KR101565656B1 - Polypeptide binding polystyrene specifically and use thereof - Google Patents

Abstract

The present invention relates to polystyrene binding specific polypeptides and their uses, and more particularly to polystyrene binding specific polypeptides comprising the amino acid sequence of SEQ ID NO: 1, i) a polystyrene binding specific polypeptide comprising the amino acid sequence of SEQ ID NO: A polypeptide consisting of the 21st amino acid sequence; ii) a peptide fragment consisting of 6-12 amino acids; iii) a polypeptide consisting of the 27th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2; iv) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2; And v) a fusion polypeptide comprising the polystyrene binding specific polypeptide fragment of claim 1, and a method for specifically detecting a target protein using the fusion polypeptide.
The polypeptide of SEQ ID NO: 1 of the present invention binds specifically to polystyrene. In addition, the fusion polypeptide includes a protein backbone module that enhances binding affinity and binding specificity with a target protein, so that it is effective for detecting and screening disease-specific peptides, To improve the efficiency of detection and screening. Therefore, the bacteriophage library screening method and protein detection method using the fusion polypeptide have high sensitivity and efficiency, and thus are highly likely to be used industrially.

Description

Polystyrene binding specific polynucleotides and polystyrene binding polystyrene specific and use thereof < RTI ID = 0.0 >

The present invention relates to polystyrene binding specific polypeptides and their uses, and more particularly to polystyrene binding specific polypeptides comprising the amino acid sequence of SEQ ID NO: 1, i) a polystyrene binding specific polypeptide comprising the amino acid sequence of SEQ ID NO: A polypeptide consisting of the 21st amino acid sequence; ii) a peptide fragment consisting of 6-12 amino acids; iii) a polypeptide consisting of the 27th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2; iv) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2; And v) a fusion polypeptide comprising the polystyrene binding specific polypeptide fragment of claim 1, and a method for specifically detecting a target protein using the fusion polypeptide.

Peptides that target diseases specific proteins and that have high affinity and specificity are of great use in diagnostic and therapeutic applications. Previously, phage display, more specifically T7 phage library, was effectively used as a screening method for peptides that specifically bind to specific proteins. However, there is a disadvantage in that the affinity of the synthesized peptide thus screened is bound to the target protein only by itself. In order to complement the weak peptide-protein interactions, studies have been made to imitate protein-protein bonds and increase affinity by inserting peptides into specific protein scaffolds. However, in this case, the peptide inserted into a specific protein skeleton does not always exhibit an increase in affinity. This is because the structure of the peptide distributed in the T7 phage library or the structure of the free peptide synthesized from the amino acid sequence differs from that of the specific protein backbone It is presumed that the tertiary structure of the inserted peptide is different.

In order to solve this problem, the inventors of the present invention disclose a fusion polypeptide comprising a protein backbone module that enhances binding affinity and binding specificity with a target protein and the fusion polypeptide in the 'T7 phage library technology' in '10-1294982' A peptide screening method specific to the target protein to be applied was devised, and the protein skeleton module was named DMID (Designed Modular Immunodiagnostics).

However, even though the fusion polypeptide containing the protein backbone module has increased the binding affinity for the target protein, there are many factors that influence the detection reaction when practically applied to molecular diagnostic techniques. In fact, The results of the detection reaction vary depending on factors.

Polystyrene is a noncrystalline polymer obtained by radical polymerization of styrene, and is a colorless transparent thermoplastic resin. Polystyrene is also called styrol resin. It is made of polystyrene which is a polymer of liquid styrene unit produced by reacting ethylene and benzene. The styrene resin is the most easily processed in plastic and has a high refractive index. In addition to being transparent and beautiful in color, it is a solid molded product (molded product) and also excellent as an electrical insulating material.

Many immunoassays require that the antigen or antibody be immobilized on a solid support. Polystyrene is the most common substance used for solid phase immunoassay, and most enzyme-linked immunosorbent assays (ELISA) use polystyrene as a solid phase. This is a very hydrophobic substance due to the linking structure of the cyclic carbon residue of the benzene ring to the linear molecule, and it can be easily modified by radiation or chemical reaction, and is widely used for the coating of protein for ELISA analysis. Most of the attachment mechanisms of antibodies and proteins to polystyrene are passive absorption, which allows attachment of proteins via hydrophobic, hydrophilic, or ionic interaction depending on the polystyrene manufacturing process. However, this also has to be accompanied by the characteristics of each protein, the size of the protein and the consideration of the coating buffer, the coating efficiency is low, and the direction of the coated protein is not constant.

Accordingly, the inventors of the present invention prepared a polypeptide of SEQ ID NO: 1 which specifically binds to polystyrene, which is one of the antigens which are difficult to produce antibodies, and which is not affected by passive absorption, and the fusion polypeptide containing the polypeptide The present inventors have found that the detection efficiency is greatly increased in the molecular diagnosis process, and thus the present invention has been completed.

Accordingly, an object of the present invention is to provide a polystyrene binding specific polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is a polypeptide comprising: i) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2;

ii) a peptide fragment consisting of 6-12 amino acids;

iii) a polypeptide consisting of the 27th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2;

iv) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2; And

v) a fusion polypeptide comprising the polystyrene binding specific polypeptide fragment of claim 1.

Yet another object of the present invention is to provide a polynucleotide encoding said fusion polypeptide.

Yet another object of the present invention is to provide a vector encoding said polynucleotide.

It is still another object of the present invention to provide a bacteriophage comprising the fusion polypeptide.

It is still another object of the present invention to provide a bacteriophage library composed of a plurality of bacteriophages comprising the fusion polypeptide.

Yet another object of the present invention is a method for identifying a target protein comprising the steps of: a) allowing interaction between a target protein and the bacteriophage library; And

and b) detecting the bacteriophage library specifically bound to the target protein. The present invention also provides a method for screening a polystyrene and a target protein specifically targeting the peptide.

Yet another object of the present invention is to provide a method for preparing a fusion polypeptide comprising: (a) attaching and coating the fusion polypeptide to a solid phase support of polystyrene;

(b) treating the target protein with the coated support of step (a) to bind to the fusion polypeptide; And

(c) detecting the target protein bound in step (b).

In order to accomplish the above object, the present invention provides a polystyrene binding specific polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

In order to accomplish another object of the present invention, there is provided a polypeptide comprising: i) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2;

ii) a peptide fragment consisting of 6-12 amino acids;

iii) a polypeptide consisting of the 27th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2;

iv) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2; And

v) a fusion polypeptide comprising the polystyrene binding specific polypeptide fragment of claim 1.

In order to achieve still another object of the present invention, the present invention provides a polynucleotide encoding said fusion polypeptide.

In order to achieve still another object of the present invention, the present invention provides a vector encoding said polynucleotide.

In order to accomplish still another object of the present invention, the present invention provides a bacteriophage comprising the fusion polypeptide.

In order to accomplish still another object of the present invention, the present invention provides a bacteriophage library composed of a plurality of bacteriophages comprising the fusion polypeptide.

In order to accomplish still another object of the present invention, the present invention provides a method for detecting a target protein, comprising the steps of: a) causing interaction between a target protein and the bacteriophage library; And

and b) detecting the bacteriophage library specifically bound to the target protein. The present invention also provides a method for screening a polystyrene and a target protein specifically targeting the bacteriophage library.

According to another aspect of the present invention, there is provided a method for preparing a fusion polypeptide comprising the steps of: (a) attaching the fusion polypeptide to a solid phase support of polystyrene;

(b) treating the target protein with the coated support of step (a) to bind to the fusion polypeptide; And

(c) detecting the target protein bound in step (b).

Hereinafter, the present invention will be described in detail.

