(4) BACK GROUND

Signal transduction through reversible protein phosphorylation is a key regulatory mechanism of both prokaryotes and eukaryotes. Typically, extracellular signals are translated into cellular responses by modulating the activity of a responsive protein depending on the phosphorylation of specific amino acid sites such as serine, threonine and tyrosine residues. The phosphorylation of proteins is carried out by specific protein kinases and is generally coupled to dephosphorylation reactions catalyzed by protein phosphatases. The covalent modifications induced through phosphorylation by Serine-Threonine Phospho Kinases (STPKs) have been shown to regulate cellular processes, such as mitosis, differentiation, stress response and nutrient acquisition. This phylogenetic compartmentalization is not rigid and eukaryotic like serine/ threonine kinases occur in a number of prokaryotic species. In early 1990s the first serine/threonine eukaryotic-like protein kinase, Pknl was identified in the proteobacterium Myxococcus Xanthus. These kinases have been implicated in the pathogenicity of several pathogenic bacteria namely; Yersinia pseudotuberculosis, Listeria monocytogenes, Pseudomonas aeruginosa Mycobacterium tuberculosis, Staphylococcus aureus, Streptomyces coelicolor Streptococcus pneumonia, Bacillus anthracis etc and also regulate various metabolic pathways. In this pathogen both BYK and STPK are highly active and are identified on the membranes and extracellular secretions. These kinases also help the bacteria to prevail in several hostile environments and also contribute in the bacteria acquiring antibiotic resistance to various antibiotics (1-6). One of the main hurdles of biologists to identify the phosphorylated proteins, several methods are being developed for the detection of phosphorylated proteins, these methods are both qualitative and quanitative. (1) Radiolabled kinase assay, (2) Electropheric mobility shift assay, (3) Fluorescence immunoassays are like Forster (fluorescence) resonance energy transfer (FRET) and (4) coupled Mass spectroscopy and western blotting technique with phospho antibodies. Radiolabeled Kinase assay

In vitro phosphorylation was carried out by using Radiolabled [? 32] ATP act as substrate for protein kinases. Radiolabled ? Phosphate group was transferred by kinases to the protein substrate which acts as another substrate for protein kinase, at the position of serine, threonine, tyrosine and histidine residues. These phosphorylated proteins are separated by SDS-PAGE and then Radioactivity analysed by Autoradiography. It is very important Qualitative assay for determination of Protein kinase activity in laboratory conditions (6, 7). Fluorescence immunoassays

One of the factors driving the development of fluorescent assays has been the rapid growth in the number of phospho antibodies available. These phosphospecific antibodies can be directed against phosphotyrosine, phospho-serine and phospho-threonine residues. The rapid expansion of the variety of phospho-antibodies has enabled the development of phospho-enzyme linked immunosorbent assays (ELISAs), fluorescence polarization assays, fluorescence resonance energy transfer (FRET) assays, time-resolved fluorescence (TRF) assays and cell-based assays.

Phospho-ELISAs can be used to assess kinase activity in cell lysates. Angeles and coworkers have described two versions of an ELISA for quantitation of trkA receptor phosphorylation from cell cultures (75). The difference between the two assays was the label readout. In the first configuration, a horseradish peroxidase-conjugated secondary antibody was used to develop a colorimetric signal that was measured by absorbance. In the second configuration, the secondary antibody was conjugated to europium, a lanthanide, and the readout was TRF, an approach similar to the dissociation enhanced lanthanide fluorescence immunoassay (DELFIA® assay, PerkinElmer, Inc). TRF assays employ lanthanide fluorescent labels, the fundamental characteristic of which is their long fluorescent decay times. The LANCE® assay format (PerkinElmer, Inc.) shown in Figure 7 combines TRF with FRET. Similar to the AlphaScreen®assay (PerkinElmer, Inc.) described below, a biotinylated peptide kinase substrate is bound to an allophycocyanin-streptavidin acceptor conjugate. The kinase to be examined is added to the target peptide along with ATP and a donor europium-labeled antiphospho peptide antibody. If the target peptide becomes phosphorylated, the europium-anti-phosphopeptide antibody binds to the phosphorylated peptide, bringing the europium label in close proximity to the allophycocyanin. Upon excitation at 340 nm, the europium-labeled donor transfers its excited energy to the allophycocyanin acceptor complex, which emits fluorescence at 665 nm. Again, the use of lanthanide chelates with long excited lifetimes avoids interference and background caused by non-specific short-lived emissions. The AlphaScreen assay is an easily automated homogenous FRET assay. The AlphaScreen assay employs two main reagents: a biotinylated peptide substrate bound to a donor streptavidin-coated fluorescent dye-containing bead, and a second fluorescent dye-containing bead coated with an antiphosphotyrosine antibody. Candidate kinase is added and, if phosphorylation of the peptide occurs, the donor bead is brought into close proximity with the acceptor bead through the binding of the phospho specific antibody on the acceptor bead. The mixture is subjected to excitation at680 nm, which induces the formation of singlet oxygen at the surface of the donor bead following conversion of ambient oxygen to a more excited singlet state by a photo-sensitizer present in the donor bead. The singlet oxygen molecules diffuse and react with a thioxene derivative present in the acceptor bead, generating chemiluminescence emitting at 370 nm. The chemiluminescence excites fluorophores contained in the acceptor bead that emit at 520-620 nm. The signal generated has a half life measured in seconds which allows signal measurement to be time gated, thereby eliminating short-lived fluorescent background. Mobility shift assays

