAGE.

Materials and Reagents

  1. 1.5 ml microcentrifuge tubes
  2. 15 ml tube (Corning, Axygen®, catalog number: SCT-15mL-25-S )
  3. 50 ml centrifuge tube (Corning, Axygen®, catalog number: SCT-50mL-25-S )
  4. Petri dish (Corning, catalog number: 70165-60 )
  5. Hitrap FF Q column: 5 ml (GE Healthcare, catalog number: 17-5156-01 )
  6. NAP-5/25 buffer exchange column (GE Healthcare, catalog number: 17-0853-02 )
  7. Centrifugal filter: 10 kDa, Amicon Ultra (EMD Millipore, catalog number: PR02967 )
  8. 0.22 µm filter (Pall, catalog number: PN 4612 )
  9. PCR tube (Bio-Sharp, catalog number: BS-02-P )
  10. 0.45 µm filter
  11. E. coli strain BL21 (DE3) (New England Biolabs, catalog number: C2527 )
  12. pGEX-6P-1-KaiC: Provided by Prof. Carl Johnson (Vanderbilt University, USA). The KaiC-coding sequence is from Synechococcus elongatus PCC 7942
  13. Bacto-tryptone (Oxoid, catalog number: LP0042 )
  14. Bacto-yeast extract (Oxoid, catalog number: LP0021 )
  15. Agar A (Beijing Dingguo Changsheng Biotechnology, catalog number: DH010 )
  16. Calcium chloride (CaCl2) (1 M; Sigma-Aldrich, catalog number: V900266 )
  17. Ampicillin (1 mg/ml; North China Pharmaceutical Group Corporation, catalog number: A102048-25g )
  18. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: 16758 )
  19. Glutathione S-transferase (GST) resin (EMD Millipore, catalog number: 70541 )
  20. PreScission protease (PSP) (GE Healthcare, catalog number: 27-0843-01 )
  21. Kanamycin (1 mg/ml; GENVIEW, catalog number: AK177-10G )
  22. 3x loading dye
  23. Tris-base (AMRESCO, catalog number: 0497 )
  24. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 7647-14-5 )
  25. Dithiothreitol (DTT) (Life Science Products&Services, catalog number: DB0058-25g )
  26. Tween-20 (Enox, catalog number: 557 )
  27. ATP (Life Science Products&Services, catalog number: AB0020-25g )
  28. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  29. EDTA (Bio Basic, catalog number: EB0185 )
  30. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  31. SDS (Life Science Products&Services, catalog number: SB0485 )
  32. Bis-acrylamide (Sigma-Aldrich, catalog number: 146072 )
  33. Ammonium persulfate (APS) (Xilong Scientific, catalog number: 51504 )
  34. TEMED (CUSABIO, catalog number: V900853 )
  35. Pierce Coomassie (Bradford) Protein Assay Kit (Sangon Biotech, catalog number: C503031 )
  36. Bromophenol blue (Sigma-Aldrich, catalog number: 115-39-9 )
  37. Methanol (Tianjin Kermel Chemical Reagent, catalog number: 32058 )
  38. Acetic acid (Heng Xing, catalog number: 81601 )
  39. Acrylamide (AMRESCO, catalog number: 0341 )
  40. Protein marker (SM0431, Fermantas) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 26610 )
  41. GSH (Reduced Glutathione) (Sangon Biotech, catalog number: A100399 )
  42. Ethanol (Heng Xing, catalog number: 32061 )
  43. PSP buffer (see Recipes)
  44. Buffer A (see Recipes)
  45. Buffer B (see Recipes)
  46. Reaction buffer (see Recipes)
  47. 1 M IPTG (see Recipes)
  48. 10x running buffer (see Recipes)
  49. 8% separation gel (see Recipes)
  50. 5% stacking gel (see Recipes)
  51. Coomassie blue staining solution (see Recipes)
  52. De-staining buffer (see Recipes)

Equipment

  1. Autoclave sterilizer (Shanghai Huaxian Medical Equipment, model: HVA-85 )
  2. Laminar flow hood (SU ZHOU AN TAI, model: SW-CJ-2F )
  3. Electric Thermostatic incubator (Shanghai Yuejin Medical Apparatus Factory, model: SHP-250 )
  4. Pipettes (Gilson, catalog numbers: 711111170000 and 711111050000 )
  5. Orbital shaker (Thermo Fisher Scientific, Thermo ScientificTM, model: SHKE4000-1CE )
  6. High-speed refrigerated centrifuge (Hitachi, model: CR21G )
  7. Refrigerated centrifuge (Sigma Laborzentrifugen, model: 5811XQ 14241g )
  8. Benchtop centrifuge (Eppendorf, model: 1024 and 5424 )
  9. Ultra-low temperature freezer (Haier, model: 906 )
  10. Microplate spectrophotometer (Thermo Fisher Scientific, model: 1500 )
  11. Analytical balance (Sartorius, model: CP225D )
  12. Ultrapure water system (Pall, model: CascadA ZX )
  13. Membrane filter system (EMD Millipore, USA)
  14. Ultrasonic cell processor (Scientz Biotechnology, model: SCIENTZ-IID )
  15. FPLC system (GE Healthcare, model: AKTA Purifier 100 )
  16. Precision pH meter (Mettler Toledo, model: EL-20 )
  17. Electrophoresis system (Bio-Rad Laboratories, model: Mini-Protean Tetra )
  18. Decolorization shaker (Haimen Kylin-Bell Lab Instruments, model: TS-8 )
  19. Gel imaging system (Kodak, model: Gel Logic 200 )
  20. PCR system (Biometra, model: T-Gradient Thermoblock )