The present invention provides a polystyrene binding specific polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

The polystyrene is an amorphous polymer generally obtained by radical polymerization of styrene, which is a colorless transparent thermoplastic resin and is also referred to as a styrol resin. The polystyrene may be a homopolymer of a styrenic monomer such as styrene, methylstyrene or glycidyl-substituted styrene, a mutual copolymer in the form of a random or block, a copolymer of an unsaturated monomer having a different weight in a majority, Wherein the other unsaturated monomer includes acrylic acid, methacrylic acid, maleic acid, itaconic acid, methyl acrylate, ethyl acrylate, methyl methacrylate, maleic acid anhydride, glycidyl methacrylic acid, Unsaturated organic acids such as alkyl maleic acid imides or derivatives thereof, vinyl esters such as vinyl acetate and vinyl butyrate, aromatic vinyl compounds such as styrene and methyl styrene, or vinyl trimethyl methoxy silane, methacryloyloxypropyl trimethoxy Vinyl silane such as silane, or a non-conjugate such as dicyclopentadiene and 4-ethylidene-2-norbornene Diene and the like can be used. In the case of copolymerization, the? -Olefin and other monomers are not limited to two kinds, but may be composed of plural kinds.

The polypeptide of SEQ ID NO: 1 of the present invention binds specifically to polystyrene.

Such an effect is well illustrated in an embodiment of the present invention.

In one embodiment of the present invention, the phage clone (SEQ ID NO: 1) inserted with the 4R1 phage clone exhibited excellent binding ability to polystyrene, and the binding to polystyrene was completely inhibited by blocking of skim milk. Respectively.

The polypeptide of the present invention is characterized by comprising the amino acid sequence of SEQ ID NO: 1. In addition, the polypeptide of the present invention includes not only a peptide having a native amino acid sequence but also an amino acid sequence variant thereof within the scope of the present invention. The variant of the peptide of the present invention means a peptide having one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1 having different sequences by deletion, insertion, non-conservative or conservative substitution, substitution of amino acid analog, or combination thereof. Amino acid exchanges that do not globally alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979).

In some cases, the polypeptides of the present invention may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like.

The peptides of the present invention can be prepared by chemical synthesis known in the art (Creighton, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemistry (see for example Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press , Boca Raton Florida, 1997; A Practical Approach, Atherton and Sheppard, Eds., IRL Press, Oxford, England, 1989).

 The peptides of the present invention can also be produced by genetic engineering methods. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (commercially available from Biosearch or Applied Biosystems). The constructed DNA sequence is operatively linked to the DNA sequence and contains one or more expression control sequences (e.g., promoters, enhancers, etc.) that regulate the expression of the DNA sequence , And the host cells are transformed with the recombinant expression vector formed therefrom. The resulting transformant is cultivated under conditions and conditions suitable for the expression of the DNA sequence to recover a substantially pure peptide encoded by the DNA sequence from the culture. The recovery can be carried out using methods known in the art (for example, chromatography). By "substantially pure peptide" herein is meant that the peptide according to the invention is substantially free of any other proteins derived from the host. Genetic engineering methods for peptide synthesis of the present invention can be found in the following references: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N. Y., Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif., 1991; And Hitzeman et al., J. Biol. Chem., 255: 12073-12080,1990.

Preferably, the polypeptide of the present invention can be prepared by a method for screening a target protein-specific peptide using a bacteriophage library containing a fusion polypeptide described in Korean Patent No. 10-1294982.

The present invention provides a polypeptide comprising: i) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2;

ii) a peptide fragment consisting of 6-12 amino acids;

iii) a polypeptide consisting of the 27th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2;

iv) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2; And

v) a fusion polypeptide comprising the polystyrene binding specific polypeptide fragment of claim 1.

The fusion polypeptide of the present invention is based on the fusion polypeptide comprising the protein skeleton module of the present invention (registered trademark 10-1294982) and is the amino acid sequence represented by SEQ ID NO: 2. The protein backbone module refers to a protein structure that does not affect the in vivo immune system and can contain an active polypeptide. The protein skeleton module is excellent in in vivo activity due to its small molecular weight and does not affect the activity of the active polypeptide contained therein. It does not affect the in vivo immune system and is derived from a protein rich in the living body, And there should be no occurrence of individual-specific effects. The protein skeleton module incorporating an active polypeptide to improve the stability and characteristics of the active polypeptide has been developed for the first time by the inventors of the present invention. The present inventors have proposed DMID (Designed Modular Immunodiagnostics) in the earlier patent application (10-2011-0035613) . The protein backbone module was designed based on Stabilin-2 derived polypeptide fragments abundant in humans. Stabilin-2 is a transmembrane receptor involved in lymphocyte induction, cell adhesion, scavenging receptor function, and angiogenesis. This protein has various domains such as 7 fasciclin, 16 epidermal growth factor (EGF) -like, 2 laminin-type EGF-like domains, and a C-type lectin-like hyaluronan-binding Link module.

The fusion polypeptide of the present invention comprises a polystyrene binding specific polypeptide fragment (hereinafter abbreviated as PS1) represented by SEQ. ID. NO: 1 to a fusion polypeptide comprising a protein skeleton module of the same inventor (registered trademark 10-1294982) And further comprising: The PS1-loaded fusion polypeptides greatly enhance the efficiency of the peptide screening method and the target protein detection method specific to the target protein described below.

Accordingly, the fusion polypeptide of the present invention comprises: i) a polypeptide consisting of the 1st to 21st amino acid sequences of the amino acid sequence shown in SEQ ID NO: 2;

ii) a peptide fragment consisting of 6-12 amino acids;

iii) a polypeptide consisting of the 27th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2;

iv) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2; And

v) a fusion polypeptide comprising the polystyrene binding specific polypeptide fragment of claim 1.

The amino acid sequence represented by SEQ ID NO: 2 is the amino acid sequence of the protein skeleton module (DMID), and various peptide fragments may be inserted into specific regions of the sequence to screen. The protein skeletal module (DMID) refers to the sequence of the # 4 to # 5 domains of EGF-like domain of stabilin-2.

The fusion polypeptide of the present invention is not limited thereto, but may be a structure in which the entire amino acid sequence or a specific domain of the protein skeleton module (DMID) shown in SEQ ID NO: 2 is repeated several times. The fusion polypeptide of the present invention is not limited to any sequence form (for example, deletion, repetition, substitution) as long as the structure or sequence of the # 4 to # 5 domain of the EGF like domain is used, 4 may be a structure repeated twice.

The peptide fragment is not particularly limited in its size, but may be preferably composed of 6 to 12 amino acids.

The site into which the peptide fragment is inserted is not particularly limited as long as it retains the structural characteristics of the protein backbone module and can be inserted preferably between the 20th to 30th amino acid sequences of the protein backbone module. Depending on the structure and size of the inserted peptide fragments, some amino acids of the protein backbone module may be deleted. More preferably, the 22th to 26th amino acids of the protein backbone module are removed and the desired peptide fragment is inserted into the removed backbone of the protein backbone module. Depending on the structure and size of the inserted polypeptide fragment, some amino acids of the protein backbone module may be deleted. Preferably, 'ii) a peptide fragment consisting of 6-12 amino acids' may be an amino acid sequence that specifically binds to a specific protein (or target protein).

The polystyrene binding specific polypeptide fragment is characterized by comprising the amino acid sequence of SEQ ID NO: 1, and may be a peptide having a native amino acid sequence as well as an amino acid sequence variant thereof.

The site into which the polystyrene binding specific polypeptide fragment is inserted is not particularly limited as long as it retains the structural characteristics of the protein backbone module and can be inserted preferably between the 20th to 30th amino acid sequences of the protein backbone module. Depending on the structure and size of the inserted polypeptide fragment, some amino acids of the protein backbone module may be deleted.

The fusion polypeptide of the present invention may include a linker between the peptide and the polypeptide fragment and the protein backbone module. The linker is inserted in the process of producing the polynucleotide encoding the fusion polypeptide of the present invention The size and the kind of the sequence are not particularly limited. Preferably two to ten amino acids. The linker may also include a restriction enzyme recognition site. Restriction enzyme recognition site refers to the site cleaved by restriction enzyme treatment, and the sequence varies depending on the type of restriction enzyme. The restriction enzyme recognition site may be inserted in the process of producing the fusion polypeptide of the present invention. Preferably, the restriction enzyme recognition site includes a restriction enzyme recognition site which is not present in the protein backbone module. For example, Glutamic acid-leucine or valine-aspartic acid.

