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In vitro Autophosphorylation and Phosphotransfer Assay of Cyanobacterial Histidine Kinase 2
蓝藻组胺酸激酶2的体外自磷酸化和磷酸根传递转分析   

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本实验方案简略版
Frontiers in Plant Science
Feb 2016

Abstract

This is a detailed protocol of an autophosphorylation and phosphotransfer activities of Synechocystis sp. PCC 6803 full-length Histidine Kinase 2 (Hik2) protein described by Ibrahim et al., 2016. In this protocol, radioactively labelled ATP was used to study an autophosphorylation and phosphotransfer activity of the full-length Hik2 protein.

Keywords: Histidine Kinase 2 (组氨酸激酶2), Autophosphorylation (自磷酸化), Phosphotransfer (磷酸转移), Rre1 (Rre1), RppA (RppA), Synechocystis sp. PCC 6803 (集胞藻PCC 6803)

Background

Protein phosphorylation is an important post-translational modification of proteins that takes place in every living organism. The activity of protein kinases, the enzyme that catalyses the phosphorylation of proteins, was first described by Burnett and Kennedy in 1954, where they showed phosphorylation of casein by a liver enzyme (Burnett and Kennedy, 1954). However, its significance was not appreciated until the 1970s and 1980s (Cohen, 2002). The transfer of the γ-phosphate from an ATP molecule to proteins can be studied using coupled assays or directly with radioactively labelled ATP. Kinase assays based on incorporation of 32P can easily be followed by autoradiography, whereas coupled assays require monitoring of indirect reporter enzyme-catalysed colorimetric or chemiluminescence signals. The work presented here was conducted using radioactive ATP. Serine/threonine-type protein kinases dominate in eukaryotes, while in prokaryotes histidine kinases are the primary protein kinases involved in signal transduction. A histidine kinase catalyses the transfer of only γ-phosphate from an ATP molecule to its conserved histidine residue and transfers phosphoryl group to its response regulator (see Figure 1), but cannot catalyse the transfer of α-phosphate of ATP (Pernestig et al., 2001). Therefore [α-32P]ATP can be used as a negative control when characterising the autophosphorylation activity of putative histidine kinases.


Figure 1. Domain architecture of two-component system. The sensor domain is indicated by oval, the dimerisation and phosphoaccepting (DHp) domain by a cylinder, and the catalytic and ATP-binding (CA) domain by a triangle; receiver (Rec) domain by a hexagon; effector (Effe) domain by a pentagon.

Materials and Reagents

  1. Eppendorf tubes
  2. 50 ml Falcon tubes
  3. Pipette tips  
  4. Chelating Sepharose Fast Flow (GE Healthcare, catalog number: 17057501 )
  5. PD-10 desalting columns (GE Healthcare, catalog number: 17085101 )
  6. 1 ml cuvette
  7. Polyethylene bags (Thermo Fisher Scientific, Fisher Scientific, catalog number: 01817200 )
  8. BL21-DE3 E.coli cells containing Hik2, Rre1, and RppA clones. Each protein should be prepared fresh for each assay.
  9. pET-21b vector (Invitrogen)
  10. One Shot® TOP10 chemically competent E. coli (Thermo Fisher Scientific, InvitrogenTM, catalog number: C404006 )
  11. BL21-(DE3) chemical competent cells
  12. NdeI endonuclease (New England BioLabs, catalog number: R0111S )
  13. XhoI endonuclease (New England BioLabs, catalog number: R0146S )
  14. KpnI
  15. Primers were purchased from Eurofins MWG Operon, Germany.
  16. Deoxynucleoside triphosphate set (Sigma-Aldrich, catalog number: DNTP-RO )
  17. Phusion® high-fidelity DNA polymerase (New England BioLabs, catalog number: M0530S )
  18. RNase/DNase free water
  19. GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K0691 )
  20. Tris-HCl
  21. Bovine serum albumin (BSA) (New England BioLabs, catalog number: B9000S )
  22. DNA loading dye (6x) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0611 )
  23. Agarose
  24. Fermentas Gel Extraction Kit
  25. T4-ligase (New England BioLabs, catalog number: M0202S )
  26. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518-25G )
  27. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Melford Laboratories, catalog number: MB1008 )
  28. Imidazole
  29. Bradford reagent (Sigma-Aldrich, catalog number: B6916-500ML )
  30. 500 μCi [γ-32P]-ATP (6,000 Ci mmol-1) (PerkinElmer, catalog number: NEG502Z500UC )
  31. Adenosine 5’-triphosphate disodium salt hydrate (Sigma-Aldrich, catalog number: A2383-5G )
  32. Luria broth (LB), low salt, granulated (Melford Laboratories, catalog number: GL1703 )
  33. KCl
  34. MgSO4
  35. MgCl2
  36. Glucose
  37. NaCl
  38. PMSF
  39. Glycerol
  40. SDS
  41. β-2-mercaptoethanol
  42. 30% acrylamide/bis-acrylamide
  43. APS
  44. TEMED
  45. Precision Plus Protein All Blue Standards (Bio-Rad Laboratories, catalog number: 161-0373 )
  46. LB medium (see Recipes)
  47. Super optimal broth with catabolic repressor (SOC) (see Recipes)
  48. Lysis buffer/wash buffer 1 (see Recipes)
  49. Wash buffer 2 (see Recipes)
  50. Wash buffer 3 (see Recipes)
  51. Elution buffer (see Recipes)
  52. PD-10 desalting column equilibration buffer (see Recipes)
  53. 5x kinase reaction buffer (see Recipes)
  54. 5x ATP mix (see Recipes)
  55. SDS-PAGE Laemmli sample buffer (see Recipes)
  56. SDS-PAGE (see Recipes)
  57. 1x SDS-PAGE running buffer (see Recipes)