Software

  1. ImageJ (version 1.8.0_77, NIH, USA)
  2. Excel (version 2012, Microsoft, USA)

Procedure

  1. Transformation of E. coli cells with plasmid the pGEX-6P-1-KaiC
    1. Place the calcium competent BL21(DE3) cells and the pGEX-6P-1-KaiC plasmid in ice water.
    2. Measure the plasmid concentration spectrophotometrically and add 0.1 µg of pGEX-6P-1-KaiC into 100 µl of BL21(DE3) cells in a 1.5 ml tube.
    3. Mix the plasmid and the cells gently and set down in ice water for 30 min.
    4. Place the tube in a 42 °C water bath for 90 sec before placing it in ice water for 60 sec.
    5. Add 400 µl of LB (Luria-Bertani) medium (without antibiotics) to the tube, and let the cells recover in a shaker at 37 °C, 150 rpm for 45 min.
    6. Spin down the cells at 6,000 x g (30 sec).
    7. Discard supernatant and re-suspend cells in 100 µl of fresh LB medium (without antibiotics).
    8. Spread cells evenly on solid LB medium supplemented with 100 µg/ml of ampicillin.
    9. Incubate the plate at 37 °C overnight until colonies form.

  2. Expression of KaiC
    1. Pick a single colony of the transformed BL21(DE3) cells containing pGEX-6P-1-KaiC and inoculate it into 3 ml liquid LB medium supplemented with 100 µg/ml of ampicillin in a sterilized 15 ml tube.
    2. Grow the cells overnight at 37 °C, 220 rpm.
    3. Transfer 1 ml of the overnight culture to 1 L of sterilized liquid LB medium containing 100 µg/ml of ampicillin in a 3 L flask.
    4. Grow culture until OD600 reaches ~0.6 at 37 °C, 220 rpm, lower the temperature to 28 °C and induce the expression of kaiC by adding 200 µl of 1 M IPTG.
    5. After 16 h of induction, collect the cells by centrifugation for 5 min at 7,000 x g, 4 °C.
    6. Keep the pelleted cells on ice for instant processing or store at -80 °C.

  3. GST affinity purification
    1. Re-suspend the cells from 1 L cell culture thoroughly with 40 ml of PSP buffer in a 100 ml glass beaker.
    2. Place the beaker in ice water, and disrupt the cells by sonication for 45 min at 35% output power (wait for 2 sec after every 1 sec of sonication).
    3. Transfer the solution to a fresh 50 ml tube, and centrifuge for 40 min at 4 °C, 16,000 x g.
    4. During the centrifugation, take 1 ml of the GST resin and wash it with 5 ml of sterilized ddH2O and then 3 ml of PSP buffer.
    5. Mix the GST resin on an orbital shaker with the supernatant from the centrifuge tube (step C3) for 2 h at 4 °C.
    6. Re-suspend the resin with 5 ml of PSP buffer (4 °C) and elute the buffer to remove unbound proteins.
    7. Add the PreScission protease to the resin and keep the solution at 4 °C for 16 h to allow the enzymatic digestion of the GST-tagged KaiC.
    8. Keep the supernatant (or the eluent) containing KaiC at 4 °C for further purification steps.
    Notes:
    1. Use an orbital shaker (40 rpm) when incubating the resin mixture at 4 °C.
    2. Store 10 µl of the sample at each step (including the steps in the following anion exchange purification) for evaluation for the first time at -20 °C.

  4. Anion exchange purification
    1. Set up the FPLC machine and turn on the 280 nm UV lights for detection.
    2. Install a 5 ml Hitrap FF Q column on the FPLC machine, and wash it with 25 ml of ddH2O and then 25 ml of buffer A at 4.0 ml/min.
    3. Exchange buffer of the KaiC protein from the last step with buffer A using a NAP-25 buffer exchange column, and load the sample onto the column at 2.0 ml/min.
    4. Wash the column with buffer A until the absorbance at 280 nm reaches the baseline.
    5. Wash the column with 10% buffer B (90% buffer A) to remove non-specifically bound proteins.
    6. Set up a linear gradient elution program with 10-50% buffer B for 70 min, and collect the protein peak (~ 40 min) which contains KaiC.
    7. Keep the KaiC sample at 4 °C.
    Notes:
    1. All buffers and samples used for FPLC purification must be filtered through 0.22 µm filters.
    2. The linear gradient elution step is strongly recommended for the first purification trial.

