# Patent

## Title
Method and agent for immobilizing protein via protein bound to silicon oxide-containing substance

## Abstract

Disclosed is a method for immobilizing a target protein to the surface of silicon oxide-containing substance such as glass without modifying the surface of silicon oxide-containing substance by using a protein binding strongly to silicon oxide-containing substance. A protein capable binding strongly to silicon oxide-containing substance such as glass has been found, and thus a target protein can be directly bound and immobilized to the surface of silicon oxide-containing substance via the found protein. In addition, a fusion protein of the found protein and a target protein can be bound and immobilized to silicon oxide-containing substance.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase patent application of International Application No. PCT/JP2006/322388, with an international filing date of Nov. 9, 2006, which claims priority to Japanese Patent Application No. 326502/2005 filed on Nov. 10, 2005, Japanese Patent Application No. 005061/2006 filed on Jan. 12, 2006, and Japanese Patent Application No. 135572/2006 filed on May 15, 2006; all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to a method for immobilizing a target protein to a silicon oxide-containing substance by using a protein that binds strongly to the silicon oxide-containing substance, and relates to an immobilizing agent including the protein.
BACKGROUND ART
Nanobiotechnology, a new field of technology that unites nanotechnology and biotechnology, has been created and been developing rapidly. The nanobiotechnology is highly expected since it contributes to production of bioelectronics element and biosensor, and to development of bio-chip for DNA and protein, for example.
As described above, bioelectronics element and biosensor technique are extremely important in the nanobiotechnology. DNA chips and protein chips are highly expected. For these chips, a sensor need be produced and controlled in consideration of molecular size, because the chips should sense a single molecule such as DNA and protein. Therefore, development of a biosensor by using semiconductor processing technique has been started.
Specifically, for example, such a biosensor is now under development that an antibody for a virus to be detected is immobilized on a silicon nanowire and that can detect the a virus electrically when even one virus attaches to the antibody.
As described above, in order to further facilitate the development of biosensors using semiconductor processing technique, it is considered essential to develop techniques of placing and immobilizing a desired protein easily and accurately onto a substrate (supporter), such as silicon or glass.
There has been some methods for binding and immobilizing protein on a surface of glass; (1) binding by physical adsorption, (2) immobilizing the protein by using a cross linker which make the protein covalently bind to a surface of a carrier on the glass surface modified with a silane coupling agent or the like.
Method (1) is a method in which the binding is carried out by utilizing electric charge and/or hydrophobicity of the protein. This method (1), however, can not be the one which adsorbs a desired protein, because intensity of binding varies depending on proteins. Also, even if the protein is bound to the glass surface, there is a possibility that the protein molecules are bound at various sites to the glass surface. This often makes differences in protein activity.
Method (2) enables immobilization of a desired protein to the glass surface. However method (2) also causes modification at various sites of protein molecules, thereby affecting protein activity. There has been a study on modification agents generating orientation. However, it is inevitable that operation in modifying substrate surface and cross-linking proteins becomes complicated.
As a method to solve the problems described above, a method has been reported, in which a desired protein is presented on a glass surface which is modified with polyethylene containing copper ion, the protein being bound with a tag which recognizes copper ion. (See Non-Patent Citation 1)
Additionally, it has been reported that a protein to which 9 arginine residues (polyarginine tag) are added can be adsorbed directly to a glass surface and silica resin, without deteriorating its enzyme activity. (See Non-Patent Citation 2)
[Non-Patent Citation 1]


    Enzymatic activity on a chip: The critical role of protein orientation, T. Cha, A. Guo, X.-Y., Zhu, Proteomics, 5, 416-419 (2005).

[Non-Patent Citation 2]

    Fuchs, S. M. & Raines, R. T. Polyarginine as a multifunctional fusion tag. Protein. Sci. 14, 1538-1544 (2005).


DISCLOSURE OF INVENTION
The method described in Non-Patent Citation 1 also has a problem in that complicated operations are required to modify the glass surface with polyethylene containing copper ion. Also, the technique described in Non-Patent Citation 2 for binding protein to silica by adding polyarginine tag has a problem in adsorbability because long-term incubation detaches proteins from the silica surface.
Use of such protein that binds strongly and specifically to a silicon oxide-containing substance would enable direct immobilization of protein to a glass substrate without modifying the glass surface. Therefore, use of such a protein is considered to be applicable to many usage such as production of protein-tip. Unfortunately, there has been no report on such protein.
The present invention has been accomplished in view of the problems above, and an object of the present invention is to find such a protein that binds strongly to the silicon oxide-containing substance, and to provide a method for immobilizing a desired protein to a surface of, for example, glass by using the protein.
The inventors of the present invention, in order to attain the object, have found a protein that can bind strongly to silica, and found that fusion protein of this protein and green fluorescent protein (GFP), or fusion protein of this protein and luciferase can bind to the glass surface, thereby accomplishing the present invention.
A method according to the present invention is a method for immobilizing a target protein to a silicon oxide-containing substance, the method comprising: binding a first protein to the silicon oxide-containing substance, the first protein being capable of binding to the silicon oxide-containing substance in a solution containing at least 0.1M or more sodium chloride; and binding a second protein to the first protein bound to the silicon oxide-containing substance, the second protein being the target protein.
Moreover, a method according to the present invention is a method for immobilizing a target protein to a silicon oxide-containing substance, the method comprising: obtaining a fusion protein of (i) a first protein capable binding to the silicon oxide-containing substance in a solution containing at least 0.1M or more sodium chloride and (ii) a second protein, which is the target protein; and binding the fusion protein to the silicon oxide-containing substance.
An agent according to the present invention is an agent for immobilizing a target protein to a silicon oxide-containing substance, the agent comprising a protein capable of binding to the silicon oxide-containing substance in a solution containing at least 0.1M or more sodium chloride.
An agent according to the present invention is an agent for immobilizing a target protein to a silicon oxide-containing substance, the agent comprising a fusion protein of (i) a first protein capable of binding to the silicon oxide-containing substance in a solution containing at least 0.1M or more sodium chloride and (ii) a second protein, which is the target protein.
The methods and the agent according to the present invention is preferably arranged such that the silicon oxide is silica.
The methods and the agent according to the present invention is preferably arranged such that the protein (the first protein) capable binding to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride is one or more selected from the group consisting of (i) proteins having the amino acid sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, and 17; and proteins having amino acid sequences with deletion, substitution, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, and 17.
A protein according to the present invention is a protein capable of binding to a silicon oxide-containing substance in a solution containing at least 0.1M or more sodium chloride, the protein being any one of the followings (a), (b), and (c): (a) a protein having the amino acid sequence of SEQ ID NO: 13 or a protein having an amino acid sequence with deletion, substitution, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 13; (b) a protein having the amino acid sequence of SEQ ID NO: 15 or a protein having an amino acid sequence with deletion, substitution, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 15; and (c) a protein having the amino acid sequence of SEQ ID NO: 17 or a protein having an amino acid sequence with deletion, substitution, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 17.
A gene according to the present invention is a gene encoding a protein according to the present invention.
An expression vector according to the present invention is an expression vector comprising a gene according to the present invention.
An expression vector according to the present invention is an expression vector comprising a fusion gene encoding a fusion protein of a protein according to the present invention and the target protein.
The present invention makes it possible to immobilize the target protein easily and strongly on, for example, a surface of a glass substrate without losing the function of the target protein. Thus, the present invention makes it possible to easily produce a protein chip, or an inorganic-organic hybrid material such as glass modified with a protein.
For a fuller understanding of the nature and advantages of the invention, reference should be made to the ensuing detailed description taken in conjunction with the accompanying drawings.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is an electrophoresis image showing that protein binding to silica was obtained from a cell lysate of Escherichia coli K-12.
FIG. 2 is an electrophoresis image showing that protein binding to silica was obtained from a cell lysate of Pseudomonas aeruginosa PA01.
FIG. 3 is an electrophoresis image showing that protein binding to silica was obtained from a cell lysate of Pseudomonas putida KT2440.
FIG. 4 shows a structure of SBP-GFP expression vector (pET SBP-GFP).
FIG. 5 is a graph showing pH influence on binding of SBP-GFP fusion protein to silica.
FIG. 6 is a graph showing a result of Scatchard Plot Analysis of SBP-GFP fusion protein.
FIG. 7 is a fluorescence imaging analyzer image showing how GFP, SBP-GFP fusion protein, and R9-GFP fusion protein were bound to the glass surface before/after washing.
FIG. 8 is an imaging analyzer image showing relation between washing time and how GFP and SBP-GFP fusion protein were bound to the glass surface.
FIG. 9 is a CCD camera image showing how luciferase and SBP-Luc fusion protein were bound to the grass surface before/after washing.
FIG. 10 shows the result of searching a silica binding domain on a silica binding protein having an amino-acid sequence shown in SEQ ID NO: 1.
FIG. 11(A) is a schematic view showing a method for immobilizing a target protein to a slide glass in a single step.
FIG. 11(B) is an imaging analyzer image showing immobilization of SBP-GFP fusion protein to a slide glass.
FIG. 11(C) is a SDS-PAGE image of the protein unstuck from the slide glass.
FIG. 12 is an electrophoresis image demonstrating that an antibody can bind to silica via Protein A-SBP fusion protein.
FIG. 13 is a microscope image demonstrating that Bacillus subtilis can be detected by using CWB-SBP fusion protein bound to a slide glass.