Equipment

  1. PCR machine
  2. Pipette shield
  3. 2 L Erlenmeyer flask
  4. Bottle assembly, J-Lite PC-1000, polycarbonate (Beckman Coulter, catalog number: 363676 )
  5. Backment Coulter AvantiTM J-30I centrifuge (Beckman Coulter, model: Avanti J-30I )
  6. EmulsiFlex-C3 homogenizer (Abestin, model: EmulsiFlex-C3 )
  7. Bottle assembly, polycarbonate, 50 ml (Beckman Coulter, order number: 357000 )
  8. Heating block
  9. Phosphorimager (Molecular Dynamics)
  10. Fume-hood
  11. GM counters
  12. Perspex Eppendorf holders
  13. JA-30.5 Ti rotor (Beckman Coulter, model: JA-30.5 Ti Rotor )
  14. JLA-9.1000 rotor, fixed angle (Beckman Coulter, catalog number: 366754 )
  15. Gilson pipettes
  16. Plexiglas shielding
  17. Mini-PROTEAN® Electrophoresis system (Bio-Rad Laboratories, catalog number: 1658000EDU )
  18. Bio-Rad PowerPac (Bio-Rad Laboratories, catalog number: 1645050 )
  19. Phosphor cassette (Molecular Dynamics)
  20. Phosphor plate (Molecular Dynamics)
  21. Image Eraser (Molecular Dynamics)

Software

  1. ImageQuant software (Molecular Dynamics)

Procedure

  1. Cloning of Hik2, Rre1 and RppA genes
    1. Polymerase Chain Reaction (PCR): Perform PCR reaction for coding sequences corresponding to the full-length Synechocystis sp. PCC6803 Hik2 (slr1147), Rre1 (slr1783), and RppA (sll0797) from Synechocystis sp. PCC 6803 genomic DNA using primer pairs listed in Table 1. Digest the PCR product of full-length Hik2 (Hik2) with NdeI and XhoI (New England BioLabs) and clone into a pET-21b vector (Invitrogen). Digest Rre1 and RppA with KpnI and XhoI endonucleases (New England BioLabs) and clone into pETG-41A (EMBL) expression vector. Prepare the following PCR reaction in a total volume of 50 µl:
      1. Add 1 µl of dNTPs (deoxynucleoside triphosphates, 10 mM each), final concentration is 200 µM each.
      2. 4 µl of the 10x HF Phusion DNA polymerase reaction buffer.
      3. 2.5 µl of forward primer (final concentration of 0.5 µM).
      4. 2.5 µl of reversed primer (final concentration of 0.5 µM).
      5. 5 ng of template DNA.
      6. 0.5 µl of Phusion DNA polymerase (final concentration of 0.01 units).
      7. Adjust the volume to 50 µl with RNase/DNase free water.
      8. Incubate in the PCR machine. For PCR programme, see Table 2.
      9. Purify the PCR amplicon from enzymatic reaction buffers using GeneJET Gel Extraction Kit and elute the DNA sample in 50 µl of Tris-HCl (pH 8.0).

        Table 1. Primer pairs used for cloning Hik2, Rre1, and RppA genes

        Note: Base pairs in lower case are restriction site overhangs.

        Table 2. PCR cycling programme


    2. Restriction endonuclease digest: Carry out the following double digestion reaction in a total volume of 50 µl:
      1. To a 1.5 ml Eppendorf tube, add 8 µg of PCR product or 4 µg of plasmid.
      2. 1 µl of the appropriate endonucleases (40 units final).
      3. 0.25 µl of the 20 mg ml-1 BSA (final concentration of 100 µg ml-1).
      4. 5 µl of 10-fold concentrated NEB-buffer.
      5. Adjust the volume to 50 µl with RNase/DNase free water.
      6. Incubate reactions at 37 °C for 3 h.
      7. Add 10 µl of 6 fold concentrated DNA loading dye and loaded onto an agarose 1% gel.
      8. Once the DNA fragments are separated on agarose gel, cut the bands and purified from the gel using Fermentas Gel Extraction Kit. 
    3. Ligation: Carry out the following ligation reaction in a total reaction volume of 20 µl.
      1. Mix the double digested plasmid and PCR product in 1:3 molar ratios, respectively.
      2. Add 1 µl of T4-ligase (20 units final).
      3. 2 µl of 10x T4 ligase buffer.
      4. Adjust the volume to 20 µl with DNase/RNase-free water.
      5. Incubate reaction at 25 °C for 15 min or at 16 °C overnight.
    1. Recombinant transformation
      1. To a pre-chilled 1.5 ml Eppendorf tube, aliquot 50 μl One Shot TOP10 chemical competent cells.
      2. Add 0.2 μl (~2 ng) of ligation product into the cells.
      3. Incubated on ice for 30 min.
      4. Heat shock at 42 °C for 90 sec and incubated on ice for further 60 sec.
      5. Diluted to 1:10 by adding 950 μl of preheated (at 37 °C) SOC medium.
      6. Incubated at 37 °C whilst agitating at 200 rpm for 1 h.
      7. Briefly spin and discard ~800 µl of the medium. Resuspend the pellet with the remaining medium and plate onto agarose LB-Amp plate. 
      8. Incubated at 37 °C overnight. 
      9. The following day, inoculate a single colony into 5 ml LB-medium containing 100 μl ml-1 ampicillin and incubated at 37 °C overnight whilst agitating at 200 rpm.
      10. Extract the clones and confirm the presence of clone by double digesting with the above restriction and sequencing.
    1. Transform the above clone into BL21-(DE3) chemical competent cells as above for recombinant protein production.

