Abstract
Protein kinases are enzymes that phosphorylate proteins in a cell. Determination of kinase activity in reactions of phosphorylation is a very convenient way for a biochemical characterization of this group of enzymes. Here we describe a method to determine the activity of a recombinant Ser/Thr protein kinase using as a possible substrate MBP, H1, and BSA.
Materials and Reagents
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Escherichia coli (E.coli) strains - for cloning and for expression - DH5α, Rosetta (DE3 pLysS)
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Specific synthetic oligonucleotides
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PCR reagents (dNTP, MgCl2, PCR buffer, Taq-polymerase, DNA) (Thermo Fisher Scientific, catalog numbers: R0181 , EP0612 )
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Plasmids for cloning and for expression [pTZ57R/T, pET41a(+)]
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CaCl2 (Sigma-Aldrich, catalog number: C3306-250G ) (for transformation)
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Antibiotics (Ampicillin, Kanamycine, Chloramphenicol) (AppliChem, catalog numbers: A-0839 , A-1493 , A-6435 )
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Isopropyl thio-β-D-galactoside (IPTG) (Fermentas, catalog number: R0393 )
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Tris-HCl (Sigma-Aldrich, catalog number: T6791 )
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Ethylenediaminetetraacetic acid (EDTA) (AppliChem, catalog number: A-1104.0500 )
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Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815-5G )
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Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889-10G )
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Na3VO4 (Sigma-Aldrich, catalog number: S6508-10G )
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NaF (Sigma-Aldrich, catalog number: S7920-100G )
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β-glycerophosphate (Sigma-Aldrich, catalog number: G9422-50G )
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Sucrose (AppliChem, catalog number: A-1125 )
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Phenylmethylsulfonyl fluoride (PMSF) (AppliChem, catalog number: A-0999 )
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Benzamidine hydrochloride (Sigma-Aldrich, catalog number: B6506 )
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6-aminocaproic acid (Sigma-Aldrich, catalog number: A7824-25G )
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2-(N-morpholino)ethanesulfonic acid (MES) (Amresco, catalog number: Am-E169-0.05 )
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Myelin Basic Protein bovine (MBP) (Sigma-Aldrich, catalog number: M1891-5MG )
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Histone from calf thymus (H1) (Sigma-Aldrich, catalog number: H5505-100MG )
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Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 05470-1G )
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MgCl2 (AppliChem, catalog number: A-1447 )
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Brilliant Blue R Concentrate (Sigma-Aldrich, catalog number: B8647 )
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BCA Protein Assay Kit (Pierce Thermo Scientific, catalog number: 23227 )
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Sodium dodecyl sulfate, SDS (Sigma-Aldrich, catalog number: L3771 )
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TGX FastCast Acrylamide Starter Kit (12%) (Bio-Rad Laboratories, catalog number: 161-0175 )
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N,N,N',N'-tetramethylethylenediamine (TEMED) (Bio-Rad Laboratories, catalog number: 161-0800 )
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Ammonium Persulfate (APS) (Bio-Rad Laboratories, catalog number: 161-0700 )
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Illustra NAP-5 Columns (GE Healthcare Life Sciences, catalog number: 17-0853-02 )
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[γ-32P] ATP
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Homogenization buffer (see Recipes)
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Reaction mixture (see Recipes)
Equipment
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Temperature controlled shaker (Heidolph, model: Unimax 1010 )
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French Pressure Cell Press (Aminco) or glass beads (Sigma-Aldrich, catalog number: G4649 ) for cell disruption
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Thermostat
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Spectrophotometer
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Gel electrophoresis equipment (Mini-PROTEAN Tetra Handcast Systems, Bio-Rad Laboratories, catalog number: 165-8000 )
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Laminar flow box
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Refrigerated microcentrifuge preset to 4 °C (Eppendorf, model: 5415 R )
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Gel Dryer System (Bio-Rad Laboratories, catalog number: 165-1789 )
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Exposure cassette/BioMax® intensifying screen set (Sigma-Aldrich, catalog numbers: C4979 and P8432 )
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Carestream® Kodak® autoradiography GBX developer/replenisher (Sigma-Aldrich, catalog number: P7042 )
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Carestream® Kodak® autoradiography GBX fixer/replenisher (Sigma-Aldrich, catalog number: P7167 )
Procedure
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Expression and isolation of a recombinant protein kinase
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Amplify a DNA fragment carrying the gene of interest using synthetic
primers. For the more convenient cloning of the desired insert, it is
worth to construct the primers harboring specific restriction sites for
endonucleases. The easiest way to clone this PCR product is to use
T-vector pTZ57R/T. Transform DH5-α strain. Spread transformed cell onto
LB agar plates containing ampicillin, IPTG, X-GAL. Incubate overnight at
37 °C. Select white colonies that should harbor plasmid with a desired
insert and inoculate 3 ml of LB-medium. Extract pDNA from the overnight
culture. Check it for containing your full length insert either via PCR
or via restriction analysis.
