KR20130018212A - Cyclodextrin-binidng peptides, their preparation method and their use - Google Patents

Abstract

PURPOSE: A peptide which binds to cyclodextrin and a method for preparing the same are provided to ensure easy fixation on a cyclodextrin polymer. CONSTITUTION: A peptide library for conjugating cyclodextrin contains LPVRPWT(CD-01), LPLTPLP(CD-02), LPPQTLI(CD-03), or NQDVPLF(CD-04) sequence as an active ingredient. A functional group or a ligand to cyclodextrin is introduced using the peptide library. A cyclodextrin-conjugated peptide complex is a fusion protein with a certain protein, and prepared by conjugating the certain protein with the cyclodextrin. [Reference numerals] (1) Combining by adding a library to CD polymer beads; (2) Settling the beads by centrifugation; (3) Removing uncombined phage; (4) Culturing the combined phage and repeating (1) to (3) steps 5 times; (5) Analyzing the base sequences of 10 finally collected phages; (6) Determining amino acid sequence; (AA) CD polymer beads

Description

Cyclodextrin-binidng peptides, their preparation method and their use {Cyclodextrin-binidng peptides, their preparation method and their use}

The present invention relates to a peptide that binds to cyclodextrin, a manufacturing method and use thereof, and more specifically, to form a complex with a specific ligand or functional group to form a complex on the surface of a sensor chip containing cyclodextrin or a cyclodextrin for drug delivery. It relates to a peptide capable of being easily immobilized on a polymer, and a method for producing and using the same.

Cyclodextrin is a compound in which a glucose monomer is α-(1,4) bonded to form a cyclic structure, and there are α-cyclodextrin with six glucose bonds, β-cyclodextrin with seven bonds, and γ-cyclodextrin with eight bonds ( Fig. 1). The hydroxy group of cyclodextrin is located outside the ring, and the outer ring of the ring is hydrophilic, but the inside of the ring is hydrophobic (FIG. 2). Therefore, it has the property of forming a complex by binding hydrophobic guest molecules using the hydrophobicity inside the ring (FIG. 3).

These characteristics of cyclodextrin have been used to improve the solubility, stability, and bioavailability of therapeutic substances. In addition, research results of developing a target-oriented drug by binding ligands that bind well to cancer cells such as transferrin or folic acid to cyclodextrin polymers that form complexes with therapeutic substances have also been reported (Bellocq et al, 2003, Bioconjugate Chem. ., 14, 1122; Salmaso et al., 2004, 15, 997). Cyclodextrin was also used to immobilize admantane-linked cytochrome c protein on the surface of the sensor chip (Fragoso et al., Langmuir, 2002, 18, 5051).

However, in order to bind a protein to cyclodextrin, a hydrophobic guest molecule such as admantane must be linked to the protein through a chemical reaction. However, it is difficult to increase the uniformity of the admantane-protein complex by this method. On the other hand, if there is a peptide that binds to cyclodextrin, it has the advantage of being able to synthesize a single fusion protein by linking the base sequence encoding the peptide with the base sequence of the protein to be immobilized. In addition, since chemical methods for synthesis or modification of peptides are very well established, it is relatively easy to introduce ligands or functional groups into peptides through chemical methods.

Korean Patent Application Publication No. 2005-51686 relates to'cyclodextrin-based materials, compositions and uses thereof', and describes a linear biocompatible polymer containing cyclodextrin and a polymer composition containing a linking molecule. No. 2009-79199 relates to a'peptide', and describes a ring oligopeptide comprising a ring of 6 or more amino acids specifically for binding to a target ligand in an internally constrained ring oligopeptide.

However, none of the prior art describes a peptide that binds to cyclodextrin as described in the present invention, and a method for producing and using the peptide.

It is an object of the present invention to provide a peptide that binds to cyclodextrin.

In addition, an object of the present invention is to provide a method for identifying a peptide that binds to cyclodextrin.

In addition, an object of the present invention is to provide a peptide for introducing a ligand or a functional group into a cyclodextrin.

According to the present invention, a peptide having a sequence of LPVRPWT (CD-01), LPLTPLP (CD-02), LPPQTLI (CD-03) or NQDVPLF (CD-04) that binds to cyclodextrin is provided.

According to the present invention, LPVRPWT (CD-01), LPLTPLP (CD-02), LPPQTLI (CD-03) or NQDVPLF (CD-04), characterized by binding to cyclodextrin and introducing a ligand or functional group into cyclodextrin. The peptide of the sequence is provided.