  2. Protein production and purification
    1. Inoculate a single colony of BL21-DE3 E. coli cells containing Hik2 clone into 10 ml Luria broth (LB) growth medium (Sambrook et al., 1989) supplemented with 100 μg ml-1 ampicillin. Incubate at 37 °C while shaking at 200 rpm.
    2. The following morning, in 2 L Erlenmeyer flask, dilute the above starter culture at 1:100 in 1 L LB medium supplemented with 100 μg ml-1 ampicillin. 
    3. Incubate at 37 °C while shaking at 200 rpm. Monitor the optical density at 600 nm until it reaches ~0.55.
    4. Induce protein expression with 0.5 mM IPTG.
    5. Incubate the induced bacterial culture for a further 16 h at 16 °C while shaking at 150 rpm.
    6. Transfer the bacterial cells into 1 L polycarbonate bottle and harvest by centrifugation at 9,000 x g for 10 min at 4 °C.
    7. Resuspend the pellet in 20 ml of the lysis buffer/wash buffer 1 per 1 L of culture.
    8. Lyse the cells by passing through EmulsiFlex-C3 homogenizer two times and transfer the lysate to 50 ml polycarbonate bottle.
    9. Separate the lysate by centrifugation at 39,000 x g for 20 min at 4 °C. And transfer the supernatant to 50 ml Falcon tube.
    10. Equilibrate the Ni2+ affinity chromatography column with 5 ml (5-bead volume) of the lysis buffer.
    11. Apply the supernatant from step B9 to the equilibrated Ni2+ affinity chromatography column.
    12. Wash the column with 30 ml of lysis buffer/wash buffer 1 containing 25 mM imidazole.
    13. Wash with 30 ml of wash buffer 2 containing 50 mM imidazole.
    14. Wash with 5 ml of wash buff 3 containing 100 mM imidazole.
    15. Elute the protein with 1 ml of elution buffer. Repeat this step B15 three more times.

  3. Desalting: prepare the following steps in the cold room
    1. Remove the top cap of PD-10 desalting column and pour off the column storage solution.
    2. Cut the sealed end of the column at notch.
    3. Fill up the column with PD-10 desalting/equilibration buffer and allow the equilibration buffer to enter the packed bed completely.
    4. Repeat step C3 4 times.
    5. Discard the flow-through.
    6. Add maximum 2.5 ml of sample to the column. For sample volumes less than 2.5 ml, add desalting/equilibration buffer to adjust the volume to 2.5 ml after the sample has entered the packed bed completely.
    7. Let the sample or desalting/equilibration buffer enter the packed bed completely.
    8. Discard the flow-through.
    9. Place a 1.5 ml Eppendorf tube for sample collection under the column.
    10. Elute with 3.5 ml desalting/equilibration buffer and collect 3 times 1 ml and finally 0.5 ml of eluate.
    11. Use the eluate for autophosphorylation immediately.

  4. Determination of protein concentration using Bradford assay
    1. Add 33 μl of Hik2 protein solution into 1.5 ml Eppendorf tube. To the second 1.5 ml Eppendorf tube add 33 µl of elution buffer and label this tube as blank.
    2. To each tube, add 1 ml of Bradford reagent and incubated at room temperature (~22 °C) for 10 min.
    3. Transfer the samples into 1 ml cuvette.
    4. Measure the absorbance for each sample at 595 nm and calculate protein concentration from BSA standard curve.

  5. In vitro autophosphorylation assay: prepare the following in ice
    1. Add 2 μM of purified Hik2 protein to 1.5 ml Eppendorf tube.
    2. Add 5 μl of 5-fold concentrated kinase reaction buffer.
    3. Adjust the volume to 20 μl with dH2O.
    4. Equilibrate the samples to 22 °C in heating block.
    5. Mix gently by vortexing for 1-2 sec.
    6. Initiate the autophosphorylation reaction by the addition of 5 μl of 5-fold concentrated ATP mix. Immediately start the stopwatch and let the autophosphorylation reaction proceed for 15 sec at 22 °C.
    7. Terminate the reaction by adding 6 μl of 5-fold concentrated Laemmli sample buffer (Laemmli, 1970).
    8. Separate the reaction products on a 12% reducing SDS-PAGE.
    9. Once protein separation is complete, rinse the gel 2-3 times with SDS running buffer and transfer the gel into a sealed polyethylene bag.
    10. Place the sealed bag against the phosphor screen and expose overnight.
    11. Visualise the incorporated γ-phosphate is using autoradiography.

  6. Phosphotransfer analysis
    1. Carry out an autophosphorylation reaction by mixing 12 µM of Hik2 protein in a total reaction volume of 150 µl containing 30 µl of the 5x concentrated kinase reaction buffer and 30 µl of 5x concentrated ATP mix.
    2. Incubate the reaction at 30 °C for 10 min.
    3. In the meantime, prepare 25 µM of each of the response regulator (MBP-Rre1 or MBP-RppA) in a total volume of 62.5 µl containing 12.5 µl of the 5x concentrated kinase reaction buffer.
    4. Prepare a control reaction lacking response regulator in the same way, except that replace response regulator protein solution with an equal volume of water.
    5. Remove 12.5 µl of the autophosphorylated Hik2 protein and add 3 µl of Laemmli sample buffer to stop the reactions. Label this tube as T0.
    6. For each phosphotransfer reaction, take 62.5 µl of the autophosphorylated radiolabeled Hik2 protein from step F1 and mix it with 62.5 µl of the response regulator or with the water control from steps F3 and F4. Kinase and response regulator should be present at a concentration of 1 µM and 5 µM, respectively. 
    7. Mix briefly by vortexing and incubated at 30 °C. 
    8. Remove 25 µl samples at 20, 40, 60, and 90 min and add 6 µl of Laemmli sample buffer to stop the reactions. 
    9. Resolve the proteins on 15% SDS-PAGE and visualized the presence of γ-32P using autoradiography.