BEST MODE FOR CARRYING OUT THE INVENTION
Obtaining and Identification of Protein Binding Strongly to Silicon Oxide-Containing Substance
The inventors have conceived use of protein capable of binding directly to a glass surface, for the purpose of completing a method that can immobilize a protein to the glass surface with no need of complicated operation, such as modification of the glass surface and modification and purification of the protein to be immobilized, unlike a conventional method. Unfortunately, there has been only one report of a protein binding directly to the glass surface. The protein is one to which a polyarginine tag is added. (Non-Patent Citation 2). There is also a problem that adsorption of the protein is weak. The inventors have found a protein binding strongly to a silicon oxide-containing substance, as a result of diligent works.
A protein used in the present invention is a protein capable of binding to the silicon oxide-containing substance in a solution containing at least 0.1 M or more sodium chloride. The “protein capable of binding to silicon oxide-containing substance in a solution containing at least 0.1 M or more sodium chloride” is herein referred to as “protein strongly bindable to silicon oxide-containing substance”. The protein may be derived from any living organisms, such as bacteria, yeast, plant, animal, and the like.
The “silicon oxide-containing substance” herein is a substance containing at least oxygen (O) and silicon (Si). Thus, the “silicon oxide-containing substance” may be a substance consisting of only oxygen and silicon, and a substance comprising oxygen, silicon, and an element other than oxygen and silicon. The element other than oxygen and silicon is not particularly limited. The “silicon oxide-containing substance” may be silica deoxide (silica), glass, asbestos, quartz, crystal, silica sand, amphibole, pyroxene, mica, talc, or feldspar, for example. Additionally, the organic silicon oxide-containing substance such as silicone is encompassed to the “silicon oxide-containing substance”.
The term “protein” herein is used interchangeably with “polypeptide” or “peptide”. The term “protein” encompasses a fragment of a protein. Further, the term “protein” encompasses a fusion protein. The “fusion protein” is a protein in which part (fragment) or whole of at least two heteroproteins are bound to each other.
For example, the protein bindable to silicon oxide-containing substance which protein used in the present invention can be obtained by the following method. However, how to obtain the protein bindable to silicon oxide-containing substance is not limited to this. A protein capable of binding to silicon oxide-containing substance in a solution containing at least 0.1M or more sodium chloride can be suitably used in the present invention.
That is, the protein bindable to silicon oxide-containing substance which protein used in the present invention can be obtained by adding the silicon oxide-containing substance to a solution containing one or more types of proteins, collecting the silicon oxide-containing substance, washing the collected silicon oxide-containing substance in a solution containing at least 0.1M or more sodium chloride, and then isolating the protein(s) binding to the silicon oxide-containing substance even after the washing.
As the solution containing at least one or more types of proteins (hereinafter referred to as “protein solution”), for example, a cell lysate can be suitably used. For example, a random peptide library derived from a phage library, or synthesized peptide library can be suitably used. However, the present invention is not limited to this. The protein solution may contain a substance other than a protein.
The protein solution may be prepared by a known method that is appropriately selected according to a material to be used. For example, the cell lysate can be prepared by a method of physically crushing cells by means of a homogenizer, ultrasonic waves, or the like, a method of crushing cells by using an enzyme or a surface activating agent, a method of crushing cells by a combined use of enzyme or a surface activating agent with a physical method, or other method.
The silicon oxide-containing substance to be added is not particularly limited. For example, the inventors of the present invention added 10 mg of silicon powder to 1 ml of bacteria-derived cell lysate (see Examples).
After the addition of silicon oxide-containing substance to the protein solution, it is preferable to sufficiently mix a mixture solution of the protein and the silicon oxide-containing substance. Mixing conditions are not particularly limited. For example, the mixture solution is mixed by inversion at 4° C. for 15 to 30 minutes.
The collection of the silicon oxide-containing substance can be performed, for example, by centrifuging the mixture solution at such revolutions that allow the silicon oxide-containing substance to precipitate, and then removing a supernatant from the mixture solution. As an alternative, the collection of the silicon oxide-containing substance can be performed by filtering the mixture solution through a filter having an appropriate pore size. However, the present invention is not limited to these methods. By a collection operation, it is possible to remove a protein which does not bind to silicon oxide-containing substance.
The washing is performed to remove a protein binding non-specifically and weakly to silicon oxide-containing substance. The washing is performed, for example, by a method of adding a solution containing at least 0.1M or more sodium chloride to the silicon oxide-containing substance collected as above, sufficiently mixing the mixture solution by pipetting or the like, and then performing centrifugation or filtering as described above. Repeating this operation several times enhances washing effect. Further, preparation of the protein solution by using a solution containing at least 0.1M or more sodium chloride makes it possible to enhance the washing effect (effect of removing non-specific binding).
A washing solution is not particularly limited as long as it contains at least 0.1M or more sodium chloride. However, the washing solution is preferably a buffer solution having near neutral pH. The “solution containing at least 0.1M or more sodium chloride” excludes a sodium chloride concentration of below 0.1M at which the protein binds non-specifically and weakly to the silicon oxide-containing substance.
The protein strongly bindable to silicon oxide-containing substance which protein is used in the present invention should be a protein capable of binding to the silicon oxide-containing substance even when washed with the solution containing at least 0.1M sodium chloride. However, with a washing solution containing a higher sodium chloride concentration, it is possible to obtain a protein more strongly bindable to silicon oxide-containing substance. For example, to obtained a protein more strongly bindable to silica, it is preferable to use 0.2M sodium chloride, more preferable to use 0.5M sodium chloride, further preferable to use 1M sodium chloride. Further, it is possible to obtain a protein exhibiting a higher binding specificity by using a washing solution to which a surface activating agent is added.
In order to obtain a bacteria-derived protein bindable to the silicon oxide-containing substance, the inventors of the present invention used, as a washing buffer solution, 25 mM Tris-HCl buffer solution (pH7.5) containing 1M sodium chloride and 0.5% polyoxyethylene sorbitan monolaurate (product name: Tween 20®) (see Examples).
As a method of releasing the protein from the silicon oxide-containing substance to which the protein is strongly bound, the following methods are examples: a method of using a surface activating agent such as dodecyl sodium sulfate; a method of decreasing pH; and a method of increasing the salt concentration in the solution (increasing the sodium chloride concentration to a concentration of approximately 2M). The present invention is not limited to these examples. The inventors of the present invention used a solution containing 1% dodecyl sodium sulfate and 2% mercaptoethanol (see Examples).
Identification of the thus obtained protein bindable strongly to silicon oxide-containing substance can be performed by a known method. For example, the protein released from the silicon oxide-containing substance as above is separated by polyacrylamide gel electrophoresis, and transferred on a polyvinylidene difluoride (PVDF) film. Then, the film is stained with coomassie brilliant blue, and thereafter a band of the target protein is cut out. A tryptic digest of the cut band is analyzed by matrix-assisted laser desorption/ionization time-of flight mass spectrometer (MALDI-TOF-MS), and the target protein is identified by peptide mass fingerprint analysis. As a result, it is possible to obtain an amino acid sequence of the target protein from a known protein database. For example, it is possible to determine an amino acid sequence by using an automatic peptide sequencer.
The determination of the amino acid sequence enables obtaining of a base sequence of a gene encoding the target protein from a known gene database, for example. As an alternative, a DNA fragment encoding the target protein can be cloned with a primer or a probe designed on the basis of the amino acid sequence of the target protein. Thereby, it is possible to determine a base sequence of the DNA fragment by using a DNA sequencer.
The protein bindable strongly to silicon oxide-containing substance which protein can be suitably used in the present invention can be a protein having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17. The inventors of the present invention identified these proteins as proteins bindable to silica (silicon dioxide). Among these proteins, the proteins having the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, and 11 are all well-known, however, the inventors of the present invention found out for the first time that these proteins are capable of binding to silicon oxide-containing substance. Also, the proteins having the amino acid sequence of SEQ ID NO: 13, 15, and 17 are partial fragments of the protein having the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 13 is a fragment from position 1 and to position 60 of SEQ ID NO: 1, SEQ ID NO: 15 is a fragment from position 203 to position 273 of SEQ ID NO: 1, and SEQ ID NO: 17 is a fragment in which the fragment from position 1 to position 60 of SEQ ID NO: 1 and the fragment from position 203 to position 273 of SEQ ID NO: 1).
Also, the present invention can suitably adopts a protein with an amino acid sequence having deletion, substitution, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17, the protein being capable of binding to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride.
What is meant by “deletion, substitution, or addition of one or several amino acids” is deletion, substitution, or addition of amino acids as many as deletable, substitutable, or addable by a known mutant peptide producing method such as a site-specific mutagenesis (preferably not more than 10 amino acids, more preferably not more than 7 amino acids, and further preferably not more than 5 amino acids). Such a mutant protein is not limited to a protein that is artificially mutated by a known mutant polypeptide producing method, and it may be obtained by isolating and purifying a naturally occurring protein.