The phosphorylation sites on target substrates for protein kinases can be mapped and short peptides comprising only the phosphorylation site synthesized. When these peptides are phosphorylated and subjected to electrophoresis, their mobility changes due to the added charge of the phosphate group. Caliper Life Sciences has exploited this feature to create microfluidics chip-based kinase assays (8, 9). The assays have been designed in two continuous flow formats in which a fluorescently labeled peptide substrate and its phosphorylated product are separated electrophoretically and subsequently detected. In the first format, the kinase under investigation is combined with its peptide in the presence of ATP in a microtiter plate. After the phosphorylation reaction occurs, the reaction mixture is transferred onto a micro fluidics chip on which separation of the product and substrate occurs by electrophoresis. The product and substrate are quantified by fluorescence detection of the leading and trailing peaks. In a second format, the kinase reaction takes place directly on a micro fluidics chip followed by electrophoretic separation and detection (10). All these assays are either stringent or short time lived therefore; we combined the colorimetric detection of phosphate and SDS-PAGE techniques to detect the phosphorylated proteins.

Methodology

1. Cultural characterization of S. aureus ATCC 12600

Staphylococcus aureus ATCC 12600 strain was used in the present study, this strain was gifted by Genei pvt., Ltd., Bangalore. This strain was grown on modified Baird Parkar media at 37°C. After overnight incubation single black shiny colony with distinct zone was picked and cultured in Brain heart infusion (BHI) broth at 37°C. Thus, grown S. aureus ATCC 12600 culture was used to characterize BY kinase and serine-threonine kinase enzymes and extraction of chromosomal DNA (11-13).

2 Amplification and sequencing of BYK and STPK of genes from Staphylococcus aureus ATCC12600

Chromosomal DNA of S. aureus ATCC 12600 was extracted by the method described earlier (3) and extracted DNA was stored at -20°C for further experiments. BY kinase gene from S. aureus ATCC 12600 were amplified from the chromosomal DNA using the primers forward primer BYK 1: 5'-CAT GAC GAA TAC ACG-3' and Reverse primers BYK 2: 5'-TCA TGA TTC ATC AGT -3'. The PCR reaction was performed in Eppendrof (Master gradient) thermocycler in a volume of 50ul of PCR mixture containing 30ng of DNA, 200uM of dNTPs mix, 20pM of forward and reverse primers, 15mM Tris-HC1 (pH 8.8), 2.5mM MgCl2, and 1 U of Hotstart Taq DNA polymerase. PCR reactions was performed with following conditions; Initial denaturation 94°C for 10 minutes, and for 40 cycles contain 94°C for 60 seconds of denaturation, 38.5°C for 30 seconds of annealing, 72°C for 90 seconds of extension and final elongation at 72°C for 10 minutes. Similarly, the STPK gene was amplified using STPK 1 Forward Primer: 5'- ATGATAGGTAAAATA -3' and STPK 2 Reverse Primer: 5'-TTATACATCATCATA -3' The PCR reaction was performed in Eppendrof (Master gradient) thermocycler in ar volume of 50ul of PCR mixture containing 30ng of DNA, 200uM of dNTPs mix, 20pM of forward and reverse primers, 15mM Tris-HC1 (pH 8.8), 2.5mM MgCl2, and 1 U of Hotstart Taq DNA polymerase. PCR reactions was performed with following conditions; Initial denaturation 94°C for 10 minutes, and for 40 cycles contain 94°C 60 seconds of denaturation, 29.2°C for 45 seconds of annealing, 72°C for 90 seconds of extension and final elongation at 72°C for 10 minutes. The amplified PCR products were resolved on 1% agarose gel along with supermix DNA ladder (Bangalore Genei). After separation the gel was observed under UV Trans illuminator and considering the super mix DNA ladder as a standard the molecular sizes of the PCR products were calculated with the software provided by Vilber-lourmat gel documentation system. The purified PCR products were sequenced by dye terminating method at commercial sequencing facility of MWG Biotech India Ltd, Bangalore, India (11-13). The obtained sequences were analysed and deposited at NCBI-Gen Bank www.ncbi.nlm.nih.gov 3. Preparation of BYK from S. aureus ATCC 12600 culture.