  5. Storage of KaiC
    1. To store the purified KaiC protein, concentrate the KaiC sample from the last step (step D7) to 1-5 mg/ml with a 10 kDa centrifugal filter at 4 °C, 3,000 rpm.
    2. Exchange buffer with reaction buffer using a NAP-5 buffer exchange column. Concentrate the KaiC sample to 1-5 mg/ml, and store the protein at -80 °C.
    Notes:
    1. Determine the concentration of the sample with the Bradford assay.
    2. We suggest using the protein immediately for the following procedures. For storage add 20% glycerol, freeze in liquid nitrogen and keep at -80 °C. Please note that long-term storage (2-3 months) might result in an activity loss of KaiC.

  6. Preparation of KaiC samples for SDS-PAGE analysis
    1. Take out the KaiC protein stored at -80 °C and thaw on ice.
    2. Dilute KaiC to 0.5 mg/ml with reaction buffer supplemented with 80 µg/ml of kanamycin to a final volume of 30 µl.
    3. Transfer 4 µl of the diluted KaiC sample to clean and sterilized PCR tubes labelled as tubes 1 to 5.
    4. Add 2 µl of 3x loading dye to sample tube 1 and incubate at 100 °C for 5 min.
    5. Store sample 1 at -20 °C and transfer samples 2-5 to 4 °C.
    6. After 24 h, take out sample 2 and prepare as described above.
    7. Place the sample tubes 3-5 in a PCR machine, and set the sample temperature to 30 °C.
    8. Take out one tube every 4 h and prepare as described before.

  7. SDS-PAGE analysis of the auto-phosphatase activity of KaiC
    1. Take out the KaiC protein stored at -80 °C and thaw on ice.
    2. Dilute KaiC to 0.5 mg/ml with reaction buffer supplemented with 80 µg/ml of kanamycin to a final volume of 30 µl.
    3. Transfer 4 µl of the diluted KaiC sample to clean and sterilized PCR tubes labelled as tubes 1 to 5.
    4. Add 2 µl of 3x loading dye to sample tube 1 and incubate at 100 °C for 5 min.
    5. Store sample 1 at -20 °C and transfer samples 2-5 to 4 °C.
    6. After 24 h, take out sample 2 and prepare as described above.
    7. Place the sample tubes 3-5 in a PCR machine, and set the sample temperature to 30 °C.
    8. Take out one tube every 4 h and prepare as described before.
    Notes:
    1. The best amount of KaiC in each well is 2-3 µg in this protocol.
    2. Add 10 µl of the loading dye in the wells beside the samples to reduce the ‘smile effect’.

Data analysis

  1. Evaluation of KaiC purity
    Since protein purity is important for the activity and stability of proteins, check the purity of the purified KaiC protein by SDS-PAGE. As shown in Figure 1, the KaiC prepared with this protocol was highly pure, and the molecular weight corresponded to the predicted value of 58 kDa.


    Figure 1. SDS-PAGE analysis of purified KaiC

  2. Analysis of auto-phosphatase activity of KaiC
    To determine the auto-phosphatase activity of the purified KaiC, the samples prepared at different time points (1-5) were analyzed by SDS-PAGE. Figure 2 clearly shows the de-phosphorylation of KaiC when incubated alone at 30 °C in reaction buffer. The lower band is the de-phosphorylated KaiC, and the upper band is the phosphorylated KaiC.


    Figure 2. SDS-PAGE analysis of auto-phosphatase activity of KaiC. KaiC was incubated at 4 °C for 24 h (-24 h) before incubation at 30 °C. Samples were taken every 4 h.

  3. Quantitation of KaiC auto-phosphatase activity
    To quantitatively analyze the de-phosphorylation of KaiC, the SDS-PAGE gel was analyzed with ImageJ (Schneider et al., 2012). Open the recorded high-quality image of the SDS-PAGE gel in ImageJ. Frame the KaiC bands in the first lane, and select ‘Analyze-Gels-Select First Lane’. Move the frame to the next lane while keeping its size unchanged to cover the KaiC bands, and then use the ‘Analyze-Gels-Select Next Lane’ function. Repeat this process for all lanes. Use the function ‘Analyze-Gels-Plot Lanes’ to plot the peak areas of the KaiC bands. Then draw the baselines with the line tool to close up the independent peak areas corresponding to the KaiC bands in the gel. Use the ‘Wand Tool’ to select the closed peak areas one by one. A new window will show the calculated peak areas. Finally, calculate the percentages of the phosphorylated KaiC band in each lane, and analyze the data in Excel or other similar software (Figure 3).


    Figure 3. Quantitative analysis of the auto-phosphatase activity of KaiC. The percentage of the phosphorylated KaiC (P-KaiC) in each lane was analyzed by ImageJ.
    Note: The baseline correction in ImageJ is somehow arbitrary, but it should be kept constant for all peaks.