      Table 3. Protein molecular weight

Data analysis


Figure 2. Recombinant vectors containing Hik2, Rre1 and RppA genes. The full-length Hik2 (A), Rre1 (B), and RppA (C) open reading frames are shown by red arrow. The C-terminal poly-histidine (His-tag) and the N-terminal MBP tags are shown by magenta. The f1 and Ori origins of replication are shown by yellow colour; an ampicillin resistant gene is shown by light green; purple and dark green shows the lacI and T7/lac promoters, respectively. The direction of arrow indicates the direction of translation of genes. Vector maps were drawn using SnapGene.


Figure 3. Autophosphorylation activity of Hik2. An autoradiograph (autorad) showing autophosphorylated recombinant Hik2 protein. 2 μM of purified Hik2 protein was assayed in the presence of 2.5 μCi ATP at 22 °C. Hik2 protein was then resolved on a 12% SDS-PAGE and the incorporation of 32P was monitored by autoradiography.


Figure 4. Phosphotransfer activity of Hik2 to Rre1 and RppA response regulators. An autoradiograph (autorad) showing autophosphorylated recombinant Hik2 protein. 2 μM of purified Hik2 protein was assayed in the presence of 2.5 μCi ATP at 22 °C. Hik2 protein was then resolved on a 12% SDS-PAGE and the incorporation of 32P was monitored by autoradiography.

Notes

  1. Radiation Hazards: possible routes of internal and external contamination
    1. Internal contamination could occur while mixing samples by vortices. Samples should be mixed in the fume-hood to reduce the potential hazard posed by volatile vapour.
    2. Aerosol: The isotope stock should be opened inside a fume-hood to prevent inhalation of aerosol.
    3. Spillage: Spillage should be monitored using GM counters and the use of the laboratory spill kit.
    4. External contamination may also occur while handling the radioactive samples. Appropriate shielding materials should be used to reduce external contamination. All radiation samples should be used behind Perspex shielding.
    5. In addition, solid type Perspex Eppendorf holders should be used to further reduce exposure.

Recipes

  1. LB medium
    Dissolve 20 g of LB broth, low salt (Lennox L Broth), granulated in 1 L of dH2O
  2. Super optimal broth with catabolic repressor (SOC)
    To 10 ml LB-broth filter sterilised:
    2.5 mM KCl
    10 mM MgSO4
    10 mM MgCl2
    20 mM glucose
  3. Lysis buffer/wash buffer 1
    300 mM NaCl
    20 mM Tris-HCl, pH 7.4
    25 mM imidazole
    1 mM PMSF
  4. Wash buffer 2
    300 mM NaCl
    20 mM Tris-HCl, pH 7.4
    50 mM imidazole
  5. Wash buffer 3
    300 mM NaCl
    20 mM Tris-HCl, pH 7.4
    100 mM imidazole
  6. Elution buffer
    300 mM NaCl
    20 mM Tris-HCl, pH 7.4
    500 mM imidazole
  7. PD-10 desalting column equilibration buffer
    Tris-HCl (10 mM final, pH 7.4)
  8. 5x kinase reaction buffer (1 ml)
    250 mM Tris-HCl (pH 7.5)
    250 mM KCl
    50% glycerol
    50 mM MgCl2
  9. 5x ATP mix
    2.5 mM ATP
    25 μCi [γ-32P]-ATP (3,000 Ci mmol-1)
  10. 5x SDS-PAGE Laemmli sample buffer
    164.5 mM Tris-HCl, pH 6.8
    26.3 % (w/v) glycerol
    5.25 % (w/v) SDS
    25 % (v/v) β-2-mercaptoethanol
    0.025% bromophenol blue
  11. SDS-PAGE


  12. 1x SDS running buffer (1 L)
    0.025 M Tris base
    0.192 M glycine
    0.1% SDS
    The pH as it is should be 8.3. No need to adjust

Acknowledgments

This protocol was adapted from an earlier report (Ibrahim et al., 2016). I.M.I thanks Queen Mary University of London for a graduate teaching studentship. The author would like to express his sincere gratitude to Prof. John F. Allen and Dr. Sujith Puthiyaveetil for their comments.
Conflict of Interest Statement: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Burnett, G. and Kennedy, E. P. (1954). The enzymatic phosphorylation of proteins. J Biol Chem 211(2): 969-980.
  2. Cohen, P. (2002). The origins of protein phosphorylation. Nat Cell Biol 4(5): E127-130.
  3. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680-685.
  4. Ibrahim, I. M., Puthiyaveetil, S. and Allen, J. F. (2016). A two-component regulatory system in transcriptional control of photosystem stoichiometry: Redox-dependent and sodium ion-dependent phosphoryl transfer from cyanobacterial histidine kinase Hik2 to response regulators Rre1 and RppA. Front Plant Sci 7: 137.
  5. Pernestig, A. K., Melefors, O. and Georgellis, D. (2001). Identification of UvrY as the cognate response regulator for the BarA sensor kinase in Escherichia coli. J Biol Chem 276(1): 225-231.
  6. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular cloning: A laboratory manual. Second edition. Cold Spring Harbor, New York: Greene Publishing Associates and John Wiley & Sons. pp: 1126.