S. aureusATCC\2600 was grown in plain BHI broth up to late log phase (OD540=0.9). The culture was centrifuged at 10,000 rpm for 10 min at 4°C and the pellet was suspended in TES buffer (0.1 M Tris HC1 pH 7.4, 50mM EDTA and 10% sucrose) and incubated in ice for 30min. Then centrifuged at 10,000 rpm for 10 min, the resulted pellet was suspended in TES buffer. Then bacterial cells were disrupted by sonication at 50Hz for 15 cycles, (each cycle with 15 seconds). The sonication was carried out in ice bath. After completion of sonication the lysate was centrifuged at 15,000 rpm for 20 min at 4°C. The supernatant was collected and stored at - 70°C immediately after separation until further use (12-13). 4. Cloning of S. aureus ATCC 12600 BYK gene in to pQE-30 vector.

The amplified PCR product of 0.7kb corresponding to BY Kinase gene was electro eluted from the agarose gel using QIA quick Gel Extraction Kit (QIAGEN Inc., USA) and Gel extraction spin-50 column (Chromous biotech Pvt, Ltd, India). The eluted PCR product was made into proper blunt ends using Klenow fragment (New England Bio labs, USA) following the manufacturer's protocol. The blunt ended PCR product was cloned into the Sma I site of plasmid pQE-30 (QIAGEN Inc., USA) and was transformed into the E. coli DH5a and the obtained clones were named as SaBYKl clones (13).

4.1 Over expression of gene BYK from SaBYKl Clone.

The SaBYKl clone was grown at 37°C in LB broth containing 50ng/ml ampicillin up to OD540nm = 0.6, 0.75mM of IPTG was added and grown for 4 h. The culture was centrifuged and sonicated as explained in section 3.The rBYK was purified from the cytosolic fraction of SaBYKl clone by passing it through nickel metal chelate column by following QIA-express expression system protocol. The obtained pure protein was dialysed against O.lMTris-HC1 pH 7.5 and was analyzed on 10%SDS-PAGE(13). 5. BY kinase enzyme assay.