Recipes

  1. PSP buffer
    50 mM Tris-HCl (pH 8.0)
    150 mM NaCl
    1 mM DTT
    0.01 % Tween-20
    1 mM ATP
    5 mM MgCl2
  2. Buffer A
    50 mM Tris-HCl (pH 8.0)
    1 mM DTT
    0.01 % Tween-20
    1 mM ATP
    5 mM MgCl2
  3. Buffer B
    50 mM Tris-HCl (pH 8.0)
    1 M NaCl
    1 mM DTT
    0.01 % Tween-20
    1 mM ATP
    5 mM MgCl2
  4. Reaction buffer
    50 mM Tris-HCl (pH 8.0)
    150 mM NaCl
    5 mM ATP
    5 mM MgCl2
    0.01 % Tween-20
    0.5 mM EDTA
  5. 1 M IPTG
    Dissolve 1 g IPTG in 4,196 μl deionized water to make 1 M solution
    Filter sterilize with syringe and 0.22 μm filter
  6. 10x running buffer
    144 g of glycine
    30.3 g of Tris
    10 g of SDS
    Add ddH2O to 1 L
  7. 8% separation gel (Table 1)

    Table 1. Preparation of the 8% separation gel


  8. 5% stacking gel (Table 2)

    Table 2. Preparation of the 5% stacking gel


  9. Coomassie blue staining solution (1 L)
    1 g Coomassie blue G250
    650 ml ddH2O
    250 ml methanol
    100 ml acetic acid
  10. De-staining buffer (1 L)
    600 ml ddH2O
    300 ml methanol
    100 ml acetic acid 

Notes:

  1. All buffers should be sterilized with 0.45 µm filters.
  2. Store buffers at 4 °C for less than a week.

Acknowledgments

This work was funded by the grants to S.L. from the National Natural Science Foundation of China (91330113, 31670768), Hubei Province of China (D20161204), and China Three Gorges University. The reference work was published in Sci Rep 6: 25129.

References

  1. Chen, W., Liu, S. and Liu, S. (2013). Advances in the molecular mechanism of the core circadian oscillator of cyanobacteria. Acta Biophysica Sinica 29(11): 801-810.
  2. Dong, P., Fan, Y., Sun, J., Lv, M., Yi, M., Tan, X. and Liu, S. (2016). A dynamic interaction process between KaiA and KaiC is critical to the cyanobacterial circadian oscillator. Sci Rep 6: 25129.
  3. Egli, M. and Johnson, C.H. (2013). A circadian clock nanomachine that runs without transcription or translation. Curr Opin Neurobiol 23(5): 732-740.
  4. Egli, M. (2015). Structural and biophysical methods to analyze clock function and mechanism. Methods Enzymol 551: 223-266.
  5. Johnson, C. H., Stewart, P. L. and Egli, M. (2011). The cyanobacterial circadian system: from biophysics to bioevolution. Annu Rev Biophys 40: 143-167.
  6. Mackey, S. R., Golden, S. S. and Ditty, J. L. (2011). The itty-bitty time machine genetics of the cyanobacterial circadian clock. Adv Genet 74: 13-53.
  7. Ma, P., Woelfle, M. A. and Johnson, C. H. (2013). An evolutionary fitness enhancement conferred by the circadian system in cyanobacteria. Chaos Solitons Fractals 50: 65-74.
  8. Nakajima, M., Imai, K., Ito, H., Nishiwaki, T., Murayama, Y., Iwasaki, H., Oyama, T. and Kondo, T. (2005). Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science 308(5720): 414-415.
  9. Nishiwaki, T. and Kondo, T. (2012). Circadian autodephosphorylation of cyanobacterial clock protein KaiC occurs via formation of ATP as intermediate. J Biol Chem 287(22): 18030-18035.
  10. Pattanayak, G. and Rust, M. J. (2014). The cyanobacterial clock and metabolism. Curr Opin Microbiol 18: 90-95.
  11. Roenneberg, T. and Merrow, M. (2016). The circadian clock and human health. Curr Biol 26(10): R432-443.
  12. Rust, M. J. (2012). Orderly wheels of the cyanobacterial clock. Proc Natl Acad Sci U S A 109(42): 16760-16761.
  13. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
  14. Shi, S. Q., Ansari, T. S., McGuinness, O. P., Wasserman, D. H. and Johnson, C. H. (2013). Circadian disruption leads to insulin resistance and obesity. Curr Biol 23(5): 372-381.

简介

昼夜节律是显示内源性振荡的生物过程,周期为〜24小时。它们允许生物体预期并准备好主要由地球旋转引起的环境变化。昼夜节律由由蛋白质,DNA和/或RNA组成的昼夜节律钟驱动。蓝藻的昼夜节律时钟是最简单的研究模型之一。它们含有三种时钟蛋白质KaiA,KaiB和KaiC,其可用于体外重组实验和本方案所述的KaiC的自磷酸酶活性的测定。

