KR20120000393A - Peptides that bind to cyclodextrins, methods for their preparation and uses - Google Patents

Abstract

PURPOSE: A peptide which binds to cyclodextrin and a method for preparing the same are provided to form a complex by being conjugated with a specific protein. CONSTITUTION: A peptide which binds to cyclodextrin has LPVRPWT(CD-01), LPLTPLP(CD-02), LPPQTLI(CD-03), or NQDVPLF(CD-04). The peptide introduces a functional group or ligand to cyclodextrin by being conjugated to cyclodextrin. A method for detecting the peptide comprises: a step of searching a phage-peptide library; a step of adding the phage-peptide library to cyclodextrin polymer beads and biopanning; and a step of detecting a base sequence of a peptide with high frequency.

Description

Peptides that bind to cyclodextrins, methods for their preparation and use {Cyclodextrin-binidng peptides, their preparation method and their use}

The present invention relates to a peptide that binds to a cyclodextrin, a method for producing the same, and a use thereof, and more particularly, to form a complex with a specific ligand or a functional group to form a complex with a cyclodextrin, or a cyclodextrin for drug delivery including a cyclodextrin. Peptides to be easily immobilized on a polymer, methods for making and uses thereof.

Cyclodextrin is a compound in which a glucose monomer is α- (1,4) -bonded to form a ring structure, such as α-cyclodextrin to which six glucoses are bound, β-cyclodextrin to which seven are bound, and γ-cyclodextrin to which eight are bound ( 1). The hydroxyl group of the cyclodextrin is located outside of the ring, so that the outer part 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 combining hydrophobic guest molecules using hydrophobicity inside the ring (FIG. 3).

Such cyclodextrins have been used to improve the solubility, stability, bioavailability, etc. of therapeutic substances. In addition, the results of research on developing targeted drugs by combining ligands that bind well to cancer cells, such as transferrin and folic acid, to cyclodextrin polymers complexed with therapeutic substances have been reported (Bellocq et al, 2003, Bioconjugate Chem). , 14, 1122; Salmaso et al., 2004, 15, 997). Cyclodextrins have also been used to immobilize cytochrome c proteins bound to admantane on the sensor chip surface (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 with this method. On the other hand, if there is a peptide that binds to cyclodextrin, there is an advantage that can be synthesized as a single fusion protein by connecting the base sequence encoding the peptide and the base sequence of the protein to be fixed. 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.

Republic of Korea Patent Publication No. 2005-51686 relates to 'cyclodextrin-based materials, compositions and uses thereof,' describes a polymer composition containing a linear biocompatible polymer and a linking molecule having a cyclodextrin, No. 2009-79199 relates to 'peptides' and describes ring oligopeptides comprising at least six amino acid rings for specifically binding to a target ligand in an internally bound ring oligopeptide.

However, none of the prior art describes peptides that bind to cyclodextrins as described herein, methods for their preparation and uses.

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

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

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

According to the present invention, peptides of the LPVRPWT (CD-01), LPLTPLP (CD-02), LPPQTLI (CD-03) or NQDVPLF (CD-04) sequences that bind to cyclodextrins are provided.

According to the present invention, LPVRPWT (CD-01), LPLTPLP (CD-02), LPPQTLI (CD-03) or NQDVPLF (CD-04), characterized in that a ligand or a functional group is introduced into the cyclodextrin by binding to the cyclodextrin. Provide peptides of the sequence.

When the peptide of the present invention is used, an effect of easily introducing a ligand or a functional group into the cyclodextrin can be obtained.

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

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

Phage-peptide library technology is a technique for searching for ligands that bind to a specific target molecule from a population of bacteriophages that exhibit peptides of random amino acid sequence in a coat protein of fibrous bacteriophages such as M13 or fd. . Figure 4 shows the structure of the fibrous bacteriophage, which can be seen the structure of a peptide of the random amino acid sequence in the N-terminal portion of protein 3 of the coat protein.

According to the present invention, a Ph.D-7 phage-peptide library with seven random amino acids was searched for a peptide that binds to cyclodextrin. Ph.D-7 is a straight line of peptides consisting of seven residues. The random amino acid sequence portion of Ph.D-7 is shown in FIG. 6 and the portion marked with 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 the phage-peptide library is put on the solid surface to which the target molecule is attached and then washed, the remaining phages remain. By recovering the bound phage and infecting it with a host bacterium, phages having the same properties are amplified. When the above process is repeated using the amplified phage, the ratio of the specifically bound phage increases, finally separating only the desired peptide. It becomes possible. When the phage binds to the target molecule through biopanning, the amino acid sequence of the peptide expressed in the phage can be estimated from the base sequence of the DNA inserted before gene 3, the gene of protein 3.