Restriction enzymes are one of the enzymes that are the basis of molecular biology and have the function of recognizing and cleaving a specific nucleotide sequence. They are used for cloning various genes, restriction fragment length polymorphism (RFLP) and single nucleotide polymorphism It is widely used in fields such as genotyping and genetic mapping.

The restriction enzyme EcoR I used in the present invention is the first restriction enzyme isolated from Escherichia coli. When the nucleotide sequence of 5'-GATTC-3 'exists in any one of double-stranded DNA, G and A, and HindIII is the third restriction enzyme from Haemophilus influenzae strain. If a nucleotide sequence of 5'-A AGCTT-3 'exists in any one of double-stranded DNA, And A are cut off. Sac I recognizes the sequence of 5'-GAGCT C-3 'in any single strand of double-stranded DNA and cleaves between T and C, and SalI cleaves a single strand of double stranded DNA If there is a nucleotide sequence called 5'-G TCGAC-3 ', it recognizes its sequence and cuts between G and T. BamHI recognizes the sequence of 5'-G GATCC-3 'in any single strand of double-stranded DNA and cleaves between G and G. XHoI recognizes the sequence of 5'-C TCGAG-3 'on any single strand of double-stranded DNA and cleaves between C and T. PstI recognizes the sequence of 5'-CTGCA G-3 'in any single strand of double-stranded DNA and cuts between A and G.

In addition, the fusion polypeptide of the present invention can be further attached with a labeling substance or the like used in a known detection method. Any substance capable of detecting the fusion polypeptide is not limited, but preferably a marker peptide can be further attached thereto have. The marker peptide may be an S-tag or a His-tag, and preferably a His-tag may be used. The S-tag means the sequence of the catalytic domain of the S ribonuclease (RNase S) protein, which means the sequence of LysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSer (SEQ ID NO: 29). The His-tag refers to a peptide fragment consisting of 1 to 10 histidine. Preferably, the His-tag may be comprised of 6 to 10 histidine.

The histidine residues are one of the tags most frequently used as a tag necessary for purification after expressing the recombinant protein. The histidine residues should have high specificity and minimal influence on the desired protein structure. Preferably, the peptide may consist of 1 or 10 consecutive peptides of Histidine. Since the size is small and does not affect the original protein structure, it is convenient that the recombinant protein is not cut separately after it is prepared. Depending on the type of vector, the tag can be made either at the N-terminus (at the front) or at the C-terminus (at the back) of the target protein, depending on the structure of the protein. Preferably, the histidine residue can be located between ii) a peptide fragment consisting of 6-12 amino acids and v) the polystyrene binding specific polypeptide fragment.

Such a fusion polypeptide of the present invention may preferably be composed of the amino acid sequence shown in SEQ ID NO: 28.

On the other hand, the present invention provides a polynucleotide encoding the fusion polypeptide.

The polynucleotide of the present invention is characterized by encoding the fusion polypeptide of the present invention. The polynucleotide of the present invention comprises a nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having at least 70% homology thereto, ii) a polynucleotide encoding the peptide fragment and v) a polystyrene binding-specific polypeptide fragment .

The present invention also provides an expression vector into which the polynucleotide is inserted.

The expression vector refers to an expression vector prepared by a person skilled in the art to insert the polynucleotide of the present invention into a vector according to any method known in the art and express the fusion polypeptide of the present invention using appropriate transcription / translation control sequences.

The expression vector of the present invention means a plasmid, virus or other medium known in the art into which a polynucleotide sequence encoding the protein of the present invention can be inserted or introduced. A polynucleotide sequence according to the present invention may be operably linked to an expression control sequence, wherein the operably linked gene sequence and expression control sequence comprise an expression vector comprising a selection marker and a replication origin, Lt; / RTI >"Operablylinked" may be a gene and expression control sequence linked in such a way as to enable gene expression when the appropriate molecule is coupled to the expression control sequence. "Expression control sequence" means a DNA sequence that regulates the expression of a polynucleotide sequence operably linked to a particular host cell. Such regulatory sequences include promoters for conducting transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation.

The plasmids derived from Escherichia coli (pBR322, pBR325, pUC118, pUC119, pET30a, pET30c and pGEX-GST) and the plasmids derived from Bacillus subtilis (pUB110 And pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50), and Ti plasmids. Animal viruses such as retroviruses, adenoviruses or vaccinia viruses, insect viruses such as baculoviruses, a binary vector such as pPZP, pGA, and pCAMBIA series can be used. One skilled in the art can select a vector suitable for introducing the polynucleotide sequence of the present invention. Preferably the vector of the invention may be pET-32a or pET-29b.

The present invention also provides a bacteriophage comprising the fusion polypeptide.

The recombinant T7 phage-DMID cassette (SEQ ID NO: 4) and the T7 phage-DMID- (X7) peptide library (SEQ ID NO: 6) in the applicant's (Patent No. 10-1294982) Have been prepared and are known.

Since the bacteriophage of the present invention contains PS1 (polystyrene-specific binding polypeptide) in the fusion peptide expressed in the envelope, the effect of screening the target protein is better than that of the inventor's base (Patent No. 10-1294982) The bacteriophage can be immobilized on the bacterium.

Bacteriophage is a virus collectively referred to as a host cell of bacteria. Here, bacteria are collectively referred to as bacteria and archaea. Also called phage. The bacteriophage of the present invention may be selected from the group consisting of? Phage, T2 phage, T4 phage, T7 phage, T12 phage, R17 phage, M13 phage, MS2 phage, G4 phage, P1 phage, Enterobacteria phage P2, P4 phage, PhiX174 phage, , Preferably a bacteriophage having E. coli as a host, more preferably a T7 phage.

In the present invention, T7 phage is a medium size E. coli phage having a molecular weight of 26 x ^{10 <} And a very short tail attached to the head. The 39,936 bp of T7 DNA is known and clustered according to function and expression time. The T7 phage used in the present invention was obtained from Novagen's T7Select10-3 Cloning Kit. The EcoRI and HindIII restriction sites of 7Select10-3, a vector encoding the capsid 10A / B protein of T7 phage contained therein, A bacteriophage library can be constructed by inserting a polynucleotide capable of expressing the polypeptide.

The present invention also provides a bacteriophage library composed of a plurality of bacteriophages comprising the fusion polypeptide. The bacteriophage library of the present invention is characterized by being composed of a plurality of bacteriophages comprising a fusion polypeptide, wherein the fusion polypeptides are different from each other and may have various nucleotide base sequences. It can also be mass amplified and used for biopanning of the target peptide.

The bacteriophage library of the present invention expresses the fusion polypeptide of the present invention in a phage envelope by a phage surface presentation technique. The phage display technique used in the present invention is one of techniques for searching for protein-protein interaction between proteins and proteins. The phage display technique is a technique for fusing a DNA library to the N-terminal end of a bacteriophage coat protein, In which the DNA encoding the fusion protein is packaged in phage particles to display heterologous peptides on the phage particle surface (Smith GP, Science 228: 1315-1317, 1985). The library of fusion proteins incorporated into the phage can then be screened through a binding component (ligand) to the target. The combined phage are then re-infected with E. coli bacteria and then amplified, and the binding component is accumulated through the repetition of this selection (Parmley et al. Gene 73: 305-318, 1988; Barrett Natl. Acad. Sci. USA, 90: 4141-4145; Marks et al., J. Mol. Biol., 222: 581- 597, 1991). The iterative process of affinitybased selection involves the use of an antibody, an epitope, an antigen, a bioactive peptide, an enzyme inhibitor, an enzyme, a DNA-binding protein, an isolated protein domain, or a protein sequence such as a receptor ligand Enrichment of related clones separated from a large library.