背景 行星地球的旋转导致〜24小时的昼夜振荡。为了适应并有效地利用环境的这种节奏变化,大多数(如果不是全部)生物体具有约24小时的内源性活动​​节律,这被称为昼夜节律。昼夜节律为这些生物提供进化优势。昼夜节律的长期破坏是非常有害的(Ma et al。,2013)。在人类中,包括癌症,高血压和睡眠障碍在内的许多疾病与昼夜节律紊乱密切相关(Shi等人,2013; Roenneberg和Merrow,2016)。
 昼夜节律由称为昼夜节律钟的内生节律发生器控制。功能性昼夜节律钟具有三个功能:接受环境信息,将环境提示转变为振荡信号,并将这些信号转发到下游调制器(Pattanayak和Rust,2014)。蓝细菌是具有良好研究的昼夜节律钟的最简单的生物体,其中振荡发生器由三种蛋白质控制:KaiA,KaiB和KaiC(Mackey等人,2011; Johnson& et al。 2011; Chen等人,2013; Egli和Johnson,2013)。 KaiC是一种多功能蛋白,具有自身激酶,自身磷酸酶和ATP酶活性(Egli,2015)。自激酶活性导致KaiC中两个关键残基T432和S431的磷酸化,而自身磷酸酶活性导致其去磷酸化(Rust,2012)。单独培养时,KaiC主要表现为磷酸酶活性(Nishiwaki和Kondo,2012)。 KaiA可以刺激KaiC的自身激酶活性,KaiB对抗KaiA的功能,使KaiC的磷酸化状态以〜24h的节律振荡(Dong等人,2016)。
    2005年,Nakajajima等人。通过将纯化的蛋白质KaiA,KaiB和KaiC在含有ATP和Mg 2+的缓冲液(Nakajima等人)中混合来成功重构体外KaiABC振荡器。,2005)。简单的程序使得KaiABC系统成为研究昼夜节律钟分子机制的非常有吸引力的模型。在该方案中,重组系统的主要部分,描述了KaiC的自身磷酸酶活性的体外测定,其中通过SDS-PAGE分析KaiC的磷酸化状态。

关键字:KaiA, KaiC, 生物钟, 磷酸化, 振荡器

材料和试剂

  1. 1.5 ml微量离心管
  2. 15ml管(Corning,Axygen ,目录号:SCT-15mL-25-S)
  3. 50ml离心管(Corning,Axygen ,目录号:SCT-50mL-25-S)
  4. 培养皿(康宁,目录号:70165-60)
  5. Hitrap FF Q柱:5 ml(GE Healthcare,目录号:17-5156-01)
  6. NAP-5/25缓冲液交换柱(GE Healthcare,目录号:17-0853-02)
  7. 离心过滤器:10kDa,Amicon Ultra(EMD Millipore,目录号:PR02967)
  8. 0.22μm过滤器(Pall,目录号:PN 4612)
  9. PCR管(Bio-Sharp,目录号:BS-02-P)
  10. 0.45μm过滤器
  11. E。大肠杆菌菌株BL21(DE3)(New England Biolabs,目录号:C2527)
  12. pGEX-6P-1-KaiC:由Carl Johnson教授(美国范德比尔特大学)提供。 KaiC编码序列来自细胞聚球蓝细菌PCC 7942
  13. Bacto-tryptone(Oxoid,目录号:LP0042)
  14. 细菌酵母提取物(Oxoid,目录号:LP0021)
  15. 琼脂A(北京定格长盛生物科技,目录号:DH010)
  16. 氯化钙(CaCl 2)(1M; Sigma-Aldrich,目录号:V900266)
  17. 氨苄青霉素(1mg/ml;华北制药集团公司,目录号:A102048-25g)
  18. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Sigma-Aldrich,目录号:16758)
  19. 谷胱甘肽S-转移酶(GST)树脂(EMD Millipore,目录号:70541)
  20. PreScission蛋白酶(PSP)(GE Healthcare,目录号:27-0843-01)
  21. 卡那霉素(1mg/ml; GENVIEW,目录号:AK177-10G)
  22. 3x加载染料
  23. 三碱基(AMRESCO,目录号:0497)
  24. 氯化钠(NaCl)(Sigma-Aldrich,目录号:7647-14-5)
  25. 二硫苏糖醇(DTT)(生命科学产品与服务,目录号:DB0058-25g)
  26. Tween-20(Enox,目录号:557)
  27. ATP(Life Science Products& Services,catalog number:AB0020-25g)
  28. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  29. EDTA(Bio Basic,目录号:EB0185)
  30. 甘油(Sigma-Aldrich,目录号:G5516)
  31. SDS(生命科学产品与服务,目录号:SB0485)
  32. 双丙烯酰胺(Sigma-Aldrich,目录号:146072)
  33. 过硫酸铵(APS)(西龙科学,目录号:51504)
  34. TEMED(CUSABIO,目录号:V900853)
  35. Pierce考马斯(Bradford)蛋白测定试剂盒(Sangon Biotech,目录号:C503031)
  36. 溴苯酚蓝(Sigma-Aldrich,目录号:115-39-9)
  37. 甲醇(天津凯美尔化学试剂,目录号:32058)
  38. 乙酸(恒兴,目录号:81601)
  39. 丙烯酰胺(AMRESCO,目录号:0341)
  40. 蛋白质标记(SM0431,Fermantas)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:26610)
  41. GSH(谷胱甘肽还原)(Sangon Biotech,目录号:A100399)
  42. 乙醇(恒兴,目录号:32061)
  43. PSP缓冲(见配方)
  44. 缓冲液A(参见食谱)
  45. 缓冲液B(参见食谱)
  46. 反应缓冲液(见配方)
  47. 1 M IPTG(见配方)
  48. 10x运行缓冲区(请参阅配方)
  49. 8%分离凝胶(参见食谱)
  50. 5%堆积凝胶(见配方)
  51. 考马斯蓝染色溶液(参见食谱)
  52. 去污缓冲液(见配方)