When the peptide of the present invention is used, it is possible to obtain an effect of easily introducing a ligand or a functional group into cyclodextrin.

1 shows the molecular structure of cyclodextrin.
Figure 2 shows the three-dimensional structure of cyclodextrin.
3 shows a reaction of a guest molecule binding to cyclodextrin.
Figure 4 shows the structure of the fibrous bacteriophage.
5 is a diagram showing the principle of biopanning.
Figure 6 shows the structure of the random amino acid sequence in the Ph.D-7 phage-peptide library.
7 shows a biopanning method according to the present invention.
8 shows the result of measuring the affinity of peptides using a quartz crystal microbalance (QCM) biosensor.
9 shows the results of affinity measurement of peptides using cyclodextrin polymer beads and a filter.

According to the present invention, the step of searching for a phage-peptide library, repeating the biopanning process by adding the phage-peptide library to a cyclodextrin polymer bead, and a peptide with a high frequency of occurrence among the peptides recovered in the biopanning process It provides the peptide of the present invention by a method comprising the step of confirming the nucleotide sequence of.

Phage-peptide library technology is a technology that searches for a ligand that binds to a specific target molecule from a group consisting of bacteriophages showing a peptide with a random amino acid sequence on the coat protein of a fibrous bacteriophage such as M13 or fd. . FIG. 4 shows the structure of a fibrous bacteriophage, showing the structure of a peptide having a random amino acid sequence at the N-terminal portion of protein 3 among coat proteins.

According to the present invention, a Ph.D-7 phage-peptide library with 7 random amino acids was searched to find a peptide that binds to cyclodextrin. Ph.D-7 is a straight line of peptides consisting of 7 residues. The portion of the random amino acid sequence of Ph.D-7 is shown in Fig. 6 and the portion marked with an X is the random sequence.

Biopanning is a method of finding phages that bind to specific target molecules. The principle of the biopanning method is as described below (see FIG. 5). When a phage-peptide library is put on the solid surface to which the target molecule is attached and then washed off, only binding phages remain. When the bound phages are recovered and infected with host bacteria, phages with the same properties are amplified, and if the above process is repeated using the amplified phages, the ratio of phages that bind specifically increases. You will be able to. When a phage that binds to a target molecule is found through biopanning, the amino acid sequence of the peptide expressed in the phage can be estimated from the nucleotide sequence of the DNA inserted before gene 3, the gene for protein 3.

According to the present invention, in order to find a peptide that binds to cyclodextrin, as shown in FIG. 7, first, a phage-peptide library was added to the cyclodextrin polymer beads to bind, and then only the beads were settled by centrifugation. Unbound phage in the solution was removed, and the beads were washed with a buffer solution. And finally, the phage bound to the beads was recovered and amplified in E. coli.

After repeating the above-described biopanning process 5 times, 10 of the finally recovered phages were randomly selected to determine the nucleotide sequence having the code for the peptide. As a result, LPVRPWT (CD-01), LPLTPLP (CD-02), LPPQTLI (CD-03), and NQDVPLF (CD-04) sequences were identified as peptides with high incidence. The amino acid sequence with an increased frequency of occurrence through the repeated biopanning process as described above means that the property of binding to cyclodextrin is excellent.

The peptide of the present invention obtained from the above can form a fusion protein such as an antibody, a hormone protein, an enzyme protein, and the like.

In addition, the peptide of the present invention can be combined with cyclodextrin to introduce functional groups or ligands such as fluorescein, folic acid, biotin, and the like.

Hereinafter, the present invention will be described in more detail by the following examples. However, the scope of the present invention is not limited thereto.