The bacteriophage and bacteriophage library of the present invention are characterized in that the protein skeleton module is incorporated into the bacteriophage together with ii) a peptide fragment consisting of 6-12 amino acids and v) a polystyrene binding specific polypeptide fragment. In the case of the conventional phage display technique, various kinds of peptide fragments constituting the library were expressed with the phage coat protein without the skeletal module. As a result, peptide fragments that showed high affinity at the time of screening of the library can not always guarantee an increase in affinity which is improved because of the structure thereof when they are included in the protein skeleton module.

However, since the bacteriophage and the bacteriophage library of the present invention are expressed in a state that the peptide fragment is contained in the protein skeleton module from the stage of being expressed on the envelope, even if only the peptide fragment and the protein skeleton module are manufactured after the screening, Affinity can be maintained.

In addition, the bacteriophage library of the present invention can screen for peptides that specifically bind to a target protein and maintain high affinity, and can be used as therapeutic polypeptides, targets for drug intervention, related targets such as GPCR diopaning ), Or a peptide having a potency to be used as a biomarker for monitoring disease.

In addition, the bacteriophage library of the present invention specifically binds polystyrene as well as the target protein. Therefore, at the time of bio-panning, the bacteriophage binds to the support of the polystyrene material in a predetermined direction, thereby enhancing the screening efficiency by imparting a directionality to the position of the polypeptide capturing the target protein.

The present invention also provides a method for detecting a target protein, comprising: a) causing interaction between a target protein and the bacteriophage library; And

and b) detecting the bacteriophage library specifically bound to the target protein. The present invention also provides a method for screening a polystyrene and a target protein specifically targeting the bacteriophage library.

The method for detecting the bacteriophage library specifically bound to the step of performing the interaction may be performed by a phage library screening method comprising known biopanning, phage amplification, and plaque assays, and a nucleotide sequence analysis method for confirming the phage library screening method.

The above-mentioned target protein refers to a protein that specifically binds to the peptide library constructed in the present invention, and may be preferably a disease-specific protein. Here, the type of disease is not limited. Preferably, the target protein may be cardiac troponin I (cTnI).

In addition, the target protein used in the present invention may be prepared by preparing a recombinant protein or by purchasing a commercial protein.

(A) attaching and coating the fusion polypeptide to a solid phase support of a polystyrene material;

(b) treating the target protein with the coated support of step (a) to bind to the fusion polypeptide; And

(c) detecting the target protein bound in step (b).

The solid phase support may be, but is not limited to, a known support or substrate that has been proposed for fixation, separation, and the like. It may also have the form of particles, sheets, gels, filters, membranes or microtitre strips, tubes or plates, fibers or capillaries, and the biochip may be used as a solid support.

The solid support is not limited thereto. Preferably it is a polystyrene material.

Many immunoassays require that the antigen or antibody be immobilized on a solid support. Polystyrene is the most common substance used in solid phase immunoassay, and most enzyme-linked immunosorbent assays (ELISA) use polystyrene as a solid phase. Is mostly passive absorption, and its efficiency is low.

Accordingly, the present invention relates to (a) coating the fusion polypeptide on a solid phase support of a polystyrene material.

The fusion polypeptide has a polystyrene-specific binding site in addition to the target protein binding site. Due to the polystyrene-specific binding site, the fusion polypeptide is attached to the polystyrene even when passive absorption can not occur.

Such an effect is well illustrated in an embodiment of the present invention.

In one embodiment of the present invention, the protein (pET29b-RASSRLW-PS1) containing the polystyrene binding specific sequence (PS1) shown in SEQ ID NO: 1 and the protein The attachment capacities of the proteins were compared under conditions in which passive absorbtion can not occur and conditions in which passive absorbtion is induced (see FIGS. 5 and 6). As a result, it was confirmed that polystyrene was adhered even when the PS1 - loaded DMID protein could not passively absorb.

Therefore, the fusion peptide plays a role of replacing the role of an antibody in detecting a target substance and diagnosis of disease. More preferably, the fusion peptide enables antibody-free immunodiagnostics.

(b) treating the target protein with the coated support of step (a) to bind to the fusion polypeptide; , The target protein refers to a protein that specifically binds to the fusion peptide produced in the present invention, and may be preferably a disease-specific protein. Here, the type of disease is not limited. Preferably, the target protein may be cardiac troponin I (cTnI).

(c) detecting the target protein bound in step (b) may be performed by a method known to those skilled in the art. For example, it may be a detection method according to a label, without limitation thereto, and in the case of using a fluorescent substance as a detectable label, immunofluorescence staining may be used. When an enzyme is used as a detectable label, the absorbance can be measured by a coloring reaction of the substrate through an enzyme reaction, and in the case of a radioactive substance, the amount of emitted radiation can be measured.

The protein detection method of the present invention is not limited thereto, but can be applied to a solid phase immunoassay, more preferably to an enzyme-linked immunosorbent assay (ELISA) More preferably sandwich ELISA (sandwich ELISA).

In one embodiment of the present invention, the protein detection method was applied to a sandwich ELISA to detect a target protein (see FIG. 8). In addition, a method of detecting a target protein by coating a solid support phase with a fusion polypeptide loaded with the PS1 and a method of detecting a target protein by coating a solid support phase with a polypeptide not having PS1 were compared (see Fig. 7) . As a result, it was confirmed that the fusion polypeptide containing PS1 enhanced the detection efficiency by giving the direction of the substance capturing the target substance as well as the coating efficiency in the ELISA using the polystyrene. That is, it is known that the DMID protein including the RASSRLW sequence has high specificity and affinity for the protein in the case of the same inventor (Patent No. 10-1294982). The fusion polypeptide of the present invention, It was found that the detection effect on the target protein (cTnI) was significantly higher than that when PS1 was not loaded. Thus, the fusion polypeptide of the present invention is remarkably effective in molecular diagnosis.

The term "detection" in the present invention means both quantitative and qualitative measurement of an antigen or antibody in a sample.

The polypeptide of SEQ ID NO: 1 of the present invention binds specifically to polystyrene. Also, i) a polypeptide consisting of the 1st to 21st amino acid sequences of the amino acid sequence shown in SEQ ID NO: 2; ii) a peptide fragment consisting of 6-12 amino acids; iii) a polypeptide consisting of the 27th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2; iv) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2; And v) the fusion polypeptide comprising the polystyrene binding specific polypeptide fragment of claim 1 is specific for binding to the target protein and polystyrene. That is, the fusion polypeptide includes a protein backbone module that enhances binding affinity and binding specificity with a target protein, and is effective for detecting and screening disease-specific peptides. In addition, the fusion polypeptides can be attached to polystyrene to capture a target substance, Providing directionality to the site enhances the efficiency of detection and screening. Therefore, the bacteriophage library screening method and protein detection method using the fusion polypeptide are excellent in sensitivity and efficiency.

FIG. 1 shows the structure of a cassette containing DMID in T7 phage 10A Capsid (DMID: a protein backbone module of the present invention (10-2011-0035613)).
FIG. 2 is a diagram showing a structure including a DMID and a peptide library in T7 phage 10A capsid (DMID: a protein skeleton module developed by the present inventor (10-2011-0035613)).
FIG. 3 is a graph showing the polystyrene binding ability of the candidate 4R1, 4R12, and 4R22 phage clones discovered by ELISA assay (PS4R1: candidate 4R1 phage binding to polystyrene, PS4R12: candidate 4R12 phage for polystyrene, PS4R22: polystyrene T7D (-): T7 DMID insertless phage, PS_blk: polystyrene plate blocked with skim milk, PS: non-blocked polystyrene plate).
4 is a schematic diagram showing the positions of the cTnI and polystyrene (PS) specific binding sequences in the pET29b-RASSRLW-DMID vector and the pET29b-RASSRLW-PS1 vector.
5 shows the polystyrene binding ability of the pET29b-RASSRLW-DMID protein and the pET29b-RASSRLW-PS1 protein in an ELISA assay under the condition that no passive adsorption can occur in the polystyrene (PBST_4 ° C / 30 min: 0.1% tween 20 Containing PBS solution, 30 min at 4 [deg.] C).
FIG. 6 shows the polystyrene binding ability of pET29b-RASSRLW-DMID protein and pET29b-RASSRLW-PS1 protein in an ELISA assay under the condition of inducing passive adsorption to polystyrene (PBS_25 ° C / 2hr: Solution, 2 hours at < RTI ID = 0.0 > 25 C).
Figure 7 shows the results of ELISA assay with pET29b-RASSRLW-DMID protein and pET29b-RASSRLW-PS1 protein to compare detection sensitivities with cTnI protein.
8 is a schematic diagram showing a detection method of cTnI protein using pET29b-RASSRLW-PS1 protein.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Selection of Polystyrene Binding Specific Polypeptides

