# Patent

## Title
Biochip for the detection of phosphorylation and the detection method using the same

## Abstract

The present invention relates to a biochip for detecting phosphorylation and a method for detecting phosphorylation using the same, more precisely a biochip prepared by integrating a protein produced from the recombination of a substrate of kinase selected from the group consisting of PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase) and the elevated protein such as Selenomonas ruminantium membrane protein on a matrix surface coated with an active group, a kit for detecting phosphorylation composed of the said biochip and a cofactor labeled with a radio-isotope and a method for detecting phosphorylation using the same. The biochip for detecting phosphorylation of the present invention using a radio-isotope facilitates the detection of phosphorylation with a minimum amount of a sample by simple processes, compared with the conventional method using an antibody. Since this method can analyze a large amount of samples in a shorter period of time, it can be effectively used for the analysis of kinase activity.

## Background
CROSS-REFERENCES TO RELATED APPLICATION
This patent application claims the benefit of priority under 35 U.S.C §119 of Korean Patent Application No. 10-2008-0059603 filed on Jun. 24, 2008, the contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a biochip for detecting phosphorylation and a method for detecting phosphorylation using the same, more precisely a biochip prepared by integrating a fusion protein produced from the recombination of a substrate of a kinase selected from the group consisting of PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase), and the elevated protein such as Selenomonas ruminantium membrane protein on a matrix surface coated with an active group, a kit for detecting phosphorylation composed of the said biochip and a cofactor labeled with a radio-isotope and a method for detecting phosphorylation using the same.
2. Description of the Related Art
Recent biotechnology industry is characterized by the fusion of biology with related technologies in the fields of electronics, computer science and mechanical engineering. Biological studies require a new system to approach an individual biological subject as a whole and also require a novel approaching method performing experiments in a large scale with a small amount of samples. Accordingly, a biochip is expected to play a crucial role in the field of bioinformation analysis using genes, proteins and cells and in use of the bioinformation.
According to the completion of human genome project, studies on micro-analysis system such as DNA chip or protein chip for the analysis of genes or proteins are actively undergoing in the overall biological industry. Biochip market is expected to be growing greatly and has been a major target of development in bio-industry in Korea.
Protein chip or peptide chip is a kind of automatic analysis device for simultaneous analysis of tens or thousands of proteins or peptides fixed on a small matrix, which is the next generation biosensor different from DNA chip in analysis mechanism and applying field. Protein chip is a key technique to disclose functions of biomolecules interacting specifically to specific proteins and to develop a novel method for preventing and treating disease, particularly those diseases untreatable so far by the conventional arts, based on functional analysis and network analysis of protein.
Protein chip techniques are largely divided into three categories; protein microarray techniques related to chip production, analysis techniques for quantitatively measuring and comparing interactions between proteins by observing proteins fixed on array and application techniques of protein chip. The first core technique, protein microarray chip is prepared by different methods according to techniques to analyze protein chip. For example, in the case of using SPR (Surface Plasmon Resonance), proteins have to be fixed on a thin metal film such as a thin gold film. Therefore, a thin gold film construction method and a protein fixation technique have to be developed together. In the case of using a fluorescent material, proteins are fixed directly on a slide glass for analysis. So, the proteins have to be labeled with fluorescent materials.
The second core technique, the analysis technique of protein chip includes SPR, mass spectrometry, fluorometry, electrochemical analysis method and nano-imaging technique such as Ellipsometry, which has been developed very competitively so far. Fluorometry based on DNA chip analysis has been most widely used so far, but each method has been recognized to have merits and demerits, and which protein chip analysis method is most appropriate for disease is still undecided.
The last core technique, the application technique of protein chip has the biggest potential for the development and still leaves a lot to discover.
The newly developed techniques related to protein chip are classified into four categories as follows.
(1) A technique to analyze interaction between DNA and protein on the chip by using DNA microarray. On the chip, single-stranded oligonucleotide is converted into double-stranded oligonucleotide, to which specific DNA sequence restriction enzyme is reacted. Then, DNA-protein interaction was examined by measuring digestion. So, this technique is effective in identifying a novel DNA binding protein and in disclosing the characteristics thereof (Bulyk, M. L. et al., Nature. Biotechnol., 17:573-577, 1999).
(2) A technique to analyze diverse enzymes including restriction enzyme, peroxidase, phosphatase and protein kinase, and antigen-antibody reaction on the chip (US Patent Publication No. 2002/0055186A1; WO 01/83827A1; Braunwalder A. et al., Anal. Biochem., 234:23-26, 1996; Houseman B. et al., Nature Biotechnol., 20:270-274, 2002; Ruud M. et al., Nature Biotechnol., 18:989-994, 2000). In particular, this technique can be applied in mass-analysis, biochemical analysis, new drug candidate analysis and disease diagnosis by examining protein-protein interaction, kinase-peptide substrate reaction and protein-ligand binding reaction. However, when a kinase specific substrate peptide or a low-molecular weight protein is fixed, bovine serum albumin (BSA) is necessarily added in order to prevent non-specific fixation, which buries the protein fixed thereon. Besides, when different antibodies are fixed on the chip to be reacted with fluorescein labeled antigen mixture, only 60% of the antibodies were quantified and only 23% of the antibodies were qualified (MacBeath G. et al., Science, 289:1760-1763, 2000; Haab B. et al., Genome Biol. 2: research 0004, 2001).