Manufacturing example 1: Preparation of peptide

Add 10 mg of β-cyclodextrin in the form of polymer beads (purchased from Sigma) into a 1.5 ml microtube and TBST (Tris-buffered saline + Tween-20, 50mM Tris-Cl, pH 7.5, 150mM NaCl, 0.1% Tween-20) 1 ml was added and hydrated at room temperature for 30 minutes. To this, 10 μl of Ph.D-7 phage peptide library purchased from New England Biolab (2×10 ¹³ pfu/ml, 2×10 ⁹ independent sequences) was added and bound while shaking at room temperature for 1 hour. After the β-cyclodextrin was settled, the supernatant was removed, and the β-cyclodextrin beads were washed three times with TBST. Finally, after removing the supernatant, 1 ml of Escherichia coli (E. coli) XL1-blue cultured in a liquid medium was added to the tube, and E. coli was infected with phages bound to β-cyclodextrin for 30 minutes. The number of phages recovered from β-cyclodextrin was determined by diluting some of the infected E. coli in turn and incubating them on a top agar plate so that plaques were formed at the site of the infected E. coli. Then, it was added to E. coli cultured in the remaining infected 20 ml liquid medium and incubated for 5 hours to amplify the phage. After amplification, the cells in the precipitate were removed by centrifugation, and the phage in the supernatant was used for the next biopanning.

After biopanning was repeated 5 times in the same way, 10 plaques obtained in the last biopanning were randomly selected and phage cultured. DNA was isolated from the cultured phage, and a random nucleotide sequence inserted in front of gene 3 of each DNA was determined and converted into an amino acid sequence. As a result, LPVRPWT (CD-01), LPLTPLP (CD-02), LPPQTLI (CD-03), NQDVPLF The (CD-04) sequence appeared twice each, and the AVTVMTP (CD-05) and TDAHASV (CD-06) sequences appeared once each. In the first Ph.D-7 library, there are 2×10 ⁹ types of phage, and the number of phages is 2×10 ¹² , so the number of phages of one type is about 1,000. When you choose randomly from among 10 phages selected with the same probability of being gripped, double the 2.5 × 10 ^{- 17} is extremely low. Therefore, the fact that the four amino acid sequences from CD-01 to CD-04 appeared twice after 5 biopannings means that the phages having that sequence were amplified much more strongly than other phages. And the fact that these phages were strongly amplified when biopanning was based on their binding properties to β-cyclodextrin, shows that these phages have good binding properties to β-cyclodextrin.

Example One: Cyclodextrin Affinity evaluation for

To measure the affinity of the three peptides CD-01, CD-02, and CD-04 for β-cyclodextrin, a peptide obtained by adding two glycine residues, one lysine residue, and a biotin group to the carboxy terminus of the three amino acid sequences Was synthesized. For example, CD-01 has the structure of LPVRPWTGGK-biotin. Then, each of the three synthesized peptides was combined with an avidin protein to form a complex.

In the first method to measure affinity, a quartz crystal microbalance (QCM) biosensor manufactured in a laboratory was used. QCM was purchased from Crystal Sunlife, Japan. The size of the square crystal microbalance is 8×8 mm, the diameter of the circular gold electrode is 5 mm, and the natural frequency of the crystal microbalance is about 10 MHz. Since the frequency of the crystal microbalance decreases in proportion to the mass increase, it can be used as a biosensor by using the principle that the frequency changes as the analyte is bound if a receptor to which the analyte is bound is fixed on the surface of the crystal microbalance. I can.

To clean the electrode surface of the crystal microbalance, immerse the crystal microbalance in a 60℃ piranha solution (H ₂ SO ₄ :H ₂ O ₂ = 7:3) for 1 minute, rinse with distilled water and ethanol, and nitrogen gas. And dried. Next, add a crystal microbalance to a solution in which 1-octadecanethiol (Sigma-Aldrich, USA) is dissolved in ethanol at 2mM, and stir for 12-15 hours to form a self-assembled monolayer on the electrode surface. SAM) was allowed to form. The sensor chip on which the SAM was formed was taken out, rinsed with ethanol, dried with nitrogen gas, and mounted on the cell.

In order to bind with the SAM, dipalmitoyl phosphatidyl cholin (DPPC) was mixed with octanoic hydrazide at a molar ratio of 2:1 to prepare liposomes. Here, DPPC was used as a base lipid to form a liposome, and octanoic hydrazide was used as a hydrocarbon compound having a functional functional group to make the hydrazide group appear in the liposome.

The prepared liposome was reacted with the sensor chip on which the self-assembled monolayer was formed in the following manner. A QCM formed with 1-octadecanethiol SAM was mounted in a well-shaped cell. The surface of the SAM formed of 1-octadecanethiol was washed three times with 100 μL of 40 mM octyl glucoside, and immediately followed by 100 μL of a liposome solution, and left at 50° C. for 30-60 minutes. The well-shaped cell was connected to a frequency meter, and after removing the liposome solution, 100 μL of 0.1 M sodium hydroxide (NaOH) was added and left for 30 seconds. And it was washed three times with phosphate buffered saline (PBS).