<1-1> Phage plaque assay

To the M9LB culture, the host cell BLT5403 E. coli is cultured until OD600 = 1.0, and then 250 μl is added to a 10 ml tube and stored at 4 ° C. Prepare the phage to be diluted at various dilutions and keep it at 4 ℃ until use. Top aga is dissolved at 100 ° C and maintained at 37 ° C. After mixing 100 μl of phage solution into 250 μl of prepared host BLT5403 E. coli, mix 4 ml of final aged Top ages and transfer to aga plate containing 50 μg / ml Ampicillin. The hard aga plate is incubated at 37 ° C for 3-4 hours, and the number of plaques formed is checked.

<1-2> Detection of polystyrene target phage library

T7 phage DMID- (X7) peptide library was used to identify phage clones specifically adhered to polystyrene. The production of the T7 phage DMID- (X7) peptide library was carried out by the same method as that of the 'registered patent 10-1294982' by the inventors of the present invention. Prepare a 96-well plate of polystyrene (Corning 96 Well EIA / RIA Clear Flat Bottom Polystyrene High Bind Microplate, Individually Wrapped, without Lid, Nonsterile (Product # 3590)). The phage library was prepared at a concentration of 1 × 10 ¹⁰ pfu / ml, in which the buffer was a Tris solution containing 500 mM NaCl, 0.1% Triton-X100, 5 mM EDTA, 1 mM DTT, 2% BSA, 0.1 mg / (pH 8.0) is used. The prepared phage was injected into a 96-well plate of polystyrene at a rate of 1 × 10 ⁹ pfu and bound at 4 ° C for 30 minutes. Thereafter, non-specific binding phage clones are removed by washing three times with TBS solution containing 0.05% Tween. Each wash is carried out for 5 minutes.

The phage clone bound to the polystyrene well was reacted with 100 μl of 1% SDS for 20 minutes to remove the phage desorbed by SDS. Finally phage not desorbed in 1% SDS was recovered with BLT5403 E. coli culture (100 μl) and amplified by infection with BLT5403 E. coli culture. The amplified phage clone is used in the next round search, and the search is repeated a total of four rounds.

<1-3> Reading the phage sequence and selecting the candidate phage clone

Of the phage clones detected in the 4th round, 30 clones were selected and the inserted nucleotide sequences were read by PCR. The primer base used in the PCR was an upstream primer 5-AGCGGACCAGATTATCGCTA-3 '(SEQ ID NO: 16) and a downstream primer 5-AACCCCTCAAGACCCGTTTA-3' (SEQ ID NO: 17), 94 ° C for 50 seconds, 50 ° C for 1 minute, Lt; / RTI &gt; for 1 minute. After reading the nucleotide sequence, the amino acids inserted into each clone were sequenced using the ClustalX program, and repeated motifs and clones were selected as candidates. The candidate groups are shown in Table 1.

Phage clone Sequence Frequency 4R1 PRAGSYRRAFRAQLKRANFPTLR 13/30 4R12 FKWRVVACRPSVHVQSRLHG 1/30 4R22 RLPRVA * 1/30

Of the total 30 clones, only 4R1 clones were repeated 13 times, and the remaining 17 clones were confirmed once, and polystyrene binding of each phage (excluding 4R12 clones) was not confirmed at all. As shown in Table 1, PRAGSYRRAFRAQLKRANFPTLR was confirmed 13 times out of 30 and selected as a candidate group for polystyrene.

<2-4> Confirmation of polystyrene binding ability of candidate phage clone (4R1) discovered by ELISA assay

Prepare wells blocked with 200 μl of 5% skim milk and unblocked wells in a polystyrene 96-well plate (Corning, NY, USA). The selected candidate phage 4R1 clones are prepared at a concentration of 1 × 10 ¹⁰ pfu / ml, and the buffer used is phosphate buffer (pH 7.4) containing 1% BSA and 0.1% Tween. The prepared phages were injected into each well of polystyrene at 1 × 10 ⁹ pfu and bound at 4 ° C for 1 hour. Thereafter, non-specific binding phages are removed by washing three times with phosphate buffer containing 0.05% Tween. Each wash is carried out for 5 minutes. The phage clones bound to the wells blocked with skim milk in polystyrene wells and polystyrene were detected with T7 phage tail fiber antibody (Merck KGaA, Darmstadt, Germany), and 100 μl of TMB substrate (Koma biotech inc., Seoul, Korea) Color reaction occurs. The TMB color reaction was terminated by the injection of 100 μl of 2N H ₂ SO ₄ , and the absorbance at 450 nm was measured to confirm the degree of binding of the candidate phage in each group. The results are shown in Fig.

As shown in FIG. 3, the 4R1 phage clone exhibited excellent binding ability to polystyrene, and its binding was completely inhibited by blocking of skim milk, indicating that it specifically binds polystyrene. T7 DMID insertless phage used as a negative control did not show binding ability to polystyrene.

In the same manner as above, the ability to adhere polystyrene to 4R12 and 4R22 phage clones was tested and compared. As shown in Fig. 3, the 4R1 phage clone showed superior adhesion ability to other phage clones.

&Lt; Example 2 >

Preparation of fusion polypeptide specific for polystyrene and target protein (cTnI) and antibody free immunoassay

&Lt; 2-1 > Cloning of the target protein (cTnI) specific binding DMID recombinant protein

The inventors of the present invention have screened a RASSRLW sequence specific to cTnI using 'T7 phage-DMID- (X7)' in '10-1294982'. For the production of DMID recombinant protein carrying this RASSRLW sequence A phage clone that specifically binds cTnI was amplified using the T7 super UP primer (5'-AGCGGACCAGATTATCGCTA-3 ', SEQ ID NO: 18) and T7 super DOWN primer (5'-AACCCCTCAAGACCCGTTTA-3', SEQ ID NO: 19) PCR, and then cloned into the BamHI and XhoI sites of the vector pET-29b (+) (Novagen; Madison, Wis.) To prepare a DMID protein expression vector containing RASSRLW, which was named pET29b-RASSRLW-DMID No. 22 and [Figure 4]). The pET29b-RASSRLW-DMID sequence is a sequence of EGF like domain # 4 # 5 linked thereto. This expression vector contains the entire amino acid sequence 1470 to 1555 of human stabilin-2, and amino acid sequences 1490 and 1496 The restriction enzyme SalI and the SacI recognition sequence are inserted between them, and the RASSRLW sequence is arranged therebetween. MKETAAAKFERQHMDSPDLGTLVPRGSMAISDPNS derived from the vector is fused to the amino terminus, and LEHHHHHH amino acid is fused to the carboxy terminus.

<2-2> Target protein ( cTnI ) And polystyrene-specific bonding DMID  Of recombinant protein Cloning

For the preparation of the DMID recombinant protein bearing PRAGSYRRAFRAQLKRANFPTLR (designated PS1), a clone pET29b-RASSRLW-EGF # 4 (see SEQ ID NO: 24) was first prepared. This is a protein consisting of one EGF-like domain (# 4), which is inserted into the C-term. In addition to the enzyme site (Pst1) so that various moieties can be inserted between Pst1 and Xho1 of the C-term .