(3) A technique to analyze massive proteins expressed on the chip from cDNA library (WO 01/83827, WO 02/50260). This technique is effective in large scale measurement of biochemical activity of a protein (Heng Zhu, et al., Nature genetics, 26:283-289, 2000).
(4) A method to analyze a sample by using the technique of regulating orientation of biomolecules at molecular level using affinity tag and forming a stable and single layer of biomolecules on the surface of the chip (US Patent Publication No. 2002/0055125A1; U.S. Pat. No. 6,406,921; Paul J. et al., JACS, 122:7849-7850, 2000; RaVi A. et al., Anal. Chem., 73:471-480, 2001; Benjamin T. et al., Tibtech., 20:279-281, 2002). For example, a protein is expressed as a His-tag fusion protein, which is reacted to a chip fixed with Ni-NTA functional group, leading to fixation. The protein is then expressed as an intein fused protein or retains the activity, which not only makes the purification easy but also makes the protein on the chip more stable and active by fixing in a regular direction on the avidin treated chip (Zhu et al., Science, 293:2101-2105, 2001; Marie-Laure L. et al., JACS 124:8768-8769, 2002). And, the protein is expressed on the chip as a fusion protein using a supporter specific protein (calmodulin, etc) and tag (poly cysteine, lysine, histidine, etc) and fixed. The protein is purified by using protein-protein interaction and further used for SPR (surface plasmon resonance) and FACS (fluorescence activated cell sorter) (Hentz et al., Anal. Chem., 68:3939-3944, 1996; Hodneland et al., PNAS, 99:5048-5052, 2002; Kukar et al., Anal. Biochem., 306:50-54, 2002; U.S. Pat. No. 6,117,976).
Kinase is a protein enzyme involved in signal transduction pathways to induce a series of reactions in vivo and hence it is considered to be an effective target for a drug. Kinase provides γ-phosphorylated group to serine, threonine and tyrosine residues of the specific sequence of a target protein from ATP provided in cells, by which it is involved in signal transduction pathway in eukaryotic cells and various diseases (Hunter, T., Cell 100:113-127, 2000; Zhang, Z. Y., Curr. Opin. Chem. Biol. 5:416-423, 2001). The conventional method to study the activity of kinase is to examine cell membrane using a radio-isotope, but this method is very slow-going and requires a great labor. Another conventional method to measure kinase and its receptor in a large scale is ELISA (enzyme-linked immunosorbent assay) or a method using antibody. ELISA is a comparatively accurate method but takes a lot of time and requires a huge amount of samples. The method using an antibody facilitates mass-analysis but requires high costs and the processes are very complicated.
Promega Co., USA provides a phosphorylation assay kit using a membrane having high absorptiveness to ATP labeled with a radio-isotope and biotinated kinase substrate. However, this kit is limited in mass-analysis. This company also provides an analysis method without using a radio-isotope but using moving difference on electrophoresis caused by changes of net charge of the substrate after phosphorylation of kinase. But, this method requires high costs for mass-analysis. Except for the said four protein chip related techniques, there is no method available for screening the activity of protein kinase in a large scale by using protein chip or peptide chip. Therefore, it is urgently required to develop a new system facilitating accurate and fast analysis with low costs.
The present inventors constructed a biochip for the detection of phosphorylation in Korean Patent Application No. 2007-0070049, in which kinase substrate is integrated on the surface of a matrix directly or as a fusion protein fused to E. coli malic enzyme, but further studies on the biochip using PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) or DNA-PK (DNA-dependent Protein Kinase) substrate are required.
Thus, the present inventors further studied and constructed a recombinant fusion protein from the fusion of PKC, cdc2-PK or DNA PK substrate with Selenomonas ruminantium membrane protein and then completed this invention by constructing a protein chip on which the recombinant fusion protein is integrated on a matrix surface coated with an active group and measuring phosphorylation by the kinase.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a biochip constructed by integrating a fusion protein prepared by the recombination of a kinase substrate selected from the group consisting of PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase) with the elevated protein, Selenomonas ruminantium membrane protein on a matrix surface coated with an active group, and a kit for detecting phosphorylation composed of the said biochip and a cofactor labeled with a radio-isotope, and a method for detecting phosphorylation using the same.
The biochip for detecting phosphorylation and the method for detecting phosphorylation using the same of the present invention increase sensitivity by using a radio-isotope, by which a smaller amount of a sample can be used by simpler processes, compared with the conventional method. In addition, the method of the present invention produces a very clear result owing to a simplified procedure excluding blocking process for preventing non-specific fixation on the chip. So, using the biochip of the present invention has advantages of saving time and having economical efficiency. According to the present invention, only a small amount of a sample is required, suggesting that the size of a spot is very small, indicating the number of samples that can be integrated on the surface can be increased, which enables fast analysis of a large amount of samples within a short period. Therefore, the method of the present invention can be effectively used for the analysis of kinase activity.