BY kinase activity was detected both in the S. aureus ATCC 12600 fraction and in the pure BYK. The reaction was determined at 30°C using novel non- radiolabled protein kinase spectrophotometric assay with synthetic peptide act as substrate on a Cyber lab spectrophotometer-USA. Protein tyrosine kinase (BYK) assay mixture contained 0.1M Tris-HC1 pH 7.5, 0.1MATP, 30^g/ml peptide (HGLDNYRGYSLG) and 0.5ug/ul enzyme fraction (either crude or pure) (as mentioned in earlier section) were mixed and incubated at 30°C for 10 minutes. The phosphorylated peptide was purified by passing through Sephadex G-25 column (1cm x 15cm), the fractions were eluted with 0.1M Tris-HC1 pH 7.5, and 150mM NaC1. The enzyme fraction appeared in the void volume, while the phosphorylated peptide was obtained in the first elution volume. The bound phosphorous was estimated by adding freshly prepared Burchell's reagent (3.4mM ammonium molybdate, 0.5mM sulphuric acid,0.5MSDS and 0.6M of L-Ascorbic acid) and incubated at 30°C for 15 minutes and the absorbance was recorded at 820nm against blank (0.1M Tris-HC1 pH 7.5 and 150mM NaC1 and Burchell's reagent). The enzyme activity was measured as amount of phosphorous added per microgram peptide at 30°C per minute per ml. For this the calibration curve was developed using standard KH2PO4 for the estimation of inorganic phosphate and free phosphate was determined by adding Burchell's reagent. The phosphorylated peptide was further demonstrated by fractionating the eluted peptide on 15% SDS-PAGE and staining the gel with Burchell's reagent the bluish green coloured band appeared in the gel indicated the peptide was phosphorylated by the enzyme fraction. Similarly, the auto-phosphorylation property of protein tyrosine kinase was also determined for this the reaction mixture composition was same except in that peptide was not added. The enzyme activity was measured as amount of phosphorous added per microgram enzyme at 30°C per minute per ml. Substrate level phosphorylation was performed by taking different substrate concentrations of 200uM, 400uM, 600uM, 800uM, lOOOuM, and 1200uM of synthetic peptide keeping the ATP concentration constant and the corresponding velocities were calculated and a graph of [S/V] vs [S] was plotted, from the graph Km was determined by Hanes-Woolf plot. For Auto phosphorylation activity of BY kinase the same enzyme assay was carried out except in that peptide was not added. Similarly Auto phosphorylation was performed by taking different substrate concentrations of 33uM, 166uM, 333uM, 500uM, 666 uM, 833 uM, 1000 uM, 1166 uM and 1300 uM and 1500 uM of s ATP keeping the protein concentration constant and the corresponding velocities were calculated and a graph of [S/V] vs [S] was plotted, from the graph Km was determined by Hanes-Woolf plot. Protein concentrations in all steps were determined by Bradford 1976 method (14-19).

6. Serine/Threonine protein kinase enzyme assay

STPK kinase activity of S. aureus ATCC 12600 extracellular filtrate was determined at 30°C using novel non- radiolabled protein kinase spectrophotometric assay with synthetic peptide act as substrate on a Cyber lab spectrophotometer-USA. Serine/Threonine Protein kinase (STPK) assay mixture contained 0.1M Tris-HC1 pH 7.5, 0.1MATP, 30Dg/ml peptide (NLCNIPCSALLSSDITASVNCAK) and lug/ul enzyme fraction (extracellar fraction and or pure enzyme) were mixed and incubated at 30°C for 10 minutes. The phosphorylated peptide was purified by passing through Sephadex G-25 column (1cm x 15cm), the fractions were eluted with 0.1M Tris-HC1 pH 7.5, and 150mM NaC1. The enzyme fraction appeared in the void volume, while the phosphorylated peptide was obtained in the first elution volume. The bound phosphorous was estimated by adding freshly prepared Burchell's reagent (as explained in earlier section and incubated at 30°C for 15 minutes and the absorbance was recorded at 820nm against blank (0.1M Tris-HC1 pH 7.5 and 150mM NaC1 and Burchell's reagent). The enzyme activity was measured as amount of phosphorous added per microgram peptide at 30°C per minute per ml. For this the calibration curve was developed using standard KH2PO4 for the estimation of inorganic phosphate and free phosphate was determined by adding Burchell's reagent. The phosphorylated peptide was further demonstrated by fractionating the eluted peptide on 15% SDS-PAGE and staining the gel with Burchell's reagent the bluish green coloured band appeared in the gel indicated the peptide was phosphorylated by the enzyme fraction. Similarly, the auto-phosphorylation property of protein tyrosine kinase was also determined for this the reaction mixture composition was same except in that peptide was not added. The enzyme activity was measured as amount of phosphorous added per microgram enzyme at 30°C per minute per ml .Substrate level phosphorylation was performed by taking different substrate concentrations of 200uM, 400uM, 600uM, 800uM, lOOOuM, and 1200uM of synthetic peptide keeping the ATP concentration constant and the corresponding velocities were calculated and a graph of [S/V] vs [S] was plotted, from the graph Km was determined by Hanes-Woolf plot. For Auto phosphorylation activity of STPK kinase the same enzyme assay was carried out except in that peptide was not added. Similarly Auto phosphorylation was performed by taking different substrate concentrations of 33uM, 166uM, 333uM, 500uM, 666 uM, 833 uM, 1000 uM, 1166 uM and 1300 uM and 1500 uM of s ATP keeping the protein concentration constant and the corresponding velocities were calculated and a graph of [S/V] vs [S] was plotted, from the graph Km was determined by Hanes-Woolf plot. Protein concentrations in all steps were determined by Bradford 1976 method (14-19).