It is well known in this field that some of the amino acids in the amino acid sequence of a protein can easily be modified without significantly affecting the structure or function of the protein. It is also known that such a mutant with no significant structural or functional change occurs not only in artificially modified proteins but in nature as well.
The mutant preferably includes substitution, deletion, or addition of an amino acid(s), which may be conservative or non-conservative. Silent substitution, silent addition, and silent deletion are preferable, and conservative substitution is particularly preferable. Neither of these modifications changes the polypeptide activities that the present invention concerns.
Representative examples of conservative substitution include: substitution of one of aliphatic amino acids Ala, Val, Leu, and Ile with another amino acid; exchange of hydroxyl residues Ser and Thr; exchange of acidic residues Asp and Glu; substitution between amide residues Asn and Gln; exchange of basic residues Lys and Arg; and substitution between aromatic residues Phe and Tyr.
The protein strongly bindable to silicon oxide-containing substance according to the present invention may include an additional peptide. Examples of such an additional peptide encompass polyhistidine tag (His-tag), and epitope-labeled peptides such as Myc and Flag.
The protein strongly bindable to silicon oxide-containing substance according to the present invention can be produced by culturing, isolating, and purifying cells that can be a supply source of the protein. As an alternative, the protein bindable to silicon oxide-containing substance according to the present invention can be produced by constructing a recombinant expression vector by a known genetic engineering technique, and introducing the recombinant expression vector into an appropriate host cell to express a recombinant protein. In particular, a partial fragment of a known protein such as the proteins having the amino acid sequence of SEQ ID NO: 13, 15, or 17 can be suitably produced as a recombinant protein (see Examples).
For easy explanation, the above protein bindable to silicon oxide-containing substance according to the present invention is hereinafter referred to as “SBP (silica material binding protein)” if necessary.
[Method for Immobilizing a Target Protein to Silicon Oxide-Containing Substance]
A method of immobilizing a target protein according to the present invention to the silicon oxide-containing substance may be embodied in any way, provided that it includes the following steps; binding a first protein (SBP) to the silicon oxide-containing substance, the first protein being bindable to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride; and binding a second protein which is the target protein to the first protein (SBP) capable of binding to the silicon oxide-containing substance. The method may include a step other than the above, and what is carried out in the step other than the above is not limited.
The wording “immobilize protein” herein means binding protein to the surface of supporter which is the silicon oxide-containing substance in the present invention.
In the step of binding the first protein (SBP) to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride, for example, (i) immersing the silicon oxide-containing substance in a SBP solution, (ii) applying or spotting the SBP solution to the surface of the silicon oxide-containing substance can be adopted as the method.
In addition, the wording “binding the first protein (SBP) to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride” expresses capability of SBP to bind to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride, in other words, capability of SBP to bind to the silicon oxide-containing substance after washing by using the solution containing at least 0.1M or more sodium chloride, and not necessarily means that the immobilization should be performed by binding SBP to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride. Thus, the binding SBP to the silicon oxide-containing substance in a sample can be carried out at any condition.
It is preferable to carry out incubation for the purpose of binding SBP to the silicon oxide-containing substance to a satisfactory extent. Conditions of the incubation are not particularly limited, however, it is preferable to incubate for a few seconds to 30 minutes at temperature between 4° C. and room temperature.
In the process described above, the SBP solution does not have to be a solution containing only purified SBP. For example, in case where SBP is expressed as a recombinant protein in an appropriate host such as Escherichia coli, the SBP solution may be a cell lysate of the host cell. This is based on the fact that proteins other than SBP can be washed away by water washing of the surface of the silicon oxide-containing substance after incubating the silicon oxide-containing substance in the cell lysate of the host cell or applying or spotting the cell lysate to the surface of the silicon oxide-containing substance. As a result, only the immobilized SBP stays on the surface of the silicon oxide-containing substance.
In the step of binding the second protein (target protein) to the first protein (SBP) capable of binding to silicon oxide-containing substance, (i) immersing the silicon oxide-containing substance binding to SBP described above to the solution containing the target protein or (ii) applying or spotting the solution containing the target protein onto the area to which SBP is to be applied or spotted can be adopted as the method.
It is preferable to incubate for the purpose of binding SBP to the target protein to a satisfactory extent. The conditions are not particularly limited, however, it is preferable to incubate for a few seconds to 30 minutes at temperature between 4° C. and room temperature.
In the present embodiment of the present invention, it is possible to select a protein capable of binding to SBP used in the process, such as an antibody specifically-binding to SBP used in the process, as a target protein.
Also, SBP can be a modified protein. For example, biotinylation of SBP makes it possible for avidinylated enzyme to bind to SBP. That is; avidination of the target protein makes it possible for the target protein to bind to SBP, and to immobilize to the silicon oxide-containing substance as a result.
In addition, another protein may interpose between SBP and the target protein. For example, biotinylated SBP may be bound to avidinylated protein A or avidinylated protein G and a desired antibody is bound to the proteins mentioned above as a target protein thereafter. Thereby, the desired antibody becomes immobilized to the silicon oxide-containing substance. The number of the proteins interposing between SBP and the target protein herein is not limited.
The Immobilization of the target protein to the surface of silicon oxide-containing substance can be done in the manner described above.
The method of immobilizing the target protein according to the present invention to the silicon oxide-containing substance may be embodied in other ways, provided that the method includes the following steps; obtaining a fusion protein of (i) the first protein (SBP) capable of binding to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride and (ii) the second protein which is a target protein; binding the fusion protein to the silicon oxide-containing substance. The method may include a step other than the above, and what is carried out in the step other than the above is not limited.
In the step of obtaining a fusion protein of (i) the first protein (SBP) capable of binding to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride and (ii) the target second protein, the fusion protein can be obtained as a recombinant protein by using a known genetic engineering technique. That is, the method may include the following steps: producing a fusion gene (hybrid gene) which SBP encoding gene and the target protein encoding gene are artificially linked, and expressing the fusion gene by (i) inserting a fusion gene to downstream of a promoter in an expression vector and (ii) introducing it to a host cell such as Escherichia coli thereafter. Specific examples of construction of the fusion protein expression vector and expression and purification of the fusion protein are given in Examples hereinafter.
In the step of binding the fusion protein to the silicon oxide-containing substance, for example, (i) immersing the silicon oxide-containing substance to a solution containing the fusion protein and (ii) applying or spotting the solution containing the fusion protein to the surface of the silicon oxide-containing substance may be adopted as the method. In addition, it is preferable to incubate for 5 to 30 minutes at temperature between 4° C. and room temperature in order to bind the fusion protein to the silicon oxide-containing substance to a satisfactory extent. However, conditions for incubating are not limited to the above. In addition, the solution containing a fusion protein is not limited to a solution that contains only a purified fusion protein in this process, as described above.
In the present embodiment of the present invention, the target protein is not limited and can be any protein. Therefore, production of a protein chip (protein array) becomes incredibly simple by obtaining SBP fusion proteins of several target proteins and spotting the fusion proteins to the surface of glass or a silicon substrate. In addition, the cell lysate of host cells, which is used for expression of the fusion protein, can be used as a fusion protein solution for spotting. This saves time and cost for producing the protein chip much more than the conventional method. Commercially produced spotters are preferably used for the production of the protein chip.
Also, several types of single-chain antibodies may be selected as the target proteins, so as to immobilized the antibodies to the substrate (thereby obtaining an antibody array). Alternatively, protein A or protein G may be selected as the target protein, thereby making it possible to bind a given antibody to these proteins.
Further, the method for immobilizing a target protein to silicon oxide-containing substance according to the present invention may be utilized to easily produce an inorganic-organic hybrid material of the silicon oxide-containing substance and a protein. For example, by binging protease to glass, it is possible to provide glass which is resistant to protein stain. Furthermore, by binding, to glass, a protein essential for cell growth, it is possible for cells to grow on the surface of the glass, which is expected to be applicable to artificial organ.
In addition, a technique for detecting bacteria by using silica nanoparticles binding to a protein has been reported in; A rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles. Zhao X, Hilliard L R, Mechery S J, Wang Y, Bagwe R P, Jin S, Tan W., Proc Natl Acad Sci USA. 101, 15027-15032, 2004. The present invention simplifies the production of complex of proteins and silica nanoparticles having quantum dot effect, which is applicable to the technique described above.
Further, by placing a protein molecule on a semiconductor material, the present invention is expected to be applicable to a technique of producing a substrate for human interface technology that enables information exchange between an organism and an electronics device.
[Immobilizing Agent and Kit According to the Present Invention]