BRIEF DESCRIPTION OF THE DRAWINGS
The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
FIG. 1 is a diagram showing the nucleotide sequence (SEQ ID NO: 17) of Mep45 derived from Selenomonas ruminantium and the amino acid sequence (SEQ ID NO: 10), the digestion site by the treatment of proteinase K, derived therefrom:
Box: promoter, SLH domain, porin homology region; and
Arrow: digestion site by the treatment of proteinase K in the whole cell.
FIG. 2 is a diagram illustrating the cloning process of the recombinant Mep45-kinase substrate fusion protein.
FIG. 3 is a diagram illustrating the phosphorylation of kinase-substrate using [γ-32P]ATP.
FIG. 4 is a diagram illustrating the phosphorylation of cdc2 protein kinase and peptide substrate using [γ-32P]ATP:
(A) a: negative control (BSA), b: cdc2 protein kinase (peptide); and
(B) a: negative control (BSA), b: cdc2 protein kinase substrate (Mep45 fusion substrate).
FIG. 5 is a diagram illustrating the phosphorylation of PKC (protein kinase C) and peptide substrate using [γ-32P]ATP:
(A) a: negative control (BSA), b: PKC (peptide); and
(B) a: negative control (BSA), b: PKC substrate (Mep45 fusion substrate).