7. Cloning of S. aureus ATCC 12600 STPK genes in to Phage cloning vector M13mpl8.

The amplified PCR product of 2.0kb corresponding to STPK gene was electro eluted from the agarose gel using QIA quick Gel Extraction Kit (QIAGEN Inc., USA) and Gel extraction spin-50 column (Chromous biotech Pvt, Ltd, India). The eluted PCR product was made into proper blunt ends using Klenow fragment (New England Bio labs, USA) following manufacturers protocol. Thus the blunt ended PCR product was cloned into the Sma I site of M13mpl8 vector and transfected into E.coli JM109 cells. The recombinant clone was named as stpk Nl and was again transfected to E.coli JM109 and pure recombinant phage DNA was isolated.

8. Cloning of The recombinant phage (stpk Nl) DNA in to plasmid cloning vector pRSETA

The recombinant phage (stpk Nl) DNA was then digested with BamHl and EcoRl and the phage digested product of 2.03 kb corresponding to STPK Kinase gene was electro eluted from the agarose gel using QIA quick Gel Extraction Kit (QIAGEN Inc., USA) and Gel extraction spin-50 column (Chromous biotech Pvt, Ltd, India).;? The eluted digested product was was cloned into the BamHl and EcoRl of plasmid pRSET-A (QIAGEN Inc., USA) and was transformed into the E. coli BL21 (DE3)pLysS cells and the obtained clone was named as VK1 clone.

8.1 Over expression of gene STPK from VK1 Clones

The pRSET-A transformants (VK1 Clones) and pRSET-A non transformants (control) were grown at 37°C in LB broth containing 50ug/ml ampicillin and 35 |xg/ml chloramphenicol when the culture reached 0.6 O.D, 0.5 mM of IPTG was added and grown for 4 h. The culture was centrifuged and sonicated as explained in section 3.The rSTPK was purified from the cytosolic fraction of VK1 clone by passing it through nickel metal chelate column by following QIA-express expression system protocol. The obtained pure protein was dialysed against O.lMTris-HC1 pH 7.5 and was analyzed on 10% SDS-PAGE.

RESULTS

The results of both substrate level phosphorylation and autophosphorylation of BYK and STPK are depicted in Table 1-4. Figures 1-2 explains the present assay clearly identifies the phosphorylated substrates and enzymes. Figures 3 and 4 explains the sequences of BYK and STPK genes cloned, sequenced and characterized (the sequences were depositied at www.ncbi.nlm.nih.gov, GenBank accession numbers are GU353130 and JN695616 respectively).

Table 3: Enzyme kinetics of BYK from S.aureus ATCC 12600 and Recombinant BYK for
substrate phosphorylation

Table-4: Enzyme kinetics of BYK from S.aureus ATCC 12600 and Recombinant BYK for

Table 5: Enzyme kinetics of STPK from S.aureus ATCC 12600 and Recombinant STPK for substrate phosphorylation

Table 6: Enzyme kinetics of STPK from S.aureus ATCC 12600 and Recombinant STPK for Auto- phosphorylation

5 BYK auto phosphorylation 6 BYK substrate phosphorylation

Figure 3 Non-radiolablaed BYkinase enzyme assay.

1) Schematic diagram of Non-radiolablaed kinase enzyme assay methodology.

2) Standard Caliberation graph of inorganic phosphate using BurchellDs reagent.

3) Expremental blue chromatogram for substrate level phosphorylation showing the separation of a reaction mixture on a Sephadex G-25 column (1cm x 15cm)column with 0.1M Tris-HC1 pH 7.5, and 150mM NaC1 as mobile phase Peak: 1 BY kinase; peak: 2 Synthetic peptide. Red graph showing intensity bluish green colour corresponding elution fractions of a reaction mixture Peak: 1 Auto phosphorylated BYK; peak: 2 phosphorylated Synthetic peptide.

4) Experimental blue chromatogram for Auto phosphorylation showing the separation of a reaction mixture on a Sephadex G-25 column (1cm x 15cm)column with 0.1M Tris-HC1 pH 7.5, and 150mM NaC1 as mobile phase Peak: 1 BYK Red graph showing intensity bluish green colour corresponding elution fractions of a reaction mixture Peak: 1 Auto phosphorylated BYK kinase .