An immobilizing agent for immobilizing the target protein to the silicon oxide-containing substance according to the present invention should include (a) the protein (SBP) capable of binding to the silicon oxide-containing substance in a solution containing at least 0.1M or more sodium chloride, or (b) the fusion protein of (i) the first protein (SBP) capable of binding to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride and (ii) a target second protein.
The immobilizing agent may contain any substance other than SBP or the fusion protein. The substance other than SBP or the fusion protein is not particularly limited. As for the protein (SBP) capable of binding to silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride and the fusion protein, refer to the above description thereof. In addition, an immobilizing agent according to the present invention can be used according to the described method for immobilizing a target protein to silicon oxide-containing substance.
The immobilizing agent according to the present invention may be realized in a form of the SBP solution, fusion protein solution, freeze-dried SBP, or freeze-dried fusion protein, for example.
A kit according to the present invention should include the immobilizing agent according to the present invention. Apart from that, the kit may have any component other than the immobilizing agent, and the kit may includes any reagent, instrument, or the like appropriately selected as needed. The kit may include an expression vector for the fusion protein of SBP and the target protein.
A kit including an immobilizing agent according to the present invention can be used as a kit for immobilizing the target protein to the silicon oxide-containing substance. This kit can be used according to the method described above for immobilizing the target protein to the silicon oxide-containing substance.
The word “kit” herein means a package including a container for containing a certain material therein. Examples of the container encompass a bottle, a plate, a tube, a dish, and the like. It is preferable that the kit has an instruction for use of the material. The instruction may have any form as being printed or written on a paper or any other media, or recorded on electronic media such as a magnetic tape, a computer read-around disk or tape, and CD-ROM.
[Protein According to the Present Invention]
A protein according to the present invention is a protein capable of binding to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride, and also is any one of the followings (a), (b), and (c):
(a) a protein having the amino acid sequence of SEQ ID NO: 13 or a protein having an amino acid sequence with deletion, substitution, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 13;
(b) a protein having the amino acid sequence of SEQ ID NO: 15 or a protein having an amino acid sequence with deletion, substitution, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 15; and
(c) a protein having the amino acid sequence of SEQ ID NO: 17 or a protein having an amino acid sequence with deletion, substitution, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 17.
These proteins described above are suitable for use in the method and the agent for immobilizing the target protein to the silicon oxide-containing substance.
As mentioned, a protein having the amino acid sequence of SEQ ID NO: 13 is a protein having the amino acid sequence between position 1 and 60 of the amino acid sequence of SEQ ID NO: 1. Likewise, the protein having the amino acid sequence of SEQ ID NO: 15 is a protein having the amino acid sequence between position 203 and 273 of the amino acid sequence of SEQ ID NO: 1, and the protein having the amino acid sequence of SEQ ID NO: 17 is a protein having the amino acid sequence in which the amino acid sequence between position 1 and 60 of the amino acid sequence of SEQ ID NO: 1 and is bonded with the amino acid sequence between position 203 and 273 of the amino acid sequence of SEQ ID NO: 1.
The inventors have confirmed that these proteins which are partial fragments of the protein having the amino acid sequence of SEQ ID NO: 1 are capable of binding to silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride. The protein having the amino acid sequence of SEQ ID NO: 17, in particular, binds to the silicon oxide-containing substance as strongly as the protein having the amino acid sequence of SEQ ID NO: 1.
A mutant protein with an amino acid sequence “with deletion, substitution, or addition of one or several amino acids” is described above. Those in the art can induce mutation in an amino acid sequence of a protein by using a known technique. For example, it is possible to mutate a given base of a gene encoding a protein according to a known technique for point mutagenesis. It is also possible to produce a deletion mutant or an addition mutant by designing a primer corresponding to a given site of a protein-encoding gene. Further, it can be determined easily by the described method whether the produced mutant is capable of binding to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride.
In addition, these proteins can be produced (obtained) according to a known genetic engineering technique by constructing a recombinant expression vector and expressing it thereafter in an appropriate host cell as a recombinant protein.
[Gene According to the Present Invention]
A gene according to the present invention can be any gene, provided that it encodes a protein according to the present invention. Specifically the followings (a), (b), or (c) are examples thereof.
(a) a gene encoding the protein having the amino acid sequence of SEQ ID NO: 13, or a gene encoding a protein with an amino acid sequence having deletion, substitution, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 13 and also capable of binding to silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride.
(b) a gene encoding a protein having the amino acid sequence of SEQ ID NO: 15, or a gene encoding a protein having an amino acid sequence with deletion, substitution, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 15 and also capable of binding to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride.
(c) a gene encoding a protein having the amino acid sequence of SEQ ID NO: 17, or a gene encoding a protein having an amino acid sequence with deletion, substitution, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 17 and also capable of binding to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride.
In the present description, the word “gene” is used interchangeably with “polynucleotide”, “nucleic acid”, or “nucleic acid molecule”, which means a nucleotide polymer. When used in the present description, the word “base sequence” is used interchangeably with “nucleic acid sequence” or “nucleotide sequence”, and is expressed as a sequence of deoxyribonucleotides (abbreviated to A, G, C, and T).
A gene according to the present invention exists as RNA (mRNA, for example) or DNA (cDNA or genomic DNA, for example). DNA can be either double strand or single strand. Single-strand DNA or RNA can be either a coding strand (known as a sense strand) or a noncoding strand (known as a antisense strand).
In addition, a gene according to the present invention may be fused to a polynucleotide encoding the tag marker (tag sequence or marker sequence) described above at its 5′ end or 3′ end.
A gene according to the present invention is preferably as a gene in the followings (i), (ii), or (iii), as one embodiment. It should be noted that the present invention is not limited to these.
(i) a gene having the base sequence of SEQ ID NO: 14
(ii) a gene having the base sequence of SEQ ID NO: 16
(iii) a gene having the base sequence of SEQ ID NO: 18
The base sequence of SEQ ID NO: 14 corresponds to the sequence between position 1 and 180 of the base sequence of SEQ ID NO: 2, and a gene having this sequence encodes the protein having the amino acid sequence of SEQ ID NO: 13. The base sequence of SEQ ID NO: 16 corresponds to the base sequence between position 607 and 819 of the base sequence of SEQ ID NO: 2, and a gene having this sequence encodes the protein having the amino acid sequence of SEQ ID NO: 15. The base sequence of SEQ ID NO: 18 corresponds to the base sequence in which the base sequence between position 1 and 180 of the base sequence of SEQ ID NO: 2 binds to the base sequence between 607 and 819 of the base sequence of SEQ ID NO: 2, and a gene having this sequence encodes a protein having the amino acid sequence of SEQ ID NO: 17.
Also, a gene according to the present invention has a base sequence that corresponds at least 80%, preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% to a complementary sequence of the sequence of SEQ ID NO: 14, 16, or 18. Beside that, a gene according the present invention is preferably a gene encoding a protein capable of binding to the silicon oxide-containing substance in the solution containing at least 0.1M or more sodium chloride.
For example, the word “a gene having a base sequence which is at least 95% identical to a reference (QUERY) base sequence of a gene encoding a protein according to the present invention” means that the base sequence in question is identical to the reference sequence of the protein according to the present invention, allowing mismatches of up to 5 bases per 100 nucleotide (base) of the reference base sequence between the base sequence in question and the reference base sequence.
It can be determined whether a given specific nucleic acid molecule is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical, for example, to the base sequence of SEQ ID NO: 14 by using a known computer program such as Best fit program (Wisconsin Sequence Analysis Package, Version 8 for Unix®, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711).
A gene according to the present invention includes not only a double strand DNA but also RNA or a single strand DNA such as a sense strand and an antisense strand which constitute a double strand DNA. Also, a gene according to the present invention may contain a sequence such as a sequence in untranslated region (UTR) or a vector sequence (including an expression vector sequence).
To obtain a gene according to the present invention, a method using amplification such as PCR can be used. For example, large amounts of DNA fragments containing a gene according to the present invention can be obtained by constructing each primer based on 5′-end sequence and 3′-end sequence (or the complementary sequence of them) of the base sequence of SEQ ID NO: 14, and running PCR thereafter with these primers and Escherichia coli genomic DNA as a template in order to amplify DNA region between the both primers. Also, a gene having the base sequence of SEQ ID NO: 18, for example, can be obtained by connecting DNA having the base sequence of SEQ ID NO: 14 and DNA having the base sequence of SEQ ID NO: 16 according to a known genetic engineering technique.
[Expression Vector According to the Present Invention]
An expression vector according to the present invention has only to contain a gene encoding a protein according to the present invention. An expression vector according to the present invention also can be a vector containing a fusion gene encoding a fusion protein of a protein according to the present invention and a target protein.
A fusion gene encoding a fusion protein can be obtained by using a known genetic engineering technique. Also, it is possible to produce a vector expressing a fusion protein by inserting a gene encoding a target protein to an expression vector containing only a gene encoding a protein according to the present invention, or inserting a gene encoding a protein according to the present invention to an expression vector containing only a gene encoding a target protein.