DESCRIPTION OF THE PREFERRED EMBODIMENTS
To achieve the above object, the present invention provides a biochip comprising a fusion protein integrated on a matrix surface coated with an active group,
wherein, the fusion protein is prepared by the fusion of a substrate of a kinase having one of amino acid sequences represented by SEQ. ID. NO: 1-NO: 3 with an elevated protein, wherein the elevated protein is Selenomonas ruminantium membrane protein.
The present invention also provides a kit for the detection of phosphorylation containing the biochip of claim 1 and [γ-32P]ATP.
The present invention further provides a detection method of phosphorylation comprising the following steps:
1) mixing a sample with [γ-32P]ATP;
2) inducing phosphorylation by treating the biochip of claim 1 with the mixed sample of step 1);
3) washing the biochip of step 2); and
4) measuring phosphorylation by observing signals from the biochip of step 3) using X-ray film and phosphorimager.
Hereinafter, the present invention is described in detail.
The present invention provides a biochip comprising a fusion protein integrated on a matrix surface coated with an active group,
wherein, the fusion protein is prepared by the fusion of a substrate of a kinase having one of amino acid sequences represented by SEQ. ID. NO: 1-NO: 3 with an elevated protein.
The material of the matrix of the biochip of the present invention is preferably selected from the group consisting of glass, plastic, metal and silicon, and glass is more preferred, but not always limited thereto.
The active group coated on the matrix of the biochip of the present invention plays a role in fixing a peptide and is preferably selected from the group consisting of amine group, aldehyde group, carboxyl group and thiol group, and aldehyde group is more preferred, but not always limited thereto. In fact, almost every active group known to those in the art as an active group capable of fixing a protein molecule on a matrix can be used.
The kinase substrate of the biochip of the present invention is preferably the substrate (AAKIQASFRGHMARKK; SEQ. ID. NO: 1, PKTPKKAKKL; SEQ. ID. NO: 2, or EPPLSQQAFADLWKK; SEQ. ID. NO: 3) for PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) or DNA-PK (DNA-dependent Protein Kinase), but not always limited thereto.
The elevated protein of the biochip herein is preferably Selenomonas ruminantium membrane protein (SrMep45, SEQ. ID. NO: 10), but not always limited thereto.
The kinase substrate herein is fixed on the biochip of the present invention as the elevated protein-substrate fusion protein, but not always limited thereto.
On the biochip of the present invention, the diameter of the integrated spot is preferably 100 ̃300 μm and the distance between the spots is preferably 300 ̃500 μm, but not always limited thereto.
The present inventors prepared a recombinant fusion protein using substrates of PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) or DNA-PK (DNA-dependent Protein Kinase) and Selenomonas ruminantium membrane protein. Then a biochip was constructed by fixing the recombinant fusion protein on the surface of a slide glass treated with aldehyde group, the functional group fixable protein only, by spotting method (see FIG. 2).
The biochip of the present invention does not need blocking process which is necessary for the conventional methods. The blocker such as BSA (Bovine Serum Albumin) causes the burial of proteins fixed on a matrix surface. BSA is used for the prevention of non-specific fixation observed during the fixation of a kinase specific peptide or a low molecular protein. So, using the biochip of the present invention makes the process simple and has advantages of saving time and having economical efficiency.
To confirm the phosphorylating conditions of kinase-substrate using [γ-32P]ATP, the biochip prepared above was treated with kinase buffer containing [γ-P]ATP and phosphorylation between Mep45-kinase substrate fusion protein and kinases was induced. Then, the phosphorylation was measured by using X-ray film and phosphorimager (see FIG. 3). As a result, there was no signal observed on the spot of BSA, the negative control, while signals were observed on both the spots of substrates of PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) or DNA-PK (DNA-dependent Protein Kinase) and the spots of substrate-SrMep45 fusion protein. The results indicate that substrates of PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) or DNA-PK (DNA-dependent Protein Kinase) and the radio-isotope [γ-32P]ATP of the present invention can be used for the measurement of phosphorylation (see FIGS. 4 and 5).
The present invention also provides a kit for the detection of phosphorylation containing the said biochip and [γ-32P] ATP.
The kit herein can measure phosphorylation of a kinase selected from the group consisting of PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase), but not always limited thereto.