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Ligate your restriction fragment with the appropriate expression vector.
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Transform E. coli expression strain with the resultant plasmid using
CaCl2 method (Sambrook et al., 1989). Spread transformed cells on the
LB-plate containing specific antibiotic that is coded by the expression
plasmid. Manipulations with bacterial cells should be carried out in a
flow box (He, 2011).
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Grow the transformed cells in 100 ml of
LB-medium until the OD595 of suspension reached 0.5. Initiate
overproduction of the recombinant protein by the addition of IPTG to a
final concentration of 0.1 mM. Induction conditions may vary strongly
(from 3 h at 37 °C to 15 h at 25 °C). Centrifuge cells at 3,500 x g for
10 min at 4 °C.
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Resuspend the collected cells in a prechilled
homogenization buffer (1 g of cells in up to 5 ml of buffer). Disrupt
the cells either using a French Pressure Cell Press (2.8 MPa) or glass
beads (use an equal volume of beads and cell suspension. Vortex
vigorously for 30 sec. To avoid warming of the sample incubate a test
tube for 30 sec on ice. Repeat this procedure 10 times). All procedures
should be carried out at 4 °C.
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Centrifuge the crude extracts
stepwise, first at 7,500 x g for 15 min to remove unbroken cells and
afterwards at 16,000 x g for 20 min for clarifying cell lysate.
Procedures should be carried out at 4 °C. Collect supernatant (soluble
protein fraction) and keep a pellet.
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Desalt the soluble protein
fraction on NAP-5 columns according manufacturer’s instruction. Elute
the protein with 20 mM MES-КОН (pH 6.5) and determine protein
concentration using BCA Protein Assay Kit. To determination protein
quantity in the pellet resuspend it with 4% of SDS and recover the
proteins by centrifugation at 16,000 x g for 20 min. Then determine
protein concentration with BCA Protein Assay Kit. Mix protein sample
with appropriate amount of Laemmli sample buffer to get the final
protein concentration 1 mg/ml (He, 2011).
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Prepare and run SDS-PAGE. Stain the gel with CBB R250 (Yan, 2011).
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Analyze both the desalted soluble fraction and proteins from the
pellet using SDS-PAGE. If your protein is soluble proceed to the protein
kinase assay. If the protein is in the pellet, it is necessary to
recover the protein from inclusion bodies or to start over induction
procedure in other conditions, trying to shift the protein into the
supernatant. Alternatively, purify the soluble proteins further by
affinity chromatography. The first possibility allows to obtain the
result much quickly. The second one requires more time and an additional
purification step but gives the better picture with the fewer non
specific bands.
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Phosphorylation in vitro
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Kinase activity assay is performed in a reaction mixture containing 20
mM MES-КОН (pH 6.5), 10 mM MgCl2, 10 µM АТP, exogenous protein
substrate, 74 kBq 2μCi [γ-32P] ATP (specific activity 148 TBq/mmol). The
quantity of the labeled ATP depends on the quantity of the protein.
Different proteins (histone Н1, basic myelin protein МВР, and BSA) can
be used as possible exogenous substrates. To calculate the volume of the
[γ-32P] ATP required you can use, for example, this site
http://www.radprocalculator.com/Decay.aspx. Or you can do it manually:
Radioactive concentration (µCi/µl) =Calibration Concentration (µCi)/2 N/14.3,
where N - number of days passed from the day of calibration, taken from
the passport of the product. 14.3 days - half-life period of the 32P.