A method for producing a recombinant expression vector can be a method using plasmid, phage, or cosmid, for example, and is not particularly limited.
Specific type of a vector is not limited, and a vector capable of expressing in a host cell can be used accordingly. That is, an expression vector can be various plasmids containing polynucleotide according to the present invention and a promoter sequence selected according to a type of a host cell for the purpose of assured expression of polynucleotide according to the present invention.
An expression vector according to the present invention contains an expression control region (a promoter, a terminator, and/or a replication origin) depending on a type of a host to be introduced. A conventional promoter (trc promoter, tac promoter, and lac promoter, for example) is used as a promoter of a bacterial expression vector. A glyceraldehyde 3-phosphate dehydrogenase promoter, and PH05 promoter, for example, is used as a promoter for yeast. In like wise, amylase and trpC, for example, is used for fungi. Additionally, a viral promoter (SV40 early promoter, SV40 late promoter, for example) is used for animal-derived cell. An expression vector can be produced according to a conventional method using a restriction enzyme and/or ligase. Transformation of a host by using an expression vector also can be performed according to a conventional method.
A target protein can be obtained and purified from a cultured host which is transformed by using the expression vector described above after culture, cultivation, or breeding, according to a conventional method (for example, filtration, centrifugation, cell lysis, gel filtration chromatography, and ion-exchange chromatography).
An expression vector preferably contains at least one selective marker. For example, Dihydrofolate reductase or a neomycin-resistant gene is such a selective marker on eucaryotic cell culture, and a tetracycline-resistant gene or an ampicillin-resistant gene is such a selective marker on culture of E. coli and other bacteria.
By using the selective marker mentioned above, it is possible to confirm whether or not polynucleotide according to the present invention is transferred to a host cell, and further, it expresses without fail in a host cell.
A host cell mentioned above is not limited, and various known cells are suitably used. Specifically, bacteria such as Escherichia coli, yeast such as budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), nematode (Caenorhabditis elegans), oocyte of African clawed frog (Xenopus laevis), and animal cell such as CHO cell, COS cell, and Bowes melanoma cell, for example, can be used.
A method for transferring an expression vector described above to a host cell, in other words, a transformation procedure is not particularly limited, and a known method such as electroporation, calcium phosphate method, liposome method, and DEAE dextran process, is suitably used.
Example 1
Identification of Silica Binding Protein (SBP)
(1) Bacterial Strains as Used
The following three types of bacterial strains were used: Escherichia coli K12; Pseudomonas aeruginosa PA01; and Pseudomonas putida KT2440.
(2) Preparation of a Cell Lysate
Each type of the bacterial strains was cultured at 37° C. for 4 hours in 2×YT medium. The cultured bacteria were centrifuged to be collected, and then the collected bacteria was suspended in 50 mM Tris-HCl having pH 7.5 with sucrose concentration of 10%. The suspension was subjected to freeze-thawing, and lysozym was added thereto at a concentration of 250 μg/ml. The resulting solution was left on ice for 30 minutes. After reacted at 37° C. for 5 minutes, the solution was left on ice for another 10 minutes, and subjected to ultrasonic homogenization until its viscosity was lost. Then, the solution was centrifuged for 15 minutes at 20,000×g, and the obtained supernatant was used as a cell lysate.
(3) Obtaining of Silica Binding Protein
The obtained cell lysate was diluted with 25 mM Tris-HCl having pH 7.5, 0.5% Tween20, and 1M NaCl so as to prepare a solution with a protein concentration of 1 mg/ml. To 1 ml of the prepared solution, 10 mg of powder silicon (JUNSEI CHEMICAL CO., LTD.) was added. The resulting solution was mixed by inversion at 4° C. for 30 minutes. After centrifugation at 5,000×g, the resultant supernatant was removed. Added to precipitate was 1 ml of solution containing 25 mM Tris-HCl having pH 7.5, 0.5% Tween20, and 1M NaCl, and the resulting solution was vortexed to dissolve. This washing operation was performed three times. To the precipitate obtained after the washing, 50 μl of SDS sample buffer (1% dodecyl sodium sulfate [SDS], 75 mM Tris-HCl pH7.5, 10% glycerol, 1% beta-mercaptoethanol) was added, and the resulting solution was incubated at 100° C. for 5 minutes. The extracted protein was separated by a typical polyacrylamide electrophoresis (Laemmli method).
FIG. 1 shows the result of Escherichia coli K12. As is apparent from FIG. 1, a band of a silica binding protein was found at the position corresponding to a molecular weight of approximately 30 kD.
FIG. 2 shows the result of Pseudomonas aeruginosa PA01. As is apparent form FIG. 2, bands of silica binding protein were found at the positions corresponding to molecular weight of approximately 30 kD and 40 kD.
FIG. 3 shows the result of Pseudomonas putida KT2440. As is apparent from FIG. 3, a band of silica binding protein was found at the position corresponding to molecular weight of approximately 15 kD and two bands of silica binding protein were found at the position corresponding to molecular weight of approximately 30 kD.
(4) Determination of Amino Acid Sequence and Base Sequence
The obtained protein was separated by polyacrylamide gel electrophoresis, and transferred on a polyvinylidene difluoride (PVDF) film. The film is stained with coomassie brilliant blue, and thereafter a band of the target protein was cut out. A portion of the film was immersed into 100% acetonitrile, and then reacted at 37° C. for 30 minutes in 100 μl of solution containing 100 mM acetic acid, 0.5% polyvinylpyrrolidone K-30, and 1% methionine. After washed with 1 ml of ultrapure water 10 times, the film portion was further washed with 100 μl of solution containing 50 mM ammonium bicarbonate and 5% acetonitrile 3 times. Then, the film portion was digested at 37° C. for 24 hours in 20 μl of 0.5 μg/ml trypsin solution (50 mM ammonium bicarbonate, 5% acetonitrile). A tryptic digest was desalted with ZipTipC18 (Millipore). The desalting was performed by a method according to Millipore's protocol. The desalted sample was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (BiflesIV:Bruker), and the protein and a gene encoding the protein was identified by peptide mass fingerprint analysis. An amino acid sequence of the identified protein was obtained from a database (DDBJ). A base sequence of the gene encoding the identified protein was also obtained from the database (DDBJ).
These results demonstrated that Escherichia coli K-12-derived silica binding protein at an approximately 30 kD molecular weight band was a protein with the amino acid sequence of SEQ ID NO: 1 (ACCESSION:NP—417776), and the protein was encoded by a gene (rplB) with the base sequence of SEQ ID NO: 2.
It was found out that Pseudomonas aeruginosa PA01-derived silica binding protein at an approximately 30 kD molecular weight band was a protein with the amino acid sequence of SEQ ID NO: 3 (ACCESSION:NP—252950), and the protein was encoded by a gene (rplB) with the base sequence of SEQ ID NO: 4.
It was found out that Pseudomonas aeruginosa PA01-derived silica binding protein at an approximately 40 kD molecular weight band was a protein with the amino acid sequence of SEQ ID NO: 5 (ACCESSION:NP—250468), and the protein was encoded by a gene (oprF) with a base sequence of SEQ ID NO: 6.
It was found out that Pseudomonas putida KT2440-derived silica binding protein at an approximately 30 kD molecular weight band was a protein with the amino acid sequence of SEQ ID NO: 7 (ACCESSION:NP—742623), and the protein was encoded by a gene (rplB) with the base sequence of SEQ ID NO: 8.
It was found out that Pseudomonas putida KT2440-derived silica binding protein at an approximately 30 kD molecular weight band was a protein with the amino acid sequence of SEQ ID NO: 9 (ACCESSION:NP—743748), and the protein was encoded by a gene (rpsB) with the base sequence of SEQ ID NO: 10.
It was found out that Pseudomonas putida KT2440-derived silica binding protein at an approximately 15 kD molecular weight band was a protein with the amino acid sequence of SEQ ID NO: 11 (ACCESSION:NP—743476), and the protein was encoded by a gene (rpsI) with the base sequence of SEQ ID NO: 12.
The following experiment was conducted using the Escherichia coli K12-derived silica binding protein at an approximately 30 kD molecular weight band with an amino acid sequence of SEQ ID NO: 1.
Example 2
Measurement of Affinity (Kd) of SBP-GFP Fusion Protein for Silica
(1) Construction of Expression Vector of SBP-GFP Fusion Protein
First, GFP expression vector was constructed. Two types of oligonucleotide primers, P1: AGAAAAGCTTAGTAAAGGAGAAGAACTTTTCACT (SEQ ID NO: 19) and P2: TCATGCGGCCGCAAGCTCATCCATGCCATGTGTA (SEQ ID NO: 20), were produced based on a known gfp gene sequence. PCR was performed with the oligonucleotide primers P1 and P2 by using pGFPuv (Clontech Laboratories, Inc.) as a template. The reaction was performed using KOD plus DNA polymerase (TOYOBO) according to TOYOBO's protocol. PCR products and expression vector pET21-b (Novagen) were treated with restriction enzymes HindIII and NotI at 37° C. for 2 hours, and then subjected to agarose gel electrophoresis. Each of the respective DNA fragments cut out from gel was ligated with Ligation High (TOYOBO) at 16° C. for 2 hours, and then transformed into Escherichia coli MV1184. From the obtained colony, a plasmid into which the target DNA fragment was inserted was extracted. The plasmid was named as pET GFP.
Next, SBP-GFP expression vector was constructed. Two types of oligonucleotide primers, P3: CATCGAATTCTATGGCAGTTGTTAAATGTAA (SEQ ID NO: 21) and P4: AGTTGAGCTCGTTTTGCTACGGCGACGTACGA (SEQ ID NO: 22), were produced based on the base sequence of SEQ ID NO: 2. PCR was performed with the oligonucleotide primers P3 and P4 by using chromosomal DNA of Escherichia coli as a template. PCR products and the pET GFP were treated with restriction enzymes EcoRI and SacI at 37° C. for 2 hours, and then subjected to agarose gel electrophoresis. Each of the respective DNA fragments cut out from gel was ligated with Ligation High (TOYOBO) at 16° C. for 2 hours, and then transformed into Escherichia coli MV1184. From the obtained colony, a plasmid into which the target DNA fragment was inserted was extracted. The plasmid was named as pET SBP-GFP. Construction of the expression vector of SBP-GFP (pET SBP-GFP) was shown in FIG. 4.
(2) Construction of Expression Vector of R9 (Polyarginine tag)-GFP Fusion Protein
An expression vector of R9 (polyarginine tag)-GFP fusion protein was constructed to obtain R9-GFP fusion protein used as a control.
For polyarginine tag, oligonucleotide, G1: CTAGCCGTCGCCGTCGTCGCCGTCGTCGTCGCAAG (SEQ ID NO: 23) and G2: AATTCTTGCGACGACGACGGCGACGACGGCGACGG (SEQ ID NO: 24), were synthesized and formed into double strand DNA. By inserting the double strand DNA into sites of restriction enzyme NheI and EcoRI on the pET GFP, an expression vector of R9 (polyarginine tag)-GFP fusion protein (pET R9-GFP) was constructed.