Preferably, the kit additionally contains protein kinase as a positive control.
The present invention further provides a detection method of phosphorylation comprising the following steps:
1) mixing a sample with [γ-32P]ATP;
2) inducing phosphorylation by treating the biochip of claim 1 with the mixed sample of step 1);
3) washing the biochip of step 2); and
4) measuring phosphorylation by observing signals from the biochip of step 3.
In the above method, the sample of step 1) can be selected from the group consisting of extracts of cells and tissues, fractions or cell culture solutions, cell lysates, crude extracts of cells or tissues, exudates such as urine, sweat, saliva and tear, and body fluids such as blood, plasma, lymph and serum, but not limited thereto and any biological sample known to those in the art can be used.
In this method, the sample and the biochip of step 2) are preferably reacted in a 30° C. or 37° C. humid chamber for 30 minutes-1 hour, and one hour reaction is more preferred, but not always limited thereto, and the reaction time can be varied according to the specificity between substrates and kinases in the sample.
In this method, the measurement of phosphorylation in step 4) is performed by using X-ray film or phosphorimager, but not always limited thereto. Sensitization time for the measurement is 12-24 hours, but not always limited thereto, and can be varied according to the specificity between substrates and kinases in the sample.
The method for detecting phosphorylation of the present invention has advantages of a simple process of chip surface treatment for substrate fixation and fast and easy measurement, compared with the conventional fluorescence ELISA, because the one-pot labeled radio-isotope can be detected as it is. According to the conventional fluorescence detection method, a specific amino acid of substrate has to be phosphorylated first, and then the phosphorylated amino acid region is reacted with a secondary antibody labeled with fluorescent materials, which is an indirect detection method that cannot provide precise quantification at a satisfactory level. However, the method for detecting phosphorylation of the present invention is characterized by direct labeling of a radio-isotope to the substrate, so that the method not only facilitates precise quantification of detection result but also facilitates detection with a minimum amount of samples with high sensitivity.
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 1
Preparation of Elevated Protein-Substrate Fusion Protein
<1-1> Cloning of SrMep45-Substrate Fusion Protein
To prepare SrMep45 (Selenomonas ruminantium membrane protein)-substrate fusion protein, the PCR product obtained by PCR amplification with chromosome DNA of Selenomonas ruminantium subsp. lactilytica, ATCC 19205) (Kanegasaki, S., and Takahashi, H., J. Bacteriol. 93, 456-463, 1967) and plasmid were cloned (FIG. 1).
For the cloning of each substrate (AAKIQASFRGHMARKK; SEQ. ID. NO: 1, PKTPKKAKKL; SEQ. ID. NO: 2, or EPPLSQQAFADLWKK; SEQ. ID. NO: 3) for PKC (Protein Kinase C), cdc2-PK (cdc2 Protein Kinase) and DNA-PK (DNA-dependent Protein Kinase), PCR was performed using pSrMep45 as a template with primers PKC-Fw-Nde (5′-CATCATATGGCTGCTAAAATTCAAGCTTCTTTTCGTGGTCATATGGCTCGTAAAAAAGCTAGCAACCCGTTCTCCGATG-3′; SEQ. ID. NO: 4), PKC-Rv-Bam (5′-GACGGATCCTTATTTTTTACGAGCCATATGACCACGAAAAGAAGCTTGAATTTTAGCAGCGAAGAAGAACTGAACGCGACCGAAG-3′; SEQ. ID. NO: 5), cdc2-MP-Fw-Nde (5′-CATCATATGCCTAAAACTCCTAAAAAAGCTAAAAAACTTGCTAGCAACCCGTTCTCCGATG-3′; SEQ. ID. NO: 6), cdc2-MP-Rv-Bam (5′-GACGGATCCTTAAAGTTTTTTAGCTTTTTTAGGAGTTTTAGGGAAGAAGAACTGAACGCGACCGAAG-3′; SEQ. ID. NO: 7), DNA-PK-MP-Fw-Nde (5′-CATCATATGGAACCTCCTCTTTCTCAACAAGCTTTTGCTGATCTTTGGAAAAAAGCTAGCAACCCGTTCTCCGATG-3′; SEQ. ID. NO: 8) and DNA-PK-MP-Rv-Bam (5′-GACGGATCCTTATTTTTTCCAAAGATCAGCAAAAGCTTGTTGAGAAAGAGGAGGTTCGAAGAAGAACTGAACGCGACCGAAG-3′; SEQ. ID. NO: 9) (Table 1). The PCR was performed using 2.0 unit Taq DNA polymerase (50 mM KCl, 10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100), 0.4 mM dNPT (deoxyribonucleotide triphosphate) and the reaction mixture containing the said primer set with Palm-cycler (Corbett Life Science, USA) as follows: at 94° C. for 5 minutes (1 cycle); and at 94° C. for 1 minute, at 55° C. for 1 minute, and at 72° C. for 1 minutes (30 cycles); and then at 72° C. for 5 minutes (1 cycle). The PCR product was electrophoresed on 0.8% agarose gel, followed by staining with SYBR Green I. The amplified right size DNA was purified by using Gel Extraction Spin Column (Bio-Rad Lab., USA). The amplified DNA was ligated to plasmid pGEM-T Easy vector, followed by sequencing. The subcloned sample was digested with NdeI and BamI, which was ligated to pET-28a predigested with the same enzymes. E. coli BL21 (DE3) was transformed with the vector. As a result, a new strain for the production of the recombinant Mep45-kinase substrate fusion protein was generated (FIG. 2).