4-PstI-His6 UP primer (5'-AACTGCAGCACCATCACCATCACCATACAGCAATCAATGCCTGTG-3 ', SEQ ID NO: 20) and EGF # 4-XhoI DOWN primer (5' -AACTCGAGGCACACAATGCCATCACC-3 ', SEQ ID NO: 21) and cloned into the PstI and XhoI restriction sites of the vector pET29b-RASSRLW-EGF # 4 to prepare a DMID protein expression vector carrying the polystyrene specific binding sequence PRAGSYRRAFRAQLKRANFPTLR , Which was named pET29b-RASSRLW-PS1 (SEQ ID NO: 26 and [Figure 4]). This expression vector contains RASSRLW, a specific binding sequence for human cTnI in the first EGF domain, followed by the EGF domain containing the polystyrene specific sequence PRAGSYRRAFRAQLKRANFPTLR. HHHHHH amino acid is fused between these two domains and MKETAAAKFERQHMDSPDLGTLVPRGSMAISDP amino acid derived from the vector is fused at the amino terminal.

More specifically, pET29b-RASSRLW-PS1 has EGF-like domain # 4 twice repeatedly. EGF # 4 containing a cTnI binding sequence (RASSRLW) and EGF # 4 containing a polystyrene binding sequence (PRAGSYRRAFRAQLKRANFPTLR *) It is a linked protein. A cTnI binding EGF # 4 was inserted between BamH1 and Pst1, and a polystyrene binding EGF # 4 was inserted between Pst1 and Xho1. However, in the polystyrene-specific binding sequence, a frame shift occurred at the insert insertion position in the phage state, and the stop codon occurred because EGF # 4 was not correctly transferred to the C-term.

<2-3> Expression of Recombinant Protein

Each of the vectors prepared in Examples <2-1> and <2-2> was transformed into E. coli cell line BL21), and cultured in a 37 ° C LB culture medium containing 50 μg / ml kanamycin. When the absorbance at 595 nm was 0.5 to 0.6, 1 mM IPTG (isopropyl-β-D - thiogalactopyranoside) to induce the expression of the recombinant protein at 20 ° C for 24 hours. The cultured Escherichia coli was resuspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF, 0.5 mM DTT) , And Ni-NTA resin (resin, Qiagen). The expressed and purified recombinant protein was used for the experiment after confirming the protein purity through SDS-PAGE .

<2-4> PS1 Equipped with DMID  protein ( RASSRLW - PS1 )of 폴리스렌  Confirm binding ability

The purified pET29b-RASSRLW-DMID protein and pET29b-RASSRLW-PS1 protein are diluted to 10, 5, 2.5, 1.25, and 0.625 μg / ml in PBST (0.1% tween 20) or PBS, respectively. 100 μl of each prepared protein is added to each well of a 96-well polystyrene plate (Corning, NY, USA), followed by reaction at 4 ° C for 30 minutes or at 25 ° C for 2 hours. After this, non-specific binding proteins are removed by washing three times with 0.05% Tween-containing phosphate buffer solution. Each wash is carried out for 5 minutes. The protein bound to the polystyrene well is detected with a His-tag antibody (Santa Cruz) with HRP, and a color reaction is carried out with 100 μl of TMB substrate. The TMB color reaction was terminated by the injection of 100 μl of 2N H ₂ SO ₄ , and the absorbance at 450 nm was measured to confirm the degree of binding to each group of polystyrene. The results are shown in Fig. 5 and Fig. 6.

As shown in FIG. 5 and FIG. 6, the DMID protein carrying the PRAGSYRRAFRAQLKRANFPTLR amino acid sequence (PS1) showed a strong binding force to polystyrene as it was in the phage state. This binding was also confirmed under the condition that passive adsorption could not occur (reaction solution containing 0.1% of tween 20, binding condition at 4 ° C for 30 min), and at the same time, the concentration dependence of pET29b-RASSRLW-PS1 protein The PS1 sequence was found to have specific binding to polystyrene. On the other hand, no binding reaction to polystyrene was observed in proteins without PS1 sequence. In the conditions of inducing passive adsorption (PBS solution without tween 20, binding conditions for 2 hours at 25 ° C), the binding pattern of both proteins to polystyrene was confirmed regardless of the presence or absence of the PS1 sequence.

<2-5> PS1 Equipped with DMID  protein ( RASSRLW - PS1 )of cTnI  Capture Capability

The purified pET29b-RASSRLW-DMID protein is diluted to a concentration of 5 μg / ml in PBS solution containing no 0.1% tween 20 and coated on polystyrene by passive adsorption method at 25 ° C for 2 hours. To match the coating to polystyrene, the pET29b-RASSRLW-PS1 protein is diluted to 5 μg / ml in PBS solution containing 0.1% tween 20 and prepared by binding to polystyrene at 4 ° C for 30 minutes. After this, each coated polystyrene well is blocked with 200 μl of 5% skim milk and washed twice with PBS. cTnI is prepared by diluting at concentrations of 20, 10, 5, 2.5, and 1.25 ng / ml. At this time, the diluted solution is a PBS solution containing 2% BSA and 0.1% tween 20. Prepared cTnI was added to DMID coated polystyrene well and allowed to bind at room temperature for 1 hour. Washing with 3 PBSTs (0.05% tween 20) removes non-specific binding. The bound cTnI is detected using the cTnI antibody (Abcam) and subjected to a color reaction with 100 μl of TMB substrate. The TMB chromogenic reaction is terminated by the injection of 100 μl of 2N H ₂ SO ₄ and the absorbance at 450 nm wavelength is measured to compare the capture ability of cTnI of each protein. The results are shown in Fig.

The inventor of the present invention has screened the RASSRLW sequence specific to cTnI in '10-1294982'. In this experiment, it was found that the DMID protein containing the PS1 sequence in the RASSRLW sequence had a better cTnI capture ability than the DMID without the sequence. The capture reaction signal for each concentration of cTnI was observed to be 4 times more intense on pET29b-RASSRLW-PS1 than on pET29b-RASSRLW-DM1, and it was confirmed that the detection concentration could be up to about 1.25 ng / ml (see FIG. 7) ). That is, the PS1 sequence bound to polystyrene contributes to enhancement of detection sensitivity by providing directionality of a substance capturing a target substance as well as improvement of coating efficiency in an ELISA using polystyrene.