The reaction is initiated by the addition of 5-100 µg of the
recombinant protein kinase and allowed to proceed for 15 min at 30 °C.
The quantity of the recombinant protein kinase in this reaction depends
on whether affinity purified or non-purified the recombinant protein
kinase is used.
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The reaction is terminated by adding half of the
reaction volume of 3x Laemmli sample buffer and samples are boiled for 5
min.
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Phosphorylated products are resolved on SDS-polyacrylamide
gels. The gels are then stained with CBB R250 as mentioned above,
dried, and subjected to autoradiography. Exposition of dried gels with
the X-Ray film varies from few hours at room temperature to few days at
-70 °C and depends on the quantity and the activity of the recombinant
protein kinase.
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If the recombinant protein kinase is
enzymatically active it can demonstrate either autophoshorylation or
phosphorylation of the substrate exogenously provided. So, it is
possible to make a decision about substrate specificity of the
recombinant protein kinase examined. According to the results obtained
we can conclude that at the phosphorylation condition described here the
examined recombinant protein kinase is capable to phosphorylate histone
H1. Since at the autoradioradiograph there is no a signal corresponding
to apparent molecular weight of the recombinant protein kinase it does
not undergo autophosphorylation.
Representative data

Figure 1. The substrate specificity of recombinant SpkE. Coomassie R-250 staining (at the top) and autoradiographs of the corresponding gels (at the bottom). As potential substrates were examined (A) histone H1 and (B) MBP. Soluble proteins were isolated from E. coli cells with pET41a(+) (1) before and (2) after induction with IPTG and E. coli cells with pET41a(+) carrying Synechocystis spkE (3) before and (4) after induction with IPTG. About 10 μg of the E. coli cells soluble protein fraction was mixed in 25 μl with 5 μg either H1 or MBP in the presence of 1.5 mCi of [γ-32P]ATP. The phosphorylation reaction was carried out for 15 min at 30 °C. As demonstrated above only histone H1 is phosphorylated by SpkE. [modified from Zorina et al. (2014)]
Recipes
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Homogenization buffer
50 mM Tris–HCl (pH 7.6)
10 mM MgCl2
2 mM EDTA
1 mM DTT
1 mM EGTA
2 mM Na3VO4
10 mM NaF
50 mM β-glycerophosphate
250 mM sucrose
1 mM PMSF
1 mM benzamidine hydrochloride
0.5 mM 6-aminocaproic acid
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Reaction mixture (total volume 25 µl)
20 mM MES-KOH (pH 6.5)
10 mM MgCl2
10 µM ATP (unlabelled)
0.25 mg/ml MBP, or 0.5 mg/ml H1, or 1 mg/ml BSA
2 µCi [γ-32P]ATP
Protein kinase
ddH2O
Acknowledgments
This work was supported by the grant from Russian Science Foundation no. 14-24-00020.
References
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He, F. (2011). Standard DNA cloning. Bio-protocol Bio101: e52.
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He, F. (2011). Laemmli-SDS-PAGE. Bio-protocol Bio101: e80.
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Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press.
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Yan, Q. (2011). Rapid coomassie protein SDS-gel staining. Bio-protocol Bio101: e83.
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Zorina, A. A., Bedbenov, V. S., Novikova, G. V., Panichkin, V. B. and Los, D. A. (2014) Involvement of serine/threonine protein kinases in the cold stress response in the cyanobacterium Synechocystis sp. PCC 6803: Functional characterization of SpkE protein kinase. Mol Biol (Moscow) 48: 390–398.
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Zorina, A., Stepanchenko, N., Novikova, G. V., Sinetova, M., Panichkin, V. B., Moshkov, I. E., Zinchenko, V. V., Shestakov, S. V., Suzuki, I., Murata, N. and Los, D. A. (2011). Eukaryotic-like Ser/Thr protein kinases SpkC/F/K are involved in phosphorylation of GroES in the Cyanobacterium synechocystis. DNA Res 18(3): 137-151.
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Zorina, A. A., Novikova, G. V. and Los, D. A. (2015). Substrate Specificity of Recombinant Ser/Thr Protein Kinase.
Bio-protocol 5(6): e1426. DOI:
10.21769/BioProtoc.1426.