TABLE 1







Target kinase matrix and primer for cloning thereof










Matrix
Primer
Sequence
SEQ. ID. NO:














PKC (Protein
PKC-Fw-Nde
5′-CAT CAT ATG GCT GCT AAA
SEQ. ID. NO: 4



Kinase C)

ATT CAA GCT TCT TTT CGT GGT


matrix

CAT ATG GCT CGT AAA AAA GCT




AGC AAC CCG TTC TCC GAT G-3′



PKC-Rv-Bam
5′-GAC GGA TCC TTA TTT TTT
SEQ. ID. NO: 5




ACG AGC CAT ATG ACC ACG AAA




AGA AGC TTG AAT TTT AGC AGC




GAA GAA GAA CTG AAC GCG ACC




GAA G-3′


 


cdc2-PK
cdc2-MP-Fw-Nde
5′-CAT CAT ATG CCT AAA ACT
SEQ. ID. NO: 6


(cdc2

CCT AAA AAA GCT AAA AAA CTT


Protein

GCT AGC AAC CCG TTC TCC GAT


Kinase)

G-3′


matrix
cdc2-MP-Rv-Bam
5′-GAC GGA TCC TTA AAG TTT
SEQ. ID. NO: 7




TTT AGC TTT TTT AGG AGT TTT




AGG GAA GAA GAA CTG AAC GCG




ACC GAA G-3′


 


DNA-PK (DNA-
DNA-PK-MP-Fw-Nde
5′-CAT CAT ATG GAA CCT CCT
SEQ. ID. NO: 8


dependent

CTT TCT CAA CAA GCT TTT GCT


Protein

GAT CTT TGG AAA AAA GCT AGC


Kinase)

AAC CCG TTC TCC GAT G-3′


matrix
DNA-PK-MP-Rv-Bam
5′-GAC GGA TCC TTA TTT TTT
SEQ. ID. NO: 9




CCA AGG ATC AGC AAA AGC TTG




TTG AGA AAG AGG AGG TTC GAA




GAA GAA CTG AAC GCG ACC GAA




G-3′









Vectors encoding the final Mep45-substrate fusion protein were pMep45PKC1, pMep45PKC2, pMep45cdc21, pMep45cdc22, pMep45DPK1 and pMep45DPK2, as shown in Table 2.