(3) Expression and Purification of SBP-GFP Fusion Protein and R9-GFP Fusion Protein

Rosetta BL21 (DE3) pLysS (Novagen) which was transformed with each expression vector produced in (1) and (2) was cultured in 2×YT medium at 28° C. until OD600 reached 0.6, and was cultured for another 8 hours after adding 0.3 mM of IPTG thereto. After the cells were collected, a cell lysate was prepared in the same manner as described in (2), and the cell lysate was purified by HisTrap HP 1 ml (Amersham Biosciences) column. The column was washed with buffer solution A (50 mM sodium phosphate pH7.4, 20% glycerol) containing 10 mM imidazole, and then eluted with buffer solution A containing 0.5M imidazole. The eluted protein was purified by Poros HS/M (Perspective Biosystems). The protein was eluted from the column in buffer solution B (20 mM Hepes-NaOH pH7.5, 1 mM EDTA, 1 mM DTT, 20% glycerol) having a linear concentration gradient of NaCl from 0 to 1M. A purification degree of each protein determined by polyacrylamide gel electrophoresis was 95% or higher.

(4) Measurement of Affinity (Kd) of SBP-GFP Fusion Protein and R9-GFP Fusion Protein for Silica

Silica of 0.1 mg was added to the protein solution (GFP, SBP-GFP, SBP-GFP) with a given concentration. After incubated for 15 minutes, silica was precipitated by centrifugation, and was washed thereafter with 1 ml of a buffer solution (25 mM Tris-HCl pH7.5 or 8.0, or Glycine-NaOH pH9.0, 0.5% Tween20, 0.5M NaCl). To know the amount of binding GFP, an amount of non-binding GFP was determined by measuring GFP fluorescence of the supernatant. The amount of binding GFP was determined by subtracting the result of the supernatant from the initial protein concentration. Affinity (Kd) and maximum binding amount (Bmax) were determined from the amount of binding GFP by using Scatchard plot analysis. Smaller value of Kd shows stronger affinity.
Effect of pH on SBP-GFP when binding to silica was shown in FIG. 5. Also, a result of Scatchard plot analysis on SBP-GFP was shown in FIG. 6.
As is apparent form FIG. 5 and FIG. 6, SBP-GFP bound to silica at pH7.0 or more (FIG. 5). Affinity (Kd) of L2-GFP for silica was 0.70, 0.55, and 0.46 nM at pH7.5, 8.0, and 9.0, respectively (FIG. 6). Maximum binding amount (Bmax) of SBP-GFP binding to silica was 25, 32, and 31 μg SBP-GFP protein/mg silica particle at pH7.5, 8.0, and 9.0, respectively (FIG. 6).
Kd value of GFP (R9-GFP) with addition of polyarginine was 120, 18, and 25 nM at pH7.5, 8.0, and 9.0, respectively (the data was not illustrated therein). Also, a maximum binding amount of R9-GFP binding to silica was 5.0, 14, and 16 μg GFP protein/mg silica particle at pH7.5, 8.0, and 9.0, respectively. As a result of this, it was found out that SBP-GFP bound to silica approximately 30 times to 200 times as strongly as R9-GFP.
Example 3
Experiment I on Binding Ability of SBP-GFP Fusion Protein to the Surface of Glass
Samples of 1 mg/ml, 0.5 mg/ml, and 0.5 mg/ml were prepared for SBP-GFP fusion protein, and R9-GFP fusion protein, and GFP (control) purified as in (3) of Example 2.
Letters were written with each solution described above containing SBP-GFP fusion protein, R9-GFP fusion protein, and GFP on the surface of slide glass (MATSUNAMI, MICRO SLIDE GLASS, white edge-polished, 1 mm thick). After leaving at 4° C. for 30 minutes, each slide glass was immersed into 25 mM Tris-HCl pH7.5, 0.5% Tween20, 1M NaCl, and then washed with moderate shaking. Each slide glass was taken out at the beginning of the washing, 24 hours after, and 1 week after, in order to observe the binding state of each protein by an imaging analyzer (Amersham Biosciences).
Images of the slide glasses before/after the washing, which were taken with a fluorescent imaging analyzer (Amersham Biosciences), were shown in FIG. 7. As is apparent from FIG. 7, the letters written with the solution containing only GFP faded completely immediately after the beginning of the washing. Letters written with the solution containing R9-GFP fusion protein also almost faded by washing 24 hours after. On the other hand, letters written with the solution containing SBP-GFP fusion protein did not fade by washing even 1 week after. As a result of this, it was found out that SBP-GFP fusion protein had high binding ability to the surface of glass compared to R9-GFP fusion protein. In addition, according to the fact that activity of SBP-GFP did not decrease even after 1 week of washing, it is considered that SBP-GFP contributes to stabilization of a protein caused by immobilization to the surface of glass.
Example 4
Experiment II of Binding Ability of SBP-GFP Fusion Protein to the Surface of Glass
As in Example 3, each solution containing SBP-GFP fusion protein and GFP was applied to a slide glass respectively, and was washed after leaving at 4° C. for 30 minutes. The washing was carried out by immersing the slide glass into 25 mM Tris-HCl pH7.5, 0.5% Tween20, 1M NaCl and shaking moderately thereafter. Each slide glass was taken out immediately after the beginning of the washing (0 hour), and also 1 hour, 3 hours, 5 hours, and 24 hours after the washing and observed by an imaging analyzer (Amersham Biosciences) to see the binding state of SBP-GFP fusion protein.
The result was shown in FIG. 8. As is apparent from FIG. 8, a spot applied with the solution containing only GFP faded immediately after the beginning of the washing, however, a spot applied with the solution containing SBP-GFP did not fade even after 24 hours of washing. This indicates that the binding is remarkably strong and is kept at least 24 hours.
Example 5
Experiment on Biding Ability of SBP-Luc Fusion Protein to the Surface of Glass
(1) Construction of Expression Vector of SBP-Luc Fusion Protein
Two types of oligonucleotide primers, L1: CCGGGTCGACATGGAAGACGCCAAAAAC (SEQ ID NO: 25) and L2: GTTGCGGCCGCCAATTTGGACTTTCCGCC (SEQ ID NO: 26), were produced based on a known luc gene sequence. PCR was performed with the olygonucleotide primers L1 and L2 by using Luciferase T7 control DNAs (Promega) as a template. The PCR products were inserted into the sites of restriction enzyme SalI and NotI on the pET SBP-GFP, and were substituted for GFP gene, so as to construct an expression vector of SBP-Luc fusion protein.
(2) Expression and Purification of SBP-Luc Fusion Protein
Purified SBP-Luc fusion protein was obtained by a method as in (3) of Example 2. A purification degree of the purified protein determined by polyacrylamide gel electrophoresis was 95% or more.
(3) The Purified SBP-Luc Fusion Protein and Luciferase (Control) were Respectively Prepared to be 10 nM Protein solution.
On the surface of slide glass (MATSUNAMI, MICRO SLIDE GLASS, white edge-polished, 1 mm thick), 3 μL of the solution containing SBP-Luc fusion protein and 0.6 μL of the solution containing Luciferase were spotted respectively. Then, 3 μL of a substrate solution (2 mM ATP, 2 mM Luciferin, 120 mM Tris-HCl pH7.4, 16 mM MgCl2) was added to each spot for measurement of Luciferase emission, and emission was observed thereafter by using a highly sensitive CCD camera (Spectral Instruments, Inc., Tucson, Ariz.). Measurement of emission was conducted immediately after spotting a protein solution or after 5 minutes of incubation thereafter, with washing in a buffer solution (25 mM Tris-HCl pH 8.0, 0.5% Tween20, 1M NaCl).
The result was shown in FIG. 9. In FIG. 9, (before washing) shows luciferase emission immediately after spotting, and (after washing) shows the same after washing in the buffer solution. As is apparent from FIG. 9, there was no difference in luciferase emission immediately after spotting between SBP-Luc fusion protein and Luciferase. However, it was observed that SBP-Luc fusion protein was bound to slide glass after washing, on the other hand, Luciferase was all rinsed away.
Example 6
Silica Binding Domain of the Silica Binding Protein with the Amino Acid Sequence of SEQ ID No: 1
(1) Production of a Deleted Mutant of a Silica Binding Protein with the Amino Acid Sequence of SEQ ID NO: 1.
To search for a silica binding domain of the silica binding protein with the amino acid sequence of SEQ ID NO: 1, four types of partially-deleted mutants, that is, the protein having the sequence between position 1 and 60 of the sequence of SEQ ID NO: 1, the protein having the sequence between position 61 and 202 of the sequence of SEQ ID NO: 1, the protein having the sequence between position 203 and 273 of the sequence of SEQ ID NO: 1, and the protein having the sequence between position 1 and 60 and also between position 203 and 273 of the sequence of SEQ ID NO: 1, were fused respectively to GFP (see FIG. 10).
To construct each expression vector of SBP(1-60)-GFP fusion protein, SBP(61-202)-GFP fusion protein, and SBP(203-273)-GFP fusion protein, four types of oligonucleotide primers, P5: AGTTGAGCTCGTCTGCTTGTGGCCACCACCGA (SEQ ID NO: 27), P6: CATCGAATTCTGCTTACCGTATTGTTGACTTC (SEQ ID NO: 28), P7: AGTTGAGCTCGTCAGCATATGCTCAGCATTGC (SEQ ID NO: 29), and P8: CATCGAATTCTCGCGTTCTGGGTAAAGCAGG (SEQ ID NO: 30), were produced based on the base sequence of SEQ ID NO: 2. PCR was performed with the nucleotide primers P3 (SEQ ID NO: 21) and P5, P6 and P7, and P4 (SEQ ID NO: 22) and P8 as a primer pair respectively by using chromosomal DNA of Escherichia coli as a template. Each DNA fragment amplified by PCR was respectively inserted into the sites of the restriction enzyme EcoRI and SacI on the pET GFP, so as to construct pET SBP(1-60)-GFP, pET SBP(61-202)-GFP, and pET SBP(203-273)-GFP.
To construct pET SBP(1-60, 203-273)-GFP, an expression vector of fusion protein of SBP(1-60, 203-273) whose middle part was deleted and GFP, inverse PCR was performed with oligonucleotide primers, P9: GTTCTGGGTAAAGCAGGTGC (SEQ ID NO: 31) and P10: CTGCTTGTGGCCACCACCGC (SEQ ID NO: 32), by using the pET SPB-GFP as a template.
(2) Expression and Purification of Each Deleted Mutant-GFP Fusion Protein
Each deleted mutant-GFP fusion protein was purified by a method as in (3) of Example 2. A purification degree of each purified protein determined by polyacrylamide gel electrophoresis was 95% or more.
(3) Measurement of Affinity (Kd) of Each Deleted Mutant-GFP Fusion Protein for Silica.
Each of a binding amount of SBP(1-60)-GFP, SBP(61-202)-GFP, SBP(203-273)-GFP, and SBP(1-60, 203-273)-GFP to silica was determined by a method as in (4) of Example 2. Affinity (Kd) and maximum binding amount (Bmax) were determined with the binding amount by using Scatchard plot analysis.
The result was shown in FIG. 10. As is apparent from the Kd value, SBP(1-60)-GFP and SBP(203-273)-GFP were bound to silica, though SBP(61-202) was not. This indicates that silica binding domain of silica binding protein with the amino acid sequence of SEQ ID NO: 1 exists in the amino acid sequence between position 1 and 60 and also between position 203 and 273. The Kd value of SBP(1-60)-GFP and SBP(203-273)-GFP were both lower than SBP-GFP, however, the Kd value of SBP(1-60, 203-273)-GFP was almost same as SBP-GFP. This result indicates that cooperative act of the two silica binding domains makes it possible for the silica binding protein having the amino acid sequence of SEQ ID NO: 1 to bind strongly to silica. In addition, maximum binding amount of SBP(1-60, 203-273)-GFP was also same as SBP-GFP.
Example 7