Before the β-cyclodextrin was immobilized on the QCM surface, an aldehyde group was formed through an oxidation reaction in β-cyclodextrin. The aldehyde group can react with a hydrazide group to form a covalent bond. 0.2 mL of 50 mM sodium m-periodate solution was mixed with 0.3 mL of β-cyclodextrin dissolved in distilled water to be 1.8 mg/mL, and reacted in the dark for 30 minutes. The unreacted sodium m-periodate was removed by dialysis against distilled water.

The surface of the QCM on which the self-assembled monolayer and the lipid monolayer were sequentially bonded was washed three times with a 100mM sodium acetate buffer solution with a pH of 5.5, and then β-cyclodextrin oxidized with sodium m-pyriodate was added to react for 1 hour. After washing three times with ultrapure water, 100 μL of 0.1M cyanoborohydride was added to reduce the double bond and reacted for 1 hour to stabilize the fixation of the receptor having an aldehyde group.

Next, in order to confirm whether the previously prepared peptide-avidin complex binds to β-cyclodextrin immobilized on QCM, each complex was diluted to 0.1 mg/ml based on avidin concentration and injected into the β-cyclodextrin immobilized QCM. At this time, after observing the frequency change by injecting one complex, a dissociation solution (0.2M Glycine-HCl, pH2.3 + 1% DMSO) was injected to completely remove the bound complex, and the next complex was injected.

As a result, all three complexes showed a higher frequency change than those in which only avidin flowed without a peptide, indicating that all of the complexes bind to β-cyclodextrin (FIG. 8). In the QCM biosensor, since the frequency change value is proportional to the mass change, a large frequency change means that a large number of peptide-avidin complexes are bound, which again means that the affinity of the peptide is high. Therefore, it is judged that the affinity of CD-01, which showed the largest frequency change among the three peptides, is the highest.

Example 2: Cyclodextrin Affinity evaluation for

In another way, in order to confirm that the three peptides bind to β-cyclodextrin, the peptides used in Example 1 were each added to the avidin-peroxidase (Av-Prx) complex to form three peptide-avidin-peroxidase complexes. 10 μg of each complex was mixed with 1.2 mg of β-cyclodextrin polymer beads in 0.4 ml PBS (phosphate-buffered saline), combined, and filtered through a filter having a pore of 0.8 micrometers. In this process, the peptide-avidin-peroxidase complex bound to the β-cyclodextrin beads remains in the filter, but the unbound complex passes through the filter and is removed. In addition, luminol and hydrogen peroxide were added to the filter to generate light by peroxidase activity. Here, for comparison, a sample containing only β-cyclodextrin beads, a sample containing only avidin-peroxidase complex and β-cyclodextrin beads, and a sample containing only peptide-avidin-peroxidase complex were made and tested at the same time.

As a result, as can be seen in FIG. 9, all three peptides showed higher values than the control sample, and in particular, CD-01 showed significantly higher values. This is consistent with the experiment using the QCM biosensor and shows that the CD-01 peptide has a high affinity, especially for β-cyclodextrin.

The present invention can be used for functionalization of a sensor chip or drug delivery using cyclodextrin.

<110> KANGNUNG-WONJU NATIONAL UNIVERSITY INDUSTRY ACADEMY COOPERATION GROUP <120> Cyclodextrin-binding peptides, their preparation method and their use <130> 20000 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> derived from phage-peptide library <400> 1 Leu Pro Val Arg Pro Trp Thr 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> derived from phage-peptide library <400> 2 Leu Pro Leu Thr Pro Leu Pro 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> derived from phage-peptide library <400> 3 Leu Pro Pro Gln Thr Leu Ile 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> derived from phage-peptide library <400> 4 Asn Gln Asp Val Pro Leu Phe 1 5

Claims (3)

For cyclodextrin binding, characterized in that it contains one or more selected from the group consisting of LPVRPWT (CD-01), LPLTPLP (CD-02), LPPQTLI (CD-03) and NQDVPLF (CD-04) sequences as an active ingredient. Peptide library. A method for introducing a functional group or ligand into a cyclodextrin using a peptide library having a property of binding to the cyclodextrin of claim 1. A cyclodextrin binding peptide complex comprising binding to a cyclodextrin and a corresponding protein by binding to a cyclodextrin while forming a fusion protein together with a specific protein.

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