TABLE 2







Cloned vector for each Mep45-substrate fusion protein











Matrix
Cloned vector
Structure







PKC substrate
pSrMep45PKC1
•-□




(SEQ ID NO: 11)




pSrMep45PKC2
•-□-•




(SEQ ID NO: 12)



cdc2-PK substrate
pSrMep45cdc21
♦-□




(SEQ ID NO: 13)




pSrMep45cdc22
♦-□-♦




(SEQ ID NO: 14)



DNA-PK substrate
pSrMep45DPK1
▴-□




(SEQ ID NO: 15)




pSrMep45DPK2
▴-□-▴




(SEQ ID NO: 16)







□: SrMep45;



•: PKC substrate;



♦: cdc2-PK substrate; and



▴: DNA-PK substrate.






<1-2> Production and Purification of Recombinant Mep45-Kinase Substrate Fusion Protein
The strain prepared in Example <1-1> was cultured in 200 ml of LB medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L) in a 500 ml Erlenmeyer flask at 30° C. with 200 rpm. The antibiotic kanamycin was added at the final concentration of 50 μg/ml. The cells were cultured until O.D.660 reached 0.4. IPTG was added at the final concentration of 0.1 mM, followed by further culture at 30° C. with 200 rpm for 3 hours. Upon completion of the culture, the cells were collected (8,000 rpm, 10 minutes, 4° C.). The collected cells were suspended in PBS (200 mM NaCl, 3 mM KCl, 2 mM KH2PO4, 1 mM Na2HPO4, pH 7.5) and lysed using ultra-sonicator. Cell debris was eliminated by centrifugation. The recombinant Mep45-kinase substrate fusion protein was purified by using Ni-chelating resin (GE Healthcare, Sweden) to 6-histidine tag. The purified protein was quantified according to Bradford method using BSA (bovine serum albumin) as a standard.
The mass of the SrMep45-substrate protein was approximately 45 kDa, which was bound to the aldehyde group on a surface of the slide to protect active site of the substrate being apt to be buried by BSA used in blocking stage.
Example 2
Construction of Biochip
To fix substrate on the aldehyde-treated slide glass (Nuricell Inc., Korea), 0.1 mg/ml of the Mep45-kinase substrate fusion protein or 1.25 μg/ml of peptide substrate (Promega, Madison, Wis.) was integrated. Particularly, the recombinant Mep45-kinase substrate fusion protein solution (10% glycerol, PBS, pH 7.5) was prepared at the concentration of 0.1 mg/ml, and this substrate solution was integrated on the aldehyde-treated slide glass at the spot intervals of 300 μm by using microarray device (Genetix Ltd, UK). The size of the spot was regulated to be 300 μm. The integrated biochip was reacted in a humid chamber at room temperature for one hour, leading to fixation.
Example 3
Confirmation of Phosphorylating Conditions between kinase and substrate using [γ-32P]ATP
The biochip constructed in Example 2 was washed three times with PBS (200 mM NaCl, 3 mM KCl, 2 mM KH2PO4, 1 mM Na2HPO4, pH 7.5), followed by reaction of the kinase and substrate on the chip. Particularly, the chip was washed with kinase buffer (40 mM Tris-HCl, 20 mM MgCl2, 0.1 mg/ml BSA, pH 7.5) once. Then, 50 μl of kinase reaction solution (kinase buffer containing 100 μM ATP, [γ-32P]ATP (0.1 ̃0.6 μCi) (GE Healthcare Life Sciences, UK) and 0.01 ̃50 unit/ml kinase of recombinant Mep45-substrate fusion protein) was distributed on the surface of the biochip. The biochip was covered with cover well, followed by reaction for one hour. One hour later, the biochip was washed with washing buffer three times, followed by washing again with PBS. Centrifugation was performed at 200×g for one minute to eliminate remaining moisture completely. The reacted biochip was sensitized on X-ray film or screen of bioimage analyzer BAS1500 (Fuji Photo Film, Tokyo) for 6-14 hours, followed by measurement of phosphorylation by kinase (FIG. 3).
<3-1> Confirmation of Phosphorylation of cdc2 Protein Kinase-Substrate
1.0 mg/ml of PKTPKKAKKL (SEQ. ID. NO: 2) and Mep45 fusion substrate were integrated on the aldehyde treated slide glass, followed by phosphorylation using 10 unit/ml of cdc2 protein kinase and 0.1 μCi/μl of [γ-32P]ATP. Phosphorylation was measured by using X-ray film or X-ray fluorescence spectrometer. BSA (Bovine Serum Albumin) was used for the negative control.
As a result, no signal was detected on the spot of BSA (negative control), while clear signal was detected on the spot of PKTPKKAKKL (SEQ. ID. NO: 2) substrate (FIG. 4A) and Mep45 fusion substrate (FIG. 4B), suggesting that the radio-isotope [γ-32P]ATP could be effectively applied for the primary detection of phosphorylation (FIG. 4).
<3-2> Confirmation of Phosphorylation of PKC-Substrate
0.3 mg/ml of AAKIQASFRGHMARKK (SEQ. ID. NO: 1) and Mep45 fusion substrate were integrated on the aldehyde treated slide glass, followed by phosphorylation using 10 unit/ml of PKC and 0.1 μCi/μl of [γ-32P]ATP. Phosphorylation was measured by using X-ray film or X-ray fluorescence spectrometer. BSA (Bovine Serum Albumin) was used for the negative control.
As a result, no signal was detected on the spot of BSA (negative control), while clear signal was detected on the spot of AAKIQASFRGHMARKK (SEQ. ID. NO: 1) substrate (FIG. 5A) and Mep45 fusion substrate (FIG. 5B), suggesting that the radio-isotope [γ-32P]ATP could be effectively applied for the primary detection of phosphorylation (FIG. 5).
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