设备

  1. 高压灭菌器(上海华西医疗器械,型号:HVA-85)
  2. 层流罩(SU周安泰,型号:SW-CJ-2F)
  3. 电热恒温箱(上海跃进医疗器械厂,型号:SHP-250)
  4. 移液器(Gilson,目录号:711111170000和711111050000)
  5. 轨道摇床(Thermo Fisher Scientific,Thermo Scientific TM,型号:SHKE4000-1CE)
  6. 高速冷冻离心机(日立,型号:CR21G)
  7. 冷冻离心机(Sigma Laborzentrifugen,型号:5811XQ 14241g)
  8. 台式离心机(Eppendorf,型号:1024和5424)
  9. 超低温冷冻机(海尔,型号:906)
  10. 微量分光光度计(Thermo Fisher Scientific,型号:1500)
  11. 分析天平(Sartorius,型号:CP225D)
  12. 超纯水系统(Pall,型号:CascadA ZX)
  13. 膜过滤系统(EMD Millipore,USA)
  14. 超声波细胞处理器(Scientz Biotechnology,型号:SCIENTZ-IID)
  15. FPLC系统(GE Healthcare,型号:AKTA净化器100)
  16. 精密pH计(Mettler Toledo,型号:EL-20)
  17. 电泳系统(Bio-Rad Laboratories,型号:Mini-Protean Tetra)
  18. 脱色摇床(海门麒麟贝尔实验室仪器,型号:TS-8)
  19. 凝胶成像系统(柯达,型号:Gel Logic 200)
  20. PCR系统(Biometra,型号:T-Gradient Thermoblock)

软件

  1. ImageJ(版本1.8.0_77,NIH,USA)
  2. Excel(2012版,微软,美国)

程序

  1. E的转型质粒pGEX-6P-1-KaiC的大肠杆菌细胞
    1. 将钙合成的BL21(DE3)细胞和pGEX-6P-1-KaiC质粒置于冰水中。
    2. 通过分光光度法测定质粒浓度,并加入0.1μgpGEX-6P-1-KaiC到1.5ml管中的100μlBL21(DE3)细胞中。
    3. 轻轻混合质粒和细胞,放入冰水中30分钟。
    4. 将管置于42°C水浴中90秒,然后放入冰水中60秒。
    5. 向管中加入400μlLB(Luria-Bertani)培养基(无抗生素),并使细胞在37℃,150rpm振荡器中回收45分钟。
    6. 以6,000 x g(30秒)旋转细胞。
    7. 弃上清,并将细胞重新悬浮于100μl新鲜LB培养基(无抗生素)中。
    8. 将细胞均匀分散在补充有100μg/ml氨苄青霉素的固体LB培养基上
    9. 将板在37℃孵育过夜直到菌落形成。

  2. 开凯的表达
    1. 挑取含有pGEX-6P-1-KaiC的转化的BL21(DE3)细胞的单个菌落,并将其接种到灭菌的15ml管中的补充有100μg/ml氨苄青霉素的3ml液体LB培养基中。
    2. 在37℃,220rpm下将细胞生长过夜。
    3. 将1ml过夜培养物转移到3L烧瓶中的1L含有100μg/ml氨苄青霉素的无菌液体LB培养基中。
    4. 生长培养直到OD 600在37℃,220rpm下达到〜0.6,将温度降低至28℃,并通过加入200μl1M IPTG诱导kaiC的表达。
    5. 诱导16小时后,以7,000 x g,4℃离心5分钟收集细胞。
    6. 将沉淀的细胞保持在冰上即时加工或储存在-80°C。

  3. GST亲和纯化
    1. 用100ml的玻璃烧杯中的40ml的PSP缓冲液将细胞从1L细胞培养物中彻底悬浮。
    2. 将烧杯放入冰水中,以35%的输出功率通过超声处理45分钟破坏细胞(每超声处理1秒后等待2秒钟)。
    3. 将溶液转移到新鲜的50ml管中,并在4℃,16,000×g离心40分钟。
    4. 在离心过程中,取1ml GST树脂,并用5ml无菌ddH 2 O和3ml PSP缓冲液洗涤。
    5. 将轨道摇床上的GST树脂与离心管的上清液(步骤C3)在4℃下混合2小时。
    6. 用5ml的PSP缓冲液(4℃)重新悬浮树脂,并洗脱缓冲液以除去未结合的蛋白质。
    7. 将PreScission蛋白酶添加到树脂中,并将溶液保持在4℃16小时,以允许酶消化GST标记的KaiC。
    8. 将含有KaiC的上清液(或洗脱液)保持在4℃,进一步纯化步骤。
    注意:
    1. 在4℃下孵育树脂混合物时,使用轨道摇床(40rpm)。
    2. 在每个步骤(包括以下阴离子交换纯化中的步骤)中储存10μl样品,首次在-20°C进行评估。