5) The Electrophoretogram showing phosphorylated BYK and Phosphorylated synthetic peptide in 10% SDS-PAGE gel and the gel was stained with BurchellDs reagent Lane LI: bluish green color phosphorylated BY kinase. Lane L2: bluish green color phosphorylated synthetic peptide

Figure 4: Non-radiolabled STPK Enzyme assay

1) Schematic diagram of Non-radiolablaed kinase enzyme assay methodology.

2) Standard Caliberation graph of inorganic phosphate using Burchell's reagent.

3) Expremental blue chromatogram for substrate level phosphorylation showing the separation of a reaction mixture on a Sephadex G-25 column (1cm x 15cm)column with 0.1M Tris-HC1 pH 7.5, and 150mM NaC1 as mobile phase Peak: 1 STP kinase ;peak: 2 Synthetic peptide. Red graph showing intensity bluish green colour corresponding elution fractions of a reaction mixture Peak: 1 Auto phosphorylated STP kinase ; peak: 2 phosphorylated Synthetic peptide.

4) Experimental blue chromatogram for Auto phosphorylation showing the separation of a reaction mixture on a Sephadex G-25 column (1cm x 15cm)column with 0.1M Tris-HC1 pH 7.5, and 150mM NaC1 as mobile phase Peak: 1 BY kinase Red graph showing intensity bluish green colour corresponding elution fractions of a reaction mixture Peak: 1 Auto phosphorylated STP kinase .

5) The Electrophoretogram showing phosphorylated STP kinase and Phosphorylated synthetic peptide in 10% SDS-PAGE gel and the gel was stained with Burchell's reagent Lane LI: bluish green color phosphorylated STP kinase. Lane L2: bluish green color phosphorylated synthetic peptide.

Figure 5 Cloning strategy of BYK gene of S. aureus ATCC12600

(1) Pure S. aureusATCC 12600 culture grown in Braid parker medium.

(2) 1% agarose gel showing the chromosomal DNA from S aureus ATCC 12600.

(3) Amplification of BY kinase gene using PCR Master Cycler gradient thermo cycler.Lane 1: Amplified PCR product of BY Kinase gene from the chromosomal DNA isolated from the S aureus ATCC 12600. Lane M: supermixMolecular size markers, (0.5,1,2,3,4,8,15 and 26.6 Kb, DNA ladder, Bangalore Genei Pvt. Ltd.)

(4) The schematic diagrams explainthe elution of PCR products of BYK from agarose gel and making the blunt ends, the gene BYK was cloned in the sma I site of the pQE-30. This was transformed into E. coli DH5a cells.

(5) Electrophoretogram showing recombinant plasmid isolated from SaBYK-1 clone. Lnae 1: Molecular size marker, lane 2 pQE-30 plasmid and lane 3 recombinant plasmid.

(6) The Electrophoretogram showing the expression of 25 kDa recombinant protein from SaBYK-1 clone in 10% SDS-PAGE gel and the gel was stained with 0.125% Coomossie brilliant blue R250. Lane M: molecular weight markers obtained from Bangalore Genei Pvt ltd. Lane LI: Un induced cell lysate of SaBYK-1 clone. Lane L2: induced cell lysate of BYK-1. Lane L4: recombinant BY kinase eluted from nickel metal chelate chromatography column was dialysed against the 0.1M Tris-HC1.

(7) The chromatogram showing the BYK gene from S. aureus ATCC 12600 and the sequence was deposited in Genbank accession number-GU353130.

Figure 6 Cloning strategy of STPK gene of S. aureus ATCC12600.

1. Pure S. aureusATCC 12600 culture grown in Braid parker medium.

2. 1% agarose gel showing the chromosomal DNA from S aureus ATCC 12600.

3. Amplification of STPK gene using PCR Master Cycler gradient thermo cycler. Lane 1: Amplified PCR product of STPK Kinase gene from the chromosomal DNA isolated from the S aureus ATCC 12600. Lane M: supermix Molecular size markers, (0.5,1, 2,3,4, 8, 15 and 26.6 Kb, DNA ladder, Bangalore Genei Pvt. Ltd.)