简介

这是集胞藻的自磷酸化和磷酸转移活性的详细方案。在该方案中,使用放射性标记的ATP来研究全长Hik2的自磷酸化和磷酸转移活性,所述磷酸转移活性是由Ibrahim等人在2016年描述的。蛋白。
关键字:组氨酸激酶2,自身磷酸化,磷酸转移,Rre1,RppA,集胞藻。 PCC 6803

[背景] 蛋白磷酸化是发生在每个活生物体中的蛋白质的重要​​翻译后修饰。蛋白激酶(催化蛋白质磷酸化的酶)的活性首先由Burnett和Kennedy在1954年描述,其中它们通过肝酶显示酪蛋白的磷酸化(Burnett和Kennedy,1954)。然而,它的意义直到20世纪70年代和80年代才被欣赏(Cohen,2002)。可以使用偶联测定或直接用放射性标记的ATP研究磷酸γ-磷酸从ATP分子转移到蛋白质。基于掺入32 P的激酶测定可以容易地进行放射自显影,而偶联测定需要监测间接报道酶催化的比色或化学发光信号。这里介绍的工作是使用放射性ATP。丝氨酸/苏氨酸型蛋白激酶在真核生物中占主导地位,而在原核生物中组氨酸激酶是参与信号转导的主要蛋白激酶。组氨酸激酶催化仅将γ-磷酸从ATP分子转移到其保守的组氨酸残基,并将磷酰基转移到其响应调节物(参见图1),但不能催化α-磷酸盐的转移的ATP(Pernestig等人,2001)。因此,当表征推定的组氨酸激酶的自磷酸化活性时,[α - 32 P] ATP可用作阴性对照。


图1.双组分系统的域架构。 传感器域由椭圆形表示,二聚化和phosphoaccepting(DHp)域由圆柱表示,催化和ATP结合(CA)域由三角形表示;接收器(Rec)域;效应子(Effe)结构域。通过五边形。


关键字:组氨酸激酶2, 自磷酸化, 磷酸转移, Rre1, RppA, 集胞藻PCC 6803

材料和试剂

  1. Eppendorf管
  2. 50ml Falcon管
  3. 移液器提示
  4. 螯合Sepharose Fast Flow(GE Healthcare,目录号:17057501)
  5. PD-10脱盐柱(GE Healthcare,目录号:17085101)
  6. 1 ml比色杯
  7. 聚乙烯袋(Thermo Fisher Scientific,Fisher Scientific,目录号:01817200)
  8. 含有Hik2,Rre1和RppA克隆的BL21-DE3大肠杆菌细胞。每个蛋白质应该为每个测定新鲜制备
  9. pET-21b载体(Invitrogen)
  10. One Shot ® TOP10化学感受能力。大肠杆菌(Thermo Fisher Scientific,Invitrogen TM ,目录号:C404006)
  11. BL21-(DE3)化学感受态细胞
  12. Nde I内切核酸酶(New England BioLabs,目录号:R0111S)
  13. Xho I内切核酸酶(New England BioLabs,目录号:R0146S)
  14. Kpn I
  15. 引物购自Eurofins MWG Operon,德国
  16. 脱氧核苷三磷酸酯(Sigma-Aldrich,目录号:DNTP-RO)
  17. Phusion 高保真DNA聚合酶(New England BioLabs,目录号:M0530S)
  18. RNase/DNase游离水
  19. GeneJET凝胶提取试剂盒(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:K0691)
  20. Tris-HCl
  21. 牛血清白蛋白(BSA)(New England BioLabs,目录号:B9000S)
  22. DNA装载染料(6x)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:R0611)
  23. 琼脂糖
  24. Fermentas凝胶提取试剂盒
  25. T4连接酶(New England BioLabs,目录号:M0202S)
  26. 氨苄青霉素钠盐(Sigma-Aldrich,目录号:A9518-25G)
  27. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Melford Laboratories,目录号:MB1008)
  28. 咪唑
  29. Bradford试剂(Sigma-Aldrich,目录号:B6916-500ML)
  30. (PerkinElmer,目录号:NEG502Z500UC)的500μCi[em]γ - [32 P] -ATP(6,000 Ci mmol -100)
  31. 腺苷5'-三磷酸二钠盐水合物(Sigma-Aldrich,目录号:A2383-5G)
  32. Luria肉汤(LB),低盐,粒化(Melford Laboratories,目录号:GL1703)
  33. KCl
  34. MgSO 4 4 /
  35. MgCl 2
  36. 葡萄糖
  37. NaCl
  38. PMSF
  39. 甘油
  40. SDS
  41. -2-巯基乙醇
  42. 30%丙烯酰胺/双丙烯酰胺
  43. APS
  44. TEMED
  45. Precision Plus Protein All Blue Standards(Bio-Rad Laboratories,目录号:161-0373)
  46. LB介质(见配方)
  47. 具有分解代谢阻遏物(SOC)的超级最佳肉汤(参见配方)
  48. 裂解缓冲液/洗涤缓冲液1(见配方)
  49. 洗涤缓冲液2(见配方)
  50. 清洗缓冲液3(参见配方
  51. 洗脱缓冲液(见配方)
  52. PD-10脱盐柱平衡缓冲液(见配方)
  53. 5x激酶反应缓冲液(见配方)
  54. 5x ATP混合物(见配方)
  55. SDS-PAGE Laemmli样品缓冲液(参见配方)
  56. SDS-PAGE(参见配方)
  57. 1x SDS-PAGE运行缓冲液(参见配方)