  4. 阴离子交换净化
    1. 设置FPLC机器,打开280nm紫外线灯进行检测。
    2. 在FPLC机上安装5ml的Hitrap FF Q柱,并用25ml的ddH 2 O和然后以4.0ml/min的速度洗涤25ml缓冲液A.
    3. 使用NAP-25缓冲液交换柱从最后一步与缓冲液A的KaiC蛋白质的交换缓冲液,并将样品以2.0ml/min加载到柱上。
    4. 用缓冲液A洗涤柱直到280nm处的吸光度达到基线。
    5. 用10%缓冲液B(90%缓冲液A)洗涤柱以除去非特异性结合的蛋白质。
    6. 设置10-50%缓冲液B线性梯度洗脱程序70分钟,收集含有KaiC的蛋白质峰(〜40分钟)。
    7. 将KaiC样品保持在4°C。
    注意:
    1. 用于FPLC纯化的所有缓冲液和样品必须通过0.22μm过滤器进行过滤。
    2. 对于第一次纯化试验,强烈推荐使用线性梯度洗脱步骤

  5. 储存开凯
    1. 为了储存纯化的KaiC蛋白质,使用10kDa离心过滤器,在4℃,3,000rpm下将KaiC样品从最后步骤(步骤D7)浓缩至1-5mg/ml。
    2. 使用NAP-5缓冲液交换柱的反应缓冲液的交换缓冲液。将KaiC样品浓缩至1-5 mg/ml,并将蛋白质储存在-80°C。
    注意:
    1. 用Bradford测定法确定样品的浓度。
    2. 我们建议立即使用蛋白质进行以下程序。为了储存,加入20%甘油,在液氮中冷冻并保持在-80℃。请注意,长期存储(2-3个月)可能会导致KaiC的活动损失。

  6. KaiC样品的制备用于SDS-PAGE分析
    1. 取出储存在-80°C的KaiC蛋白质并在冰上解冻。
    2. 用补充有80μg/ml卡那霉素的反应缓冲液将KaiC稀释至0.5mg/ml,终体积为30μl。
    3. 将4μl稀释的KaiC样品转移到标记为管1至5的清洁和灭菌的PCR管中
    4. 向样品管1中加入2μl3x负载染料,并在100℃孵育5分钟。
    5. 将样品1储存在-20°C,并将样品2-5 - 4°C转移。
    6. 24小时后取出样品2,如上所述进行准备。
    7. 将样品管3-5置于PCR机中,并将样品温度设定为30℃。
    8. 每4小时取一管,如前所述进行准备。

  7. KaiC自动磷酸酶活性的SDS-PAGE分析
    1. 取出储存在-80°C的KaiC蛋白质并在冰上解冻。
    2. 用补充有80μg/ml卡那霉素的反应缓冲液将KaiC稀释至0.5mg/ml,终体积为30μl。
    3. 将4μl稀释的KaiC样品转移到标记为管1至5的清洁和灭菌的PCR管中
    4. 向样品管1中加入2μl3x负载染料,并在100℃孵育5分钟。
    5. 将样品1储存在-20°C,并将样品2-5 - 4°C转移。
    6. 24小时后取出样品2,如上所述进行准备。
    7. 将样品管3-5置于PCR机中,并将样品温度设定为30℃。
    8. 每4小时取一管,如前所述进行准备。
    注意:
    1. 在这个方案中,每个孔中最好的开CaC量是2-3μg。
    2. 在样品旁边的孔中加入10μl加载染料,以减少"微笑效应"

数据分析

  1. KaiC纯度评估
    由于蛋白纯度对于蛋白质的活性和稳定性很重要,因此通过SDS-PAGE检查纯化的KaiC蛋白的纯度。如图1所示,用该方案制备的KaiC是高纯度的,分子量对应于58kDa的预测值。


    图1.纯化KaiC
    的SDS-PAGE分析
  2. 开凯自动磷酸酶活性分析
    为了确定纯化的KaiC的自磷酸酶活性,通过SDS-PAGE分析在不同时间点(1-5)制备的样品。图2清楚地显示了在30℃下在反应缓冲液中单独孵育时KaiC的去磷酸化。下段是去磷酸化的KaiC,上限是磷酸化的KaiC

    图2.KaiC的自动磷酸酶活性的SDS-PAGE分析。在30℃孵育前,将KaiC在4℃下孵育24小时(-24小时)。每4小时取样一次。

  3. 定量KaiC自动磷酸酶活性
    为了定量分析KaiC的去磷酸化,使用ImageJ(Schneider等人,2012)分析SDS-PAGE凝胶。在ImageJ中打开SDS-PAGE凝胶的高质量图像。在第一个通道中打开KaiC乐队,并选择"Analyze-Gels-Select First Lane"。将框架移动到下一个通道,同时保持其大小不变以覆盖开启频段,然后使用"Analyze-Gels-Select Next Lane"功能。对所有车道重复此过程。使用"Analyze-Gels-Plot Lanes"功能绘制KaiC波段的峰面积。然后用线工具绘制基线,以关闭对应于凝胶中KaiC带的独立峰面积。使用"魔杖工具"逐个选择封闭的峰值区域。一个新窗口将显示计算出的峰面积。最后,计算每个泳道中磷酸化KaiC带的百分比,并分析Excel或其他类似软件中的数据(图3)。


    图3.KaiC的自动磷酸酶活性的定量分析。通过ImageJ分析每个泳道中磷酸化KaiC(P-KaiC)的百分比。
    注意:ImageJ中的基线校正以某种方式是任意的,但对于所有峰值应保持不变。