Binding the Target Protein in the Cell Lysate to Slide Glass in a Single Step
0.2 ml of a culture solution containing Escherichia coli expressing SBP-GFP fusion protein was mixed with a same amount of B-PER cell lysate (PIRCE), and then 13 μl of 1M Tris-HCl (pH7.5) and 14 μl of 5M NaCl were added to the mixture after 10 minutes, as was shown in FIG. 11(A). A portion of the mixture (cell lysate) was plotted on a slide glass by using Stampman (Nippon Laser & Electronics Lab.). The slide glass was then washed by a washing buffer (25 mM Tris-HCl pH8.0, 0.5% Tween20, 1M NaCl).
The result of observation of emission on the slide glass determined by an imaging analyzer (Amersham Biosciences) was shown in FIG. 11(B).
The protein binding to the slide glass was taken off with SDS sample buffer by pipetting, and then subjected to SDS-PAGE. The result of SDS-PAGE was shown in FIG. 11(C). Lane 1 showed the result of the cell lysate plotted as above, and Lane 2 showed the result of the protein taken off from the washed slide glass. As is apparent from FIG. 11(C), it was found out that only SBP-GFP fusion protein was left on the washed slide glass.
That is, it was demonstrated that a protein chip could be produced by plotting a fusion protein of silica binding protein and a target protein on a slide glass and washing thereafter, without purifying the fusion.
Example 8
Binding Antibody to Silica Via Protein A-SBP Fusion Protein
(1) Construction of an Expression Vector of Protein A-SBP Fusion Protein
Two types of oligonucleotide primers, P11: TGCGGATCCTGCGCAACACGATGAAGC (SEQ ID NO: 33) and P12: TTAGAGCTCAGGTTGTTGTCTTCCTCTTT (SEQ ID NO: 34), were produced based on a known protein A gene sequence (ACCESSION: NC—003923 (Gene ID: 1004837)). PCR was performed with the nucleotide primers P11 and P12 by using chromosomal DNA of Staphylococcus aureus subsp. aureus MW2 as a template. The reaction was performed using KOD Plus DNA polymerase (TOYOBO) according to TOYOBO's protocol. PCR products and expression vector pET21-b (Novagen) were treated with restriction enzymes BamHI and SacI at 37° C. for 2 hours, and then subjected to agarose gel electrophoresis. Each of the respective DNA fragments cut out from gel was ligated with Ligation High (TOYOBO) at 16° C. for 2 hours, and then transformed into Escherichia coli MV1184. From the obtained colony, a plasmid into which the target DNA fragment was inserted was extracted. The plasmid was named as pET ProteinA.
Next, ProteinA-SBP expression vector was constructed. Two types of oligonucleotide primers, P13: GTTGTCGACATGGCAGTTGTTAAATGTAA (SEQ ID NO: 35) and P14: GTTGCGGCCGCTTTGCTACGGCGACGTACG (SEQ ID NO: 36), were produced based on the base sequence of SEQ ID NO: 2. PCR was performed with the oligonucleotide primers P13 and P14 by using a chromosome of Escherichia coli K12 as a template. PCR products and the pET ProteinA were treated with restriction enzymes SalI and NotI at 37° C. for 2 hours, and then subjected to agarose gel electrophoresis. Each of the respective DNA fragments cut out from gel was ligated with Ligation High (TOYOBO) at 16° C. for 2 hours, and then transformed into Escherichia coli MV1184. From the obtained colony, a plasmid into which the target DNA fragment was inserted was extracted. The plasmid was named as pET ProteinA-SBP.
(2) Expression and Purification of ProteinA-SBP Fusion Protein
Purified ProteinA-SBP fusion protein was obtained by a method as in (3) of Example 2. A purification degree of the purified protein determined by polyacrylamide gel electrophoresis was 95% or higher.
(3) Binding of Silica, ProteinA-SBP Fusion Protein and Antibody
Anti-Pseudomonas aeruginosa, IgG2a (Biogenesis Ltd., product name: Anti-Pseudomonas aeruginosa, Mouse-Mono (B11), product code: 7889-9007) was used as antibody.
A sample was prepared by mixing 1 mg of silica (particle diameter: 0.8 μm, SOEKAWA CHEMICAL CO., LTD., product name: silica dioxide, product code: No. 90372A), 0.73 μg (=13.3 pmol) of ProteinA-SBP fusion protein (molecular weight: approximately 55 kDa), and 2 μg (=13.3 pmol) of an antibody (molecular weight: approximately 150 kDa), in 1 ml of a buffer solution (25 mM Tris-HCl pH8, 0.5M NaCl, 0.5% Tween20). In addition, a sample containing only silica and ProteinA-SBP fusion protein and a sample containing only silica and an antibody were prepared.
The prepared sample was mixed by inversion at 4° C. for 30 minutes. After centrifugation at 10,000×g, the supernatant was removed. To the precipitate was added 1 ml of the buffer solution, and the resulting solution was vortexed to dissolve. Such a washing operation was performed three times in total. To the precipitate obtained after the washing, 20 μl of SDS sample buffer (1% dodecyl sodium sulfate [SDS], 75 mM Tris-HCl pH7.5, 10% glycerol, 1% beta-mercaptoethanol) was added, and resulting solution was incubated at 100° C. for 5 minutes. The extracted protein was separated by a typical polyacrylamide electrophoresis (Laemmli method).
To determine a position and a density of a band, 0.73 μg of ProteinA-SBP fusion protein and 2 μg of the antibody were dissolved respectively in 20 μl of SDS sample buffer, and then an equal amount of the each sample was subjected to electrophoresis.
The result was shown in FIG. 12. Lane 1 shows the result of the sample containing only ProteinA-SBP fusion protein. Lane 2 shows the result of precipitate of the sample containing only ProteinA-SBP fusion protein and silica particles. By the fact that a band in lane 2 existed at the same position and density as a band in lane 1, it is indicated that 0.73 μg (13.3 pmol) of ProteinA-SBP fusion protein was all bound to 1 mg of silica particles.
Lane 4 shows the result of the sample containing only the antibody, and bands of H chain and L chain exist in lane 4. Lane 5 shows the result of precipitate of the sample containing the antibody and silica particles. By the fact that a band of the antibody did not exist in lane 5, it can be understood that the antibody was not bound to silica particles.
Lane 3 shows the result of precipitate of the sample containing ProteinA-SBP fusion protein, an antibody, and silica particles. A band of ProteinA-SBP fusion protein, a band of H chain of the antibody, and a band of L chain of the antibody exist in lane 3. By the fact that each band shows almost same density as a band in lane 1 and bands in lane 4 respectively, it can be understand that 2 μg (13.3 pmol) of the antibody was bound to 1 mg of silica particles via 0.73 μg (13.3 pmol) of ProteinA-SBP fusion protein.
As a result of Example 8, it was indicated that a target protein could be immobilized easily by using ProteinA-SBP fusion protein, without modifying an antibody or glass.
Example 9
Detection of Bacteria by Using Cell Wall Binding Protein (CWB)-SBP Fusion Protein on Slide Glass
Two types of oligonucleotide primers, P15: CATCGAATTCTAAATTAACAGTTGCTGCAAACAA (SEQ ID NO: 37) and P16: AGTTGAGCTCGTTAAATCTTTTGCATTTACCCA (SEQ ID NO: 38), were produced based on a known Staphylococcus aureus atl gene sequence (ACCESSION: NC—003923). PCR was performed with the oligonucleotide primers P15 and P16 by using chromosomal DNA of Staphylococcus aureus subsp. aureus MW2 as a template. The reaction was performed using KOD Plus DNA polymerase (TOYOBO) according to TOYOBO's protocol. PCR products and expression vector pET21-b (Novagen) were treated with restriction enzymes EcoRI and SacI at 37° C. for 2 hours, and then subjected to agarose gel electrophoresis. Each of the respective DNA fragments cut out from gel was ligated with Ligation High (TOYOBO) at 16° C. for 2 hours, and then transformed into Escherichia coli MV1184. From the obtained colony, a plasmid into which the target DNA fragment was inserted was extracted. The plasmid was named as pET CWB.
Next, CWB-SBP expression vector was constructed. PCR was performed with two types of nucleotide primers P13 (SEQ ID NO: 35) and P14 (SEQ ID NO: 36) as used in Example 8 by using a chromosome of Escherichia coli K12 as a template. PCR products and pET CWB were treated with restriction enzymes SalI and NotI at 37° C. for 2 hours, and then subjected to agarose gel electrophoresis. Each of the respective DNA fragments cut out from gel was ligated with Ligation High (TOYOBO) at 16° C. for 2 hours, and then transformed into Escherichia coli MV1184. From the obtained colony, a plasmid into which the target DNA fragment was inserted was extracted. The plasmid was named as pET CWB-SBP.
(2) Expression and Purification of CWB-SBP Fusion Protein
Purified CWB-SBP fusion protein was obtained by a method as in (3) of Example 2. A purification degree of the purified protein determined by polyacrylamide gel electrophoresis was 95% or higher.
(3) Binding of CWD-SBP Fusion Protein and Bacillus subtilis on Slide Glass
Bacillus subtilis was cultured overnight at 37° C. in 2×YT medium (OD600=5 ̃6). Then, 1 ml of the culture solution was centrifuged at 15000 rpm for 1 minute to be collected, and the supernatant was removed. The precipitate was suspended in 1 ml of a buffer solution (20 mM Tris-HCl pH 9). Such a washing operation was performed two times in total. Also, CWB-SBP fusion protein was prepared to be a solution with a protein concentration of 0.2 mg/ml by using the buffer solution described above.
A plastic tape with a hole 6 mm in diameter was attached to a slide glass (MATSUNAMI, product name: MICRO SLIDE GLASS, white edge-polished, No. 1, product code: S-1111). Spotted to the hole was 10 μl (2 μg) of CWB-SBP fusion protein solution, and binding reaction was performed at room temperature for 1 minute. After removing extra solution, 50 μl of the buffer solution was spotted, and washed by pipetting several times. Such a washing operation was performed two times. After washing, 50 μl of the bacteria solution was spotted, and then left for 10 minutes to be bound. Extra solution was removed, and the washing operation described above was performed 3 times. The spotted place was observed by using a phase microscope after peeling away the seal. A slide glass to which only B. subtilis was spotted and CWB-SBP fusion protein was not spotted was also observed as a control.
The result was shown in FIG. 13. When CWB-SBP fusion protein is not spotted, Bacillus subtilis can't be observed on the slide glass. On the other hand, it turns out that Bacillus subtilis is immobilized to all over the slide glass under the presence of CWB-SBP fusion protein.
CWB is a protein binding to peptide glycan in surface (cell wall) of a gram-positive bacteria. Therefore, as a result of Example 9, it was indicated that a gram-positive bacteria could be detected specifically on glass by using CWB-SBP fusion protein.
Specific embodiments or examples implemented in BEST MODE FOR CARRYING OUT THE INVENTION only show technical features of the present invention and are not intended to limit the scope of the invention. Variations can be effected within the spirit of the present invention and the scope of the following claims.
Further, all of the academic documents and patent documents listed herein are incorporated by reference herein.
INDUSTRIAL APPLICABILITY
The present invention is applicable to wide-ranging technical fields including inorganic-organic hybrid material such as protein chip, nanobiodevice, and modified glass.