## Claims

1. A biochip comprising a fusion protein integrated a matrix surface coated with an active group,
wherein the fusion protein comprises an elevated protein and a kinase substrate selected from the group consisting of SEQ ID NO: 1-3, wherein the elevated protein is Selenomonas ruminantium membrane protein of SEQ ID NO:10.

2. The biochip according to claim 1, wherein the matrix is selected from the group consisting of glass, plastic, metal and silicon.

3. The biochip according to claim 1, wherein the active group is selected from the group consisting of amine group, aldehyde group, carboxyl group and thiol group.

4. The biochip according to claim 1, wherein the kinase is selected from the group consisting of PKC (protein kinase C), cdks (cyclin-dependent protein kinase) and DNA-PK (DNA-dependent protein kinase).

5. The biochip according to claim 1, wherein the fusion protein is integrated as a spot having 100-300 μm diameter and the distance between the spots is 300-500 μm.

6. A kit for the detection of phosphorylation containing the biochip of claim 1 and [γ-32P]ATP.

7. The kit for the detection of phosphorylation according to claim 6, wherein the kit additionally contains protein kinase as a positive control.

8. A detection method of phosphorylation comprising the following steps:
1) mixing a sample with [γ-32P]ATP;
2) inducing phosphorylation by treating the biochip of claim 1 with the mixed sample of step 1);
3) washing the biochip of step 2); and
4) measuring phosphorylation by observing signals from the biochip of step 3) using X-ray film and phosphorimager.

9. The detection method of phosphorylation according to claim 8, wherein the sample of step 1) is selected from the group consisting of cell culture solutions, cell lysates, crude extracts of cells or tissues, exudates, and body fluids.

10. The detection method of phosphorylation according to claim 8, wherein step 2) is performed at 30° C. or at 37° C. for 30-60 minutes.

11. The detection method of phosphorylation according to claim 9, wherein the exudates is selected from the group consisting of urine, sweat, saliva and tear.

12. The detection method of phosphorylation according to claim 9, wherein the body fluids is selected from the group consisting of blood, plasma, lymph and serum.