设备

  1. PCR机
  2. 移液管护罩
  3. 2 L锥形瓶
  4. 瓶组件,J-Lite PC-1000,聚碳酸酯(Beckman Coulter,目录号:363676)
  5. Backment Coulter Avanti TM supec J-30I离心机(Beckman Coulter,型号:Avanti J-30I)
  6. EmulsiFlex-C3匀浆器(Abestin,型号:EmulsiFlex-C3)
  7. 瓶组件,聚碳酸酯,50ml(Beckman Coulter,订货号:357000)
  8. 加热块
  9. 磷光体(分子动力学)
  10. 通风橱
  11. GM计数器
  12. Perspex Eppendorf持有人
  13. JA-30.5Ti转子(Beckman Coulter,型号:JA-30.5Ti Rotor)
  14. JLA-9.1000转子,固定角(Beckman Coulter,目录号:366754)
  15. 吉尔森移液器
  16. 有机玻璃屏蔽
  17. Mini-PROTEAN 电泳系统(Bio-Rad Laboratories,目录号:1658000EDU)
  18. Bio-Rad PowerPac(Bio-Rad Laboratories,目录号:1645050)
  19. 磷光体盒(Molecular Dynamics)
  20. 磷光板(Molecular Dynamics)
  21. 图像橡皮擦(分子动力学)

软件

  1. ImageQuant软件(分子动力学)

程序

  1. 克隆Hik2 , Rre1 和 RppA 基因
    1. 聚合酶链式反应(PCR):对对应于全长集胞藻属的孢子进行编码序列的PCR反应。来自集胞藻属物种的PCC6803 Hik2(slr1147),Rre1(slr1783)和RppA(sll0797)使用表1中列出的引物对,对PCC 6803基因组DNA进行PCR。用Nde I和Xho I(New England BioLabs)消化全长Hik2(Hik2)的PCR产物,克隆到pET-21b载体(Invitrogen)中。 Digest Rre1和RppA与Kpn I和Xho I I内切核酸酶(New England BioLabs)并克隆到pETG-41A(EMBL)表达载体中。在总体积50μl中制备以下PCR反应:
      1. 加入1μldNTP(脱氧核苷三磷酸,各10mM),最终浓度为200μM。
      2. 4μl的10×HF Phusion DNA聚合酶反应缓冲液。
      3. 2.5μl正向引物(终浓度为0.5μM)。
      4. 2.5μl反向引物(终浓度0.5μM)
      5. 5ng模板DNA。
      6. 0.5μlPhusion DNA聚合酶(终浓度0.01单位)。
      7. 用RNase/DNase游离水调节体积至50μl。
      8. 在PCR机中孵育。 PCR程序见表2.
      9. 使用GeneJET Gel Extraction Kit从酶反应缓冲液中纯化PCR扩增子,并将DNA样品在50μlTris-HCl(pH 8.0)中洗脱。

        表1.用于克隆Hik2 , Rre1 和 RppA 基因的引物对

        注意:小写的碱基对是限制性位点突出端。

        表2. PCR循环程序


    2. 限制性内切核酸酶消化:在50μl的总体积中进行以下双重消化反应:
      1. 向1.5ml Eppendorf管中加入8μgPCR产物或4μg质粒。
      2. 1μl适当的内切核酸酶(最终40单位)
      3. 0.25μl的20mg/ml BSA(最终浓度为100μg/ml)。
      4. 5μl10倍浓缩的NEB-缓冲液。
      5. 用RNase/DNase游离水调节体积至50μl。
      6. 孵育反应在37℃下3小时。
      7. 加入10μl的6倍浓缩DNA加样染料,并加载到琼脂糖1%凝胶上。
      8. 一旦DNA片段在琼脂糖凝胶上分离,切下带并使用Fermentas Gel Extraction Kit从凝胶中纯化。
    3. 连接:在20μl的总反应体积中进行以下连接反应。
      1. 混合双消化的质粒和PCR产物,分别为1:3的摩尔比。
      2. 加入1μlT4连接酶(最终20单位)。
      3. 2μl10×T4连接酶缓冲液。
      4. 用DNase/RNase-free水调节体积至20μl。
      5. 在25℃下反应15分钟或在16℃下反应过夜
    1. 重组转化
      1. 向预冷的1.5ml Eppendorf管中,等分50μlOne Shot TOP10化学感受态细胞。
      2. 向细胞中加入0.2μl(约2ng)连接产物
      3. 在冰上孵育30分钟。
      4. 在42℃热休克90秒,并在冰上孵育另外60秒。
      5. 通过加入950μl预热(在37℃)SOC培养基稀释至1:10。
      6. 在37℃下孵育,同时在200rpm下搅拌1小时。
      7. 短暂旋转并丢弃〜800μl的培养基。用剩余培养基重悬沉淀,平板在琼脂糖LB-Amp平板上。
      8. 在37℃孵育过夜。
      9. 第二天,将单个菌落接种到含有100μl/ml氨苄青霉素的5ml LB培养基中,并在37℃下孵育过夜,同时以200rpm搅拌。
      10. 提取克隆并通过用上述限制性酶切和测序双酶切确认克隆的存在
    1. 将上述克隆转化到BL21-(DE3)化学感受态细胞中,如上所述重组蛋白生产
  2. 蛋白质生产和纯化
    1. 接种BL21-DE3 E的单个菌落。将含有Hik2克隆的大肠杆菌细胞加入补充有100μg/ml的氨苄青霉素的10ml Luria肉汤(LB)生长培养基(Sambrook等人,1989)中。在37℃下孵育,同时以200rpm振摇
    2. 第二天早晨,在2L锥形烧瓶中,在补充有100μg/ml的氨苄青霉素的1L LB培养基中以1:100稀释上述发酵剂培养物。
    3. 在37℃下孵育,同时以200rpm振荡。监测600 nm处的光密度,直到它达到〜0.55
    4. 用0.5mM IPTG诱导蛋白表达
    5. 将诱导的细菌培养物在16℃下再孵育16小时,同时以150rpm振荡
    6. 将细菌细胞转移到1L聚碳酸酯瓶中,并通过在4℃下以9,000×g离心10分钟收获。
    7. 将沉淀重悬在20ml每1L培养物的裂解缓冲液/洗涤缓冲液1中。
    8. 通过EmulsiFlex-C3匀浆器两次裂解细胞,并将裂解物转移到50ml聚碳酸酯瓶中。
    9. 通过在4℃下以39,000×g离心20分钟来分离裂解物。并将上清转移到50ml Falcon管中。
    10. 用5ml(5珠体积)的裂解缓冲液平衡Ni超级亲和层析柱。
    11. 将来自步骤B9的上清液应用于平衡的Ni 2+ 2亲和层析柱
    12. 用30ml含有25mM咪唑的裂解缓冲液/洗涤缓冲液1洗涤柱子
    13. 用30ml含有50mM咪唑的洗涤缓冲液2洗涤。
    14. 用5ml含有100mM咪唑的洗涤缓冲液3洗涤
    15. 用1ml洗脱缓冲液洗脱蛋白。重复此步骤B15三次以上。