食谱

  1. PSP缓冲区
    50mM Tris-HCl(pH8.0)
    150 mM NaCl
    1 mM DTT
    0.01%Tween-20
    1 mM ATP
    5mM MgCl 2
  2. 缓冲区A
    50mM Tris-HCl(pH8.0)
    1 mM DTT
    0.01%Tween-20
    1 mM ATP
    5mM MgCl 2
  3. 缓冲区B
    50mM Tris-HCl(pH8.0)
    1 M NaCl
    1 mM DTT
    0.01%Tween-20
    1 mM ATP
    5mM MgCl 2
  4. 反应缓冲液
    50mM Tris-HCl(pH8.0)
    150 mM NaCl
    5 mM ATP
    5mM MgCl 2
    0.01%Tween-20
    0.5 mM EDTA
  5. 1 M IPTG
    将1 g IPTG溶解在4,196μl去离子水中,制成1M溶液 用注射器和0.22μm过滤器过滤灭菌
  6. 10x运行缓冲区
    144克甘氨酸
    30.3克Tris
    10g SDS
    将ddH 2 O添加到1 L
  7. 8%分离凝胶(表1)

    表1:8%分离凝胶的制备


  8. 5%堆积凝胶(表2)

    表2. 5%堆积凝胶的制备


  9. 考马斯蓝染色液(1升)
    1克考马斯蓝G250
    650ml ddH 2 O
    250毫升甲醇
    100 ml乙酸
  10. 脱色缓冲液(1L)
    600毫升ddH 2 O
    100ml乙酸 

注意:

  1. 所有缓冲液均应用0.45μm过滤器灭菌。
  2. 将缓冲液存放在4°C不到一周的时间

致谢

这项工作是由向S.L.来自中国湖北省国家自然科学基金(91330113,31670768),中国湖北省(D20161204)和中国三峡大学。参考文献发表在Sci Rep 6:25129中。

参考文献

  1. Chen,W.,Liu,S.and Liu,S。(2013)。< a class ="ke-insertfile"href ="http://xueshu.baidu.com/s?wd=paperuri%3A% 28a24d3f42065fb6a5f7f97c8aa7266b05%29&filter = sc_long_sign&tn = SE_xueshusource_2kduw22v&sc_vurl = http%3A%2F%2Fen.cnki.com.cn%2FArticle_en%2FCJFDTotal-SWWL201311002.htm&ie = utf-8&sc_us = 17893988670517441677"target ="_ blank">核心分子机制的进展蓝细菌的昼夜节律振荡器。生物物理学报29(11):801-810。
  2. Dong,P.,Fan,Y.,Sun,J.,Lv,M.,Yi,M.,Tan,X.and Liu,S。(2016)。  KaiA和KaiC之间的动态相互作用过程对蓝藻昼夜节律振荡器至关重要。 Rep 6:25129.
  3. Egli,M.和Johnson,C.H。 (2013)。没有转录的昼夜节律纳米机器或翻译。 Curr Opin Neurobiol 23(5):732-740。
  4. Egli,M。(2015)。  结构和生物物理方法分析时钟功能和机制。 方法Enzymol 551:223-266。
  5. Johnson,CH,Stewart,PL和Egli,M。(2011)。蓝藻昼夜节律系统:从生物物理学到生物学演化。 Annu Rev Biophys 40:143-167。
  6. Mackey,SR,Golden,SS and Ditty,JL(2011)。  蓝藻昼夜节律时钟的时间机器遗传学。高分辨率Genet 74:13-53。
  7. Ma,P.,Woelfle,MA和Johnson,CH(2013)。  昼夜节律系统在蓝细菌中赋予的进化适应度增强。 混沌Solitons分形 50:65-74。
  8. Nakajima,M.,Imai,K.,Ito,H.,Nishiwaki,T.,Murayama,Y.,Iwasaki,H.,Oyama,T.and Kondo,T。(2005)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15831759"target ="_ blank">体外研究蓝藻KaiC磷酸化的昼夜节律振荡/a> 科学 308(5720):414-415。
  9. Nishiwaki,T.和Kondo,T。(2012)。蓝细菌时钟蛋白KaiC的昼夜循环自磷酸化通过形成ATP作为中间体而发生。 J Biol Chem 287(22):18030-18035。
  10. Pattanayak,G。和Rust,MJ(2014)。蓝藻时钟和新陈代谢。 Curr Opin Microbiol 18:90-95。
  11. Roenneberg,T.和Merrow,M。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27218855"target ="_ blank" >昼夜节律钟和人体健康 Curr Biol 26(10):R432-443。
  12. Rust,MJ(2012)。  有序的蓝色轮子时钟。 Proc Natl Acad Sci USA 109(42):16760-16761。
  13. Schneider,CA,Rasband,WS and Eliceiri,KW(2012)。  NIH Image to ImageJ:25年的图像分析。 Nat方法 9(7):671-675。
  14. Shi,SQ,Ansari,TS,McGuinness,OP,Wasserman,DH and Johnson,CH(2013)。  昼夜节律紊乱导致胰岛素抵抗和肥胖。 Curr Biol 23(5):372-381。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Q., Yu, L., Tan, X. and Liu, S. (2017). Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity. Bio-protocol 7(4): e2140. DOI: 10.21769/BioProtoc.2140.
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