4. The schematic diagrams explain the elution of PCR products of STPK from agarose gel and making the blunt ends, the gene STPK was cloned in the sma I site of the M13MP18 phage vector. This was transfected into E.coli JM109 cells.

5. Electrophoretogram showing recombinant M13MP18 clone having STPK insert isolated from the E. coli JM109 in the Lane2 against the native Ml3 MP 18 in Lanel.The schematic diagrams explains the rM13mpl8 clone contain insert STPK and was digested with Eco RI and BamHI for making the 1 frame , the gene this was cloned in the Eco RI and BamHI site of the pRSETA vector. This was transformed into E. coli DH5a cells.

6. The Electrophoretogram showing the expression of 73 KD recombinant protein from VK-1 clone in 10% SDS-PAGE gel and the gel was stained with 0.125% Coomossie brilliant blue R250. Lane M: molecular weight markers obtained from Bangalore Genei Pvt ltd. Lane UI: Un induced cell lysate of BYK-1 clone. Lane II and 12 : induced cell lysate of VK-1. Lane N4: recombinant BY kinase eluted from nickel metal chelate chromatography column was dialysed against the 0.1M Tris-HC1. 7. The chromatogram showing the STPK gene from S. aureus ATCC12600 and the sequence was deposited in Genbank accession number -JN695616.

References:

1. Pawson T, Scott JD. Protein phosphorylation in signaling— 50 years and counting, Trends Biochem. Sci. 2005;30: 286-290.

2. Kennelly PJ and Potts M. Fancy meeting you here! A fresh look at 'prokaryotic' protein phosphorylation. J. Bacteriol. 1996; 178: 4759-4764.

3. Av-Gay Y, Everett M. The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis. Trends Microbiol.2000; 8:238-244.

4. Peirs P, De Wit L, Braibant M, Huygen K, Content J. A serine/threonine protein kinase from Mycobacterium tuberculosis. Eur. J. Biochem. 1997; 244:604—612.

5. Munoz-Dorado J, Inouye S, Inouye M. A gene encoding aprotein serine/threonine kinase is required for normal developmentof M xanthus, a Gram-negative bacterium. Cell.1991; 67:995-1006.

6. Koul A, Choidas A, Tyagi AK, Drlica K, Singh Y, Ullrich A. Serine/ threonine protein kinases PknF and PknG of Mycobacterium tuberculosis: Characterization and localization. Microbiology. 2001; 147: 2307-2314.

7. Angeles TS, Lippy JS, Yang SX. Quantitative, high-throughput cell based assays for inhibitors of trkA receptor. Anal. Biochem. 2000; 278:93-98.

8. Cohen CB, Chin-Dixon E, Jeong S, Nikiforov TT.A microchip-based enzyme assay for protein kinase A. Anal. Biochem. 1999; 273:89-97.

9. Dunne J, Reardon H, Trinh V, Li E, Farinas J. Comparison of on-chip and off-chip microfluidics kinase assay formats. Assay Drug DeveI. 2004; 2: 121-129.

10. Deribe YL, Pawson T, Dikic I. Post-translational modifications in signal integration.Ata. Struct. Mol. Biol.2010; 17: 666-672.

11. Stadhoulders J, Hassings F, Van Aalsten-Van, Mareno NO. A pour-plate method for the detection and enumeration of coagulase-positive Staphylococcus aureus in the BAIRD-PARKER Medium without egg-yolk.JVefe Milk Diary J. 1976;30:222-229.

12. Hari Prasad O, Nagarjuna V, Lakshmi Prasanna Ch, Vasu D, Yugandhar VG, Prasad UV, Srinivas Rao PVLN, Chaudhary A, Reddy OVS, Sarma PVGK. UV light Induces Transposition of blaZ gene resulting in high expression of G-lactamase in a clinical isolate of Staphylococcus aureus sensitive to ampicillin J. Pure and Apllied Microbiology. 2010; 4(2): 609-616.

13. Hari Prasad O, Nanda Kumar Y, Reddy, OVS, Chaudhary A, Sarma PVGK. Cloning, Expression, Purification and Characterization of UMP Kinase from Staphylococcus aureus. Protein J. 2012; 31:345-352.

14. Walsh R, Martin E, Darvesh S. A method to describe enzyme-catalyzed reactions by combining steady state and time course enzyme kinetic parameters. Biochim. Biophys. Acta.2010; 1800:1-5.