  3. 脱盐:在冷室中准备以下步骤
    1. 取出PD-10脱盐柱的顶盖,倒出柱存储溶液
    2. 在凹口处切割柱的密封端。
    3. 用PD-10脱盐/平衡缓冲液填充色谱柱,使平衡缓冲液完全进入填充床。
    4. 重复步骤C3 4次。
    5. 丢弃流出物。
    6. 向柱中加入最多2.5 ml的样品。对于小于2.5ml的样品体积,在样品完全进入填充床之后,加入脱盐/平衡缓冲液以将体积调节至2.5ml。
    7. 让样品或脱盐/平衡缓冲液完全进入填充床
    8. 丢弃流出物。
    9. 在柱下放置1.5ml用于样品收集的Eppendorf管
    10. 用3.5ml脱盐/平衡缓冲液洗脱,收集3次1ml,最后0.5ml洗脱液。
    11. 立即使用洗脱液进行自磷酸化。

  4. 使用Bradford测定法测定蛋白质浓度
    1. 加入33微升的Hik2蛋白溶液到1.5毫升离心管中。向第二个1.5ml Eppendorf管中加入33μl洗脱缓冲液,并将该管标记为空白
    2. 向每个试管中加入1ml Bradford试剂,并在室温(〜22℃)温育10分钟
    3. 将样品转移到1ml比色皿中
    4. 测量每个样品在595nm处的吸光度,并从BSA标准曲线计算蛋白质浓度
  5. 体外自磷酸化测定:在冰中制备以下物质
    1. 将2μM纯化的Hik2蛋白加入到1.5ml Eppendorf管中
    2. 加入5μl5倍浓缩的激酶反应缓冲液
    3. 用dH 2 O调节体积至20μl。
    4. 在加热块中将样品平衡至22℃。
    5. 通过涡旋1-2秒钟轻轻混匀。
    6. 通过加入5μl5倍浓缩的ATP混合物启动自磷酸化反应。立即启动秒表,让自磷酸化反应在22℃进行15秒
    7. 通过加入6μl的5倍浓缩的Laemmli样品缓冲液(Laemmli,1970)终止反应。
    8. 在12%还原SDS-PAGE上分离反应产物
    9. 一旦蛋白质分离完成,用SDS运行缓冲液冲洗凝胶2-3次,将凝胶转移到密封的聚乙烯袋中
    10. 将密封袋放在荧光屏上,过夜曝光
    11. 可视化所结合的磷酸盐是使用放射自显影。

  6. 磷转移分析
    1. 进行自磷酸化反应通过混合12μM的Hik2蛋白在总反应体积为150μl,包含30μl的5x浓缩的激酶反应缓冲液和30μl的5x浓缩的ATP混合物。
    2. 在30℃下孵育反应10分钟。
    3. 同时,在总体积62.5μl中制备25μM的每种响应调节剂(MBP-Rre1或MBP-RppA),其含有12.5μl5x浓缩的激酶反应缓冲液。
    4. 准备一个控制反应缺乏响应调节器相同的方式,除了替换反应调节蛋白溶液与等体积的水。
    5. 删除12.5微升自磷酸化Hik2蛋白,加入3微升Laemmli样品缓冲液停止反应。将此管标记为T0。
    6. 对于每个磷酸转移反应,取62.5μl来自步骤F1的自磷酸化放射性标记的Hik2蛋白,并将其与62.5μl响应调节剂或来自步骤F3和F4的水对照混合。激酶和反应调节剂应分别以1μM和5μM的浓度存在。
    7. 通过涡旋短暂混合并在30℃温育。
    8. 在20,40,60和90分钟取出25微升样品,加入6微升Laemmli样品缓冲液停止反应。
    9. 在15%SDS-PAGE上分离蛋白质,并使用放射自显影法显现γ - γ - 32的存在。