As described above, the polypeptide of SEQ ID NO: 1 of the present invention is specifically bound to polystyrene. Also, i) a polypeptide consisting of the 1st to 21st amino acid sequences of the amino acid sequence shown in SEQ ID NO: 2; ii) a peptide fragment consisting of 6-12 amino acids; iii) a polypeptide consisting of the 27th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2; iv) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2; And v) the fusion polypeptide comprising the polystyrene binding specific polypeptide fragment of claim 1 is specific for binding to the target protein and polystyrene. That is, the fusion polypeptide includes a protein backbone module that enhances binding affinity and binding specificity with a target protein, and is effective for detecting and screening disease-specific peptides. In addition, the fusion polypeptides can be attached to polystyrene to capture a target substance, Providing directionality to the site enhances the efficiency of detection and screening. Therefore, the bacteriophage library screening method and protein detection method using the fusion polypeptide have high sensitivity and efficiency, and thus are highly likely to be used industrially.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Polypeptide binding polystyrene specifically and use thereof <130> NP13-0054 <160> 29 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> PRT <213> Artificial Sequence <220> Polystyrene specific polypeptide (amino acid sequence) <400> 1 Pro Arg Ala Gly Ser Tyr Arg Arg Ala Phe Arg Ala Gln Leu Lys Arg   1 5 10 15 Ala Asn Phe Pro Thr Leu Arg              20 <210> 2 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> DMID amino acid sequence <400> 2 Thr Ala Ile Asn Ala Cys Glu Ile Ser Asn Gly Gly Cys Ser Ala Lys   1 5 10 15 Ala Asp Cys Lys Arg Thr Thr Pro Gly Arg Arg Val Cys Thr Cys Lys              20 25 30 Ala Gly Tyr Thr Gly Asp Gly Ile Val Cys Leu Glu Ile Asn Pro Cys          35 40 45 Leu Glu Asn His Gly Gly Cys Asp Lys Asn Ala Glu Cys Thr Gln Thr      50 55 60 Gly Pro Asn Gln Ala Ala Cys Asn Cys Leu Pro Ala Tyr Thr Gly Asp  65 70 75 80 Gly Lys Val Cys Thr Leu                  85 <210> 3 <211> 273 <212> DNA <213> Artificial Sequence <220> <223> DMID nucleotide <400> 3 ggatccacag caatcaatgc ctgtgagatc agcaatggag gttgctctgc caaggctgac 60 tgtaagagaa ccaccccagg aaggcgagtg tgcacgtgca aagcaggcta cacgggtgat 120 ggcattgtgt gcctggaaat caacccgtgt ttggagaacc atggtggctg tgacaagaat 180 gcggagtgca cacagacagg acccaaccag gctgcctgta actgtttgcc agcatacact 240 ggagatggaa aggtctgcac actctaactc gag 273 <210> 4 <211> 304 <212> DNA <213> Artificial Sequence <220> <223> DMID-ESSH cassette nucleotide <400> 4 gaattctaca gcaatcaatg cctgtgagat cagcaatgga ggttgctctg ccaaggctga 60 ctgtaagaga gagctcacca ccccaggaag ggtcgaccga gtgtgcacgt gcaaagcagg 120 ctacacgggt gatggcattg tgtgcctgga aatcaacccg tgtttggaga accatggtgg 180 ctgtgacaag aatgcggagt gcacacagac aggacccaac caggctgcct gtaactgttt 240 gccagcatac actggagatg gaaaggtctg cacactccat catcaccacc atcactaaaa 300 gctt 304 <210> 5 <211> 96 <212> PRT <213> Artificial Sequence <220> <223> DMID-ESSH cassette amino acid <400> 5 Thr Ala Ile Asn Ala Cys Glu Ile Ser Asn Gly Gly Cys Ser Ala Lys   1 5 10 15 Ala Asp Cys Lys Arg Glu Leu Thr Thr Pro Gly Arg Val Asp Arg Val              20 25 30 Cys Thr Cys Lys Ala Gly Tyr Thr Gly Asp Gly Ile Val Cys Leu Glu          35 40 45 Ile Asn Pro Cys Leu Glu Asn His Gly Gly Cys Asp Lys Asn Ala Glu      50 55 60 Cys Thr Gln Thr Gly Pro Asn Gln Ala Ala Cys Asn Cys Leu Pro Ala  65 70 75 80 Tyr Thr Gly Asp Gly Lys Val Cys Thr Leu His His His His His                  85 90 95 <210> 6 <211> 310 <212> DNA <213> Artificial Sequence <220> <223> DMID- (X7) nucleotide <400> 6 gaattctaca gcaatcaatg cctgtgagat cagcaatgga ggttgctctg ccaaggctga 60 ctgtaagaga gagctcnnkn nknnknnknn knnknnkgtc gaccgagtgt gcacgtgcaa 120 agcaggctac acgggtgatg gcattgtgtg cctggaaatc aacccgtgtt tggagaacca 180 tggtggctgt gacaagaatg cggagtgcac acagacagga cccaaccagg ctgcctgtaa 240 ctgtttgcca gcatacactg gagatggaaa ggtctgcaca ctccatcatc accaccatca 300 ctaaaagctt 310 <210> 7 <211> 92 <212> PRT <213> Artificial Sequence <220> DMID- (X7) amino acid <400> 7 Thr Ala Ile Asn Ala Cys Glu Ile Ser Asn Gly Gly Cys Ser Ala Lys   1 5 10 15 Ala Asp Cys Lys Arg Glu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Asp              20 25 30 Arg Val Cys Thr Cys Lys Ala Gly Tyr Thr Gly Asp Gly Ile Val Cys          35 40 45 Leu Glu Ile Asn Pro Cys Leu Glu Asn His Gly Gly Cys Asp Lys Asn      50 55 60 Ala Glu Cys Thr Gln Thr Gly Pro Asn Gln Ala Ala Cys Asn Cys Leu  65 70 75 80 Pro Ala Tyr Thr Gly Asp Gly Lys Val Cys Thr Leu                  85 90 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> DMIDEFwd <400> 8 gatcgaattc tacagcaatc aatgcctgt 29 <210> 9 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> DMIDSSRvs <400> 9 gtcgaccctt cctggggtgg tgagctctct cttacagtca gcctt 45 <210> 10 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> DMIDSSFwd <400> 10 gagctcacca ccccaggaag ggtcgaccga gtgtgcacgt gcaaa 45 <210> 11 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> DMIDHRvs <400> 11 gatcaagctt ttagtgatgg tggtgatgat ggagtgtgca gacctttcc 49 <210> 12 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for library construction <400> 12 gatcgagctc nnknnknnkn nknnknnknn kgtcgaccat catcaccacc atcac 55 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for library construction <400> 13 gtgatggtgg tgatgatggt cgac 24 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T7 UP FWD -100: Forward primer for library sequencing <400> 14 agcgcgctcg ccgtgctaac 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T7 Down Rvs -100: reverse primer for library sequencing <400> 15 ctgataactc agcggcagtc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> upstream primer <400> 16 agcggaccag attatcgcta 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> downstream primer <400> 17 aacccctcaa gacccgttta 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T7 super UP primer <400> 18 agcggaccag attatcgcta 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T7 super DOWN primer <400> 19 aacccctcaa gacccgttta 20 <210> 20 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> EGF # 4-PstI-His6 UP primer <400> 20 aactgcagca ccatcaccat caccatacag caatcaatgc ctgtg 45 <210> 21 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> EGF # 4-XhoI DOWN primer <400> 21 aactcgaggc acacaatgcc atcacc 26 <210> 22 <211> 133 <212> PRT <213> Artificial Sequence <220> PET29b-RASSRLW-DMID (amino acid sequence) <400> 22 Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Met Asp Ser   1 5 10 15 Pro Asp Leu Gly Thr Leu Val Pro Arg Gly Ser Met Ala Ile Ser Asp              20 25 30 Pro Asn Ser Thr Ala Ile Asn Ala Cys Glu Ile Ser Asn Gly Gly Cys          35 40 45 Ser Ala Lys Ala Asp Cys Lys Arg Glu Leu Arg Ala Ser Ser Arg      50 55 60 Trp Val Asp Arg Val Cys Thr Cys Lys Ala Gly Tyr Thr Gly Asp Gly  65 70 75 80 Ile Val Cys Leu Glu Ile Asn Pro Cys Leu Glu Asn His Gly Gly Cys                  85 90 95 Asp Lys Asn Ala Glu Cys Thr Gln Thr Gly Pro Asn Gln Ala Ala Cys             100 105 110 Asn Cys Leu Pro Ala Tyr Thr Gly Asp Gly Lys Val Cys Thr Leu His         115 120 125 His His His His His     130 <210> 23 <211> 405 <212> DNA <213> Artificial Sequence <220> PET29b-RASSRLW-DMID (nucleotide sequence) <400> 23 atggacagcc cagatctggg taccctggtg ccacgcggtt ccatggcgat atcggatccg 60 aattctacag caatcaatgc ctgtgagatc agcaatggag gttgctctgc caaggctgac 120 tgtaagagag agctccgtgc gtcgtcgcgt ctttgggtcg accgagtgtg cacgtgcaaa 180 gcaggctaca cgggtgatgg cattgtgtgc ctggaaatca acccgtgttt ggagaaccat 240 ggtggctgtg acaagaatgc ggagtgcaca cagacaggac ccaaccaggc tgcctgtaac 300 tgtttgccag catacactgg agatggaaag gtctgcacac tccatcatca ccaccatcac 360 taaaagcttg cggccgcact cgagcaccac caccaccacc actga 405 <210> 24 <211> 91 <212> PRT <213> Artificial Sequence <220> PET29b-RASSRLW-EGF # 4 (amino acid sequence) <400> 24 Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Met Asp Ser   1 5 10 15 Pro Asp Leu Gly Thr Leu Val Pro Arg Gly Ser Met Ala Ile Ser Asp              20 25 30 Pro Thr Ala Ile Asn Ala Cys Glu Ile Ser Asn Gly Gly Cys Ser Ala          35 40 45 Lys Ala Asp Cys Lys Arg Glu Leu Arg Ala Ser Ser Arg Leu Trp Val      50 55 60 Asp Arg Val Cys Thr Cys Lys Ala Gly Tyr Thr Gly Asp Gly Ile Val  65 70 75 80 Cys Leu Gln Leu Glu His His His His His                  85 90 <210> 25 <211> 297 <212> DNA <213> Artificial Sequence <220> PET29b-RASSRLW-EGF # 4 (nucleotide sequence) <400> 25 atgaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagccc agatctgggt 60 accctggtgc cacgcggttc catggcgata tcggatccga cagcaatcaa tgcctgtgag 120 atcagcaatg gaggttgctc tgccaaggct gactgtaaga gagagctccg tgcgtcgtcg 180 cgtctttggg tcgaccgagt gtgcacgtgc aaagcaggct acacgggtga tggcattgtg 240 tgcctgcagc tcgagcacca ccaccaccac cactgacacc accaccacca ccactga 297 <210> 26 <211> 135 <212> PRT <213> Artificial Sequence <220> <223> ppET29b-RASSRLW-PS1 (amino acid sequence) <400> 26 Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Met Asp Ser   1 5 10 15 Pro Asp Leu Gly Thr Leu Val Pro Arg Gly Ser Met Ala Ile Ser Asp              20 25 30 Pro Thr Ala Ile Asn Ala Cys Glu Ile Ser Asn Gly Gly Cys Ser Ala          35 40 45 Lys Ala Asp Cys Lys Arg Glu Leu Arg Ala Ser Ser Arg Leu Trp Val      50 55 60 Asp Arg Val Cys Thr Cys Lys Ala Gly Tyr Thr Gly Asp Gly Ile Val  65 70 75 80 Cys Leu Gln His His His His His Thr Ala Ile Asn Ala Cys Glu                  85 90 95 Ile Ser Asn Gly Gly Cys Ser Ala Lys Ala Asp Cys Lys Arg Glu Leu             100 105 110 Pro Arg Ala Gly Ser Tyr Arg Arg Ala Phe Arg Ala Gln Leu Lys Arg         115 120 125 Ala Asn Phe Pro Thr Leu Arg     130 135 <210> 27 <211> 509 <212> DNA <213> Artificial Sequence <220> PET29b-RASSRLW-PS1 (nucleotide sequence) <400> 27 atgaaagaaa ccgctgctgc taaattcgaa cgccagcaca tggacagccc agatctgggt 60 accctggtgc cacgcggttc catggcgata tcggatccga cagcaatcaa tgcctgtgag 120 atcagcaatg gaggttgctc tgccaaggct gactgtaaga gagagctccg tgcgtcgtcg 180 cgtctttggg tcgaccgagt gtgcacgtgc aaagcaggct acacgggtga tggcattgtg 240 tgcctgcagc accatcacca tcaccataca gcaatcaatg cctgtgagat cagcaatgga 300 ggttgctctg ccaaggctga ctgtaagaga gagctccctc gtgccggatc ctaccggcga 360 gcgtttcgtg ctcagctaaa gcgtgcgaac tttcccaccc tacgctaacc tgcctcgatg 420 tttacgaggt cgaccgagtg tgcacgtgca aagcaggcta cacgggtgat ggcattgtgt 480 gcctcgagca ccaccaccac caccactga 509 <210> 28 <211> 102 <212> PRT <213> Artificial Sequence <220> RASSRLW-PS1 (amino acid sequence) <400> 28 Thr Ala Ile Asn Ala Cys Glu Ile Ser Asn Gly Gly Cys Ser Ala Lys   1 5 10 15 Ala Asp Cys Lys Arg Glu Leu Arg Ala Ser Ser Arg Leu Trp Val Asp              20 25 30 Arg Val Cys Thr Cys Lys Ala Gly Tyr Thr Gly Asp Gly Ile Val Cys          35 40 45 Leu Gln His His His His His Thr Ala Ile Asn Ala Cys Glu Ile      50 55 60 Ser Asn Gly Gly Cys Ser Ala Lys Ala Asp Cys Lys Arg Glu Leu Pro  65 70 75 80 Arg Ala Gly Ser Tyr Arg Arg Ala Phe Arg Ala Gln Leu Lys Arg Ala                  85 90 95 Asn Phe Pro Thr Leu Arg             100 <210> 29 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> s-tag <400> 29 Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser   1 5 10 15