15. Panchal M, Muralidhar K. Purification and biological characterization of bacterially expressed recombinant buffalo prolactin.Preparative Biochemistry and Biotechnology. 2010; 40: 4276-285.

16. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685.

17. Fiske CH, Subbarao Y. The colorimetric determination of phosphorous. Biol Chem. 1925;66 :375-400.

18. Burchell A, Burchell B. Stabilization of partially-purified glucose 6-phosphatase by fluoride. Is enzyme inactivation caused by dephosphorylation? FEBS Lett. 1980 Sep 8; 118(2): 180-184.
19. Bradford, MM. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248-254.

5. CLAIMS: I/We claim

1. BY kinase enzyme assay.

BY kinase activity was detected both in the S. aureus ATCC 12600 fraction and in the pure BYK. The reaction was determined at 30°C using novel non- radiolabled protein kinase spectrophotometric assay with synthetic peptide act as substrate. Protein tyrosine kinase (BYK) assay mixture contained 0.1M Tris-HC1 pH 7.5, 0.1MATP, 30ug/ml peptide (HGLDNYRGYSLG) and 0.5^g/ul enzyme fraction (either crude or pure) (as mentioned in earlier section) were mixed and incubated at 30°C for 10 minutes. The phosphorylated peptide was purified by passing through Sephadex G-25 column (1cm x 15cm), the fractions were eluted with 0.1M Tris-HC1 pH 7.5, and 150mM NaC1. The enzyme fraction appeared in the void volume, while the phosphorylated peptide was obtained in the first elution volume. The bound phosphorous was estimated by adding freshly prepared Reagent (3.4mM ammonium molybdate, 0.5mM sulphuric acid, 0.5MSDS and 0.6M of L-Ascorbic acid) and incubated at 30°C for 15 minutes and the absorbance was recorded at 820nm against blank (0.1M Tris-HC1 pH 7.5 and 150mM NaC1 and Reagent). The enzyme activity was measured as amount of phosphorous added per microgram peptide at 30°C per minute per ml. For this the calibration curve was developed using standard KH2PO4 for the estimation of inorganic phosphate and free phosphate was determined by adding Reagent. The phosphorylated peptide was further demonstrated by fractionating the eluted peptide on 15% SDS-PAGE and staining the gel with Burchell's reagent the bluish green coloured band appeared in the gel indicated the peptide was phosphorylated by the enzyme fraction. Similarly, the auto-phosphorylation property of protein tyrosine kinase was also determined for this the reaction mixture composition was same except in that peptide was not added.

2. Serine/Threonine protein kinase enzyme assay

STPK kinase activity of S. aureus ATCC 12600 extracellular filtrate was determined at 30 C using novel non- radiolabled protein kinase spectrophotometric assay with synthetic peptide act as substrate. Serine/Threonine Protein kinase (STPK) assay mixture contained 0.1M Tris-HC1 pH 7.5, 0.1MATP, 30ug/ml peptide (NLCNIPCSALLSSDITASVNCAK) and lug/ul enzyme fraction (extracellar fraction and or pure enzyme) were mixed and incubated at 30 C for 10 minutes. The phosphorylated peptide was purified by passing through Sephadex G-25 column (1cm x 15cm), the fractions were eluted with 0.1M Tris-HC1 pH 7.5, and 150mM NaC1. The enzyme fraction appeared in the void volume, while the phosphorylated peptide was obtained in the first elution volume. The bound phosphorous was estimated by adding freshly prepared Reagent (as explained in earlier section and incubated at 30°C for 15 minutes and the absorbance was recorded at 820nm against blank (0.1M Tris-HC1 pH 7.5 and 150mM NaC1 and Reagent). The enzyme activity was measured as amount of phosphorous added per microgram peptide at 30°C per minute per ml. For this the calibration curve was developed using standard KH2P04 for the estimation of inorganic phosphate and free phosphate was determined by adding Reagent. The phosphorylated peptide was further demonstrated by fractionating the eluted peptide on 15% SDS-PAGE and staining the gel with Reagent the bluish green coloured band appeared in the gel indicated the peptide was phosphorylated by the enzyme fraction. Similarly, the auto-phosphorylation property of protein tyrosine kinase was also determined for this the reaction mixture composition was same except in that peptide was not added. The enzyme activity was measured as amount of phosphorous added per microgram enzyme at 30°C per minute per ml.