## Claims

1. A method for immobilizing a target protein to a silicon oxide-containing substance, the method comprising:
obtaining a fusion protein of (i) a first protein and (ii) a second protein, which is the target protein; and
binding the first protein of the fusion protein directly to the silicon oxide-containing substance,
wherein the first protein is selected from the group consisting of a protein having the amino acid sequence of SEQ ID NO: 1, 13, 15, and 17, or is selected from the group consisting of a protein having an amino acid sequence with deletion(s), substitution(s), or addition(s) of one to ten amino acids in the amino acid sequence of SEQ ID NO: 1, 13, 15, and 17.

2. The method according to claim 1, wherein the silicon oxide is silica.

3. The method of claim 1, wherein the first protein is selected from the group consisting of a protein having the amino acid sequence of SEQ ID NO: 1, 13, 15, and 17.

4. The method of claim 1, wherein the first protein is selected from the group consisting of a protein having an amino acid sequence with deletion(s), substitution(s), or addition(s) of one to seven amino acids in the amino acid sequence of SEQ ID NO: 1, 13, 15, or 17.

5. The method of claim 1, wherein the first protein is selected from the group consisting of a protein having an amino acid sequence with deletion(s), substitution(s), or addition(s) of one to five amino acids in the amino acid sequence of SEQ ID NO: 1, 13, 15, or 17.

6. The method of claim 1, wherein the first protein has the amino acid sequence of SEQ ID NO: 1.