Claims (17)

A polystyrene binding specific polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
i) a polypeptide consisting of the 1st to 21st amino acid sequences of the amino acid sequence shown in SEQ ID NO: 2;
ii) a peptide fragment consisting of 6-12 amino acids;
iii) a polypeptide consisting of the 27th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2;
iv) a polypeptide consisting of the 1st to 21st amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2; And
v) a fusion polynucleotide comprising a polystyrene binding specific polypeptide fragment consisting of the amino acid sequence of SEQ ID NO: 1.
The fusion polypeptide of claim 2, wherein the fusion polypeptide comprises
i) a polypeptide consisting of the 1st to 21st amino acid sequences of the amino acid sequence shown in SEQ ID NO: 2;
ii) a linker consisting of 2 to 10 amino acids;
iii) a peptide fragment consisting of 6-12 amino acids;
iv) a linker consisting of 2 to 10 amino acids;
v) a polypeptide consisting of the 27th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2;
vi) a polypeptide consisting of the 1st to 21st amino acid sequences of the amino acid sequence shown in SEQ ID NO: 2;
vii) a linker consisting of 2 to 10 amino acids; And
viii) a fusion polypeptide comprising a polystyrene binding specific polypeptide fragment comprising the amino acid sequence of SEQ ID NO: 1.
3. The fusion polypeptide of claim 2, wherein the fusion polypeptide further comprises a S-tag or His-tag marker peptide.
3. The fusion polypeptide of claim 2, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 28.
4. The method of claim 3, wherein the fusion polypeptide comprises
i) a polypeptide consisting of the 1st to 21st amino acid sequences of the amino acid sequence shown in SEQ ID NO: 2;
ii) a linker consisting of 2 to 10 amino acids;
iii) a peptide fragment consisting of 6-12 amino acids;
iv) a linker consisting of 2 to 10 amino acids;
v) a polypeptide consisting of the 27th to 42nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2;
vi) a peptide fragment consisting of 1 to 10 histidine residues;
vii) a polypeptide consisting of the 1st to 21st amino acid sequences of the amino acid sequence shown in SEQ ID NO: 2;
viii) a linker consisting of 2 to 10 amino acids; And
ix) A fusion polypeptide comprising a polystyrene binding specific polypeptide fragment comprising the amino acid sequence of SEQ ID NO: 1.
4. The fusion polypeptide of claim 3, wherein the linker is composed of glutamic acid-leucine or valine-aspartic acid.
8. A polynucleotide encoding the fusion polypeptide of any one of claims 2-7.
9. A vector encoding the polynucleotide of claim 8.
A bacteriophage comprising the fusion polypeptide of claim 2.
11. The bacteriophage of claim 10, wherein the bacteriophage is a T7 bacteriophage.
11. The bacteriophage according to claim 10, wherein the bacteriophage is Escherichia coli.
A bacteriophage library consisting of a plurality of bacteriophages comprising the fusion polypeptide of claim 2.
14. The bacteriophage library of claim 13, wherein the plurality of bacteriophages comprise different fusion polypeptides.
a) allowing interaction between the target protein and the bacteriophage library of claim 13; And
b) detecting the bacteriophage library of claim 13 which is specifically bound to the protein of interest, wherein the bacteriophage library specifically targets polystyrene and a target protein.
(a) attaching the fusion polypeptide of claim 2 to a solid phase support of polystyrene material and coating;
(b) treating the target protein with the coated support of step (a) to bind with the fusion polypeptide of claim 2; And
(c) detecting the bound protein in step (b).
17. The protein detection method according to claim 16, wherein the target protein is cardiac troponin I (cTnI).

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