      表3.蛋白分子量

数据分析


图2.含有Hik2 , Rre1 和 RppA 基因的重组载体。 开放阅读框的全长 Hik2 (A), Rre1 (B)和 RppA 红色箭头。 C-末端多组氨酸(His-标签)和N-末端MBP标签以品红色显示。 f1和Ori复制起点用黄色表示;浅绿色显示氨苄青霉素抗性基因;紫色和深绿色分别显示lacI 和T7 em/lac 启动子。箭头的方向表示基因的翻译方向。使用SnapGene绘制矢量地图。


图3. Hik2的自磷酸化活性。 显示自磷酸化重组Hik2蛋白的放射自显影(自动)。在22℃下在2.5μCiATP存在下测定2μM纯化的Hik2蛋白。然后在12%SDS-PAGE上分离Hik2蛋白,并通过放射自显影监测 P的掺入。


图4.Hik2对Rre1和RppA应答调节剂的磷酸转移活性。 显示自磷酸化重组Hik2蛋白的放射自显影(自动)。在22℃下在2.5μCiATP存在下测定2μM纯化的Hik2蛋白。然后在12%SDS-PAGE上分离Hik2蛋白,并通过放射自显影监测32 P的掺入。

笔记

  1. 辐射危害:内部和外部污染的可能途径
    1. 当通过涡流混合样品时可能发生内部污染。样品应在通风柜中混合,以减少挥发性蒸气造成的潜在危害。
    2. 气溶胶:同位素原料应在通风橱内开放,以防止吸入气溶胶。
    3. 溢出:溢出应使用GM计数器和使用实验室溢出组件进行监控。
    4. 处理放射性样品时也可能发生外部污染。应使用适当的屏蔽材料以减少外部污染。所有辐射样品应在Perspex屏蔽后使用。
    5. 此外,应使用实体类型Perspex Eppendorf持有人进一步减少曝光。

食谱

  1. LB培养基
    将20g LB肉汤,低盐(Lennox L Broth)溶解,在1L dH 2 O中制粒
  2. 具有分解代谢阻遏物(SOC)的超级最佳肉汤
    向10ml LB-肉汤过滤器灭菌:
    2.5mM KCl
    10mM MgSO 4 10mM MgCl 2/
    20mM葡萄糖
  3. 裂解缓冲液/洗涤缓冲液1
    300 mM NaCl
    20mM Tris-HCl,pH7.4 25mM咪唑 1mM PMSF
  4. 洗涤缓冲液2
    300 mM NaCl
    20mM Tris-HCl,pH7.4 50mM咪唑
  5. 洗涤缓冲液3
    300 mM NaCl
    20mM Tris-HCl,pH7.4 100mM咪唑
  6. 洗脱缓冲液
    300 mM NaCl
    20mM Tris-HCl,pH7.4 500mM咪唑
  7. PD-10脱盐柱平衡缓冲液
    Tris-HCl(10mM终浓度,pH7.4)
  8. 5x激酶反应缓冲液(1ml) 250mM Tris-HCl(pH7.5) 250 mM KCl
    50%甘油 50mM MgCl 2/v/v
  9. 5x ATP混合物
    2.5 mM ATP
    25μCi[em]γ - [sup] 32 sup] -ATP(3000Ci mmol -1
  10. 5x SDS-PAGE Laemmli样品缓冲液
    164.5mM Tris-HCl,pH 6.8
    26.3%(w/v)甘油 5.25%(w/v)SDS
    25%(v/v)β-巯基乙醇
    0.025%溴酚蓝
  11. SDS-PAGE


  12. 1×SDS运行缓冲液(1L)
    0.025M Tris碱
    0.192M甘氨酸 0.1%SDS
    pH值应为8.3。无需调整

致谢

该协议改编自早期的报告(Ibrahim等人,2016)。我感谢伦敦皇后大学毕业的教学学生。作者要对John F. Allen教授和Sujith Puthiyaveetil博士的真诚感谢表示感谢。
利益冲突声明:作者声明,研究是在没有任何商业或财务关系,可以解释为潜在的利益冲突。

参考文献

  1. Burnett,G。和Kennedy,E.P。(1954)。 蛋白质的酶促磷酸化  J Biol Chem 211(2):969-980
  2. Cohen,P。(2002)。 蛋白磷酸化的起源  Nat Cell Biol 4(5):E127-130
  3. Laemmli,U.K。(1970)。 在噬菌体T4头部装配期间切割结构蛋白。 227(5259):680-685。
  4. Ibrahim,I.M.,Puthiyaveetil,S.and Allen,J.F。(2016)。 光系统化学计量学的转录控制的双组分调节系统:Redox依赖性和钠离子依赖性磷酰基转移从蓝细菌组氨酸激酶Hik2到反应调节剂Rre1和RppA。前植物科学 7:137.
  5. Pernestig,A.K.,Melefors,O.and Georgellis,D。(2001)。 将UvrY鉴定为BarA传感器激酶的同源反应调节剂 Escherichia coli 。  J Biol Chem 276(1):225-231。
  6. Sambrook,J.,Fritsch,E.F。和Maniatis,T。(1989)。 分子克隆:实验室手册。第二版。< Cold Spring Harbor,New York: Greene Publishing Associates 和 John Wiley&儿子。 pp:1126.
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ibrahim, I. M. (2016). In vitro Autophosphorylation and Phosphotransfer Assay of Cyanobacterial Histidine Kinase 2. Bio-protocol 6(23): e2036. DOI: 10.21769/BioProtoc.2036.
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