According to the present invention, in order to find a peptide that binds to cyclodextrin, the phage-peptide library was added to the cyclodextrin polymer beads first, as shown in FIG. Unbound phages were removed from the solution and the beads were washed with buffer. Finally, the phages bound to the beads were recovered and amplified in E. coli.

After repeating the biopanning process five times as described above, ten of the finally recovered phages were randomly selected to determine the base 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 high incidence peptides. As described above, the amino acid sequence of which frequency is increased through repeated biopanning process means that the binding property to cyclodextrin is excellent.

The peptides of the present invention obtained from the above can form fusion proteins such as proteins, such as antibodies, hormone proteins, enzyme proteins 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 Peptides

10 mg of β-cyclodextrin (purchased from Sigma) in the form of polymer beads was placed in a 1.5 ml microtube and TBST (Tris-buffered saline + Tween-20, 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) 1 ml was added and hydrated for 30 minutes at room temperature. To this was added 10 μl (2 × 10 ¹³ pfu / ml, 2 × 10 ⁹ independent sequences) of Ph.D-7 phage peptides purchased from New England Biolab, followed by shaking for 1 hour at room temperature. 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 (XL) blue incubated in liquid medium was added to the tube to infect E. coli with phages bound to β-cyclodextrin for 30 minutes. The number of phages recovered from β-cyclodextrin was determined by incubating some of the infected E. coli in turn and incubating on top agar plates to form plaques in place of the infected E. coli. In addition, E. coli incubated 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.

In the same manner, the biopanning was repeated five times, and then ten of the plaques obtained in the last biopanning were randomly selected and cultured. The DNA was isolated from the cultured phage, and the random sequencing inserted in front of gene 3 of each DNA was determined and converted into the amino acid sequence. The (CD-04) sequence appeared twice each and the AVTVMTP (CD-05) and TDAHASV (CD-06) sequences appeared once each. The first Ph.D-7 library used has 2 × 10 ⁹ types of phages, and the number of phages is 2 × 10 ¹² , so the number of phages of one kind is about 1,000. When ten phages are randomly selected, the probability of selecting the same phage twice is extremely low, 2.5 × 10 ⁻¹⁷ . Therefore, four amino acid sequences from CD-01 to CD-04 appear twice after five biopannings, indicating that the phages with those sequences were amplified much more strongly than other phages. The fact that these phages were strongly amplified by biopanning based on their binding to β-cyclodextrin indicates that these phages bind well to β-cyclodextrin.

Example 1: On cyclodextrin  Affinity rating

CD-01, CD-02, CD-04 peptides with two glycine residues, one lysine residue and a biotin group added to the carboxy terminus of three amino acid sequences to determine the affinity for the β-cyclodextrin of the three peptides Was synthesized. CD-01, for example, has the structure of LPVRPWTGGK-biotin. Each of the three peptides synthesized was combined with avidin protein to form a complex.

In the first method for measuring affinity, a laboratory-manufactured Quartz Crystal Microbalance (QCM) biosensor was used. QCM was purchased from Crystal Sunlife, Japan. The size of the square quartz crystal is 8 × 8 mm, the diameter of the circular gold electrode is 5 mm, and the natural frequency of the quartz microbalance is about 10 MHz. Since the frequency of the crystal microbalance decreases in proportion to the increase in mass, if the receptor to which the analyte is bound is fixed on the surface of the microcrystal scale, the frequency changes as the analyte binds to be used as a biosensor. Can be.

To clean the electrode surface of the fertilized microbalance, soak the fertilized microbalance in 60 ° C piranha solution (H ₂ SO ₄ : H ₂ O ₂ = 7: 3) for 1 minute, rinse with distilled water and ethanol and nitrogen gas. Dried over. Next, add a microcrystalline scale to a solution of 1-octadecanethiol (1-octadecanethiol, Sigma-Aldrich, USA) in 2mM in ethanol, and stir for 12-15 hours to prepare a self-assembled monolayer (self-assembled monolayer, SAM) was formed. The sensor chip formed with SAM was taken out, rinsed with ethanol, dried with nitrogen gas, and mounted in a cell.

Liposomes were prepared by mixing octanoic hydrazide in dipalmitoyl phosphatidyl cholin (DPPC) in a 2: 1 molar ratio to bind the SAM. DPPC was used here as a substrate for forming liposomes and octanoic hydrazide was used as a hydrocarbon compound with functional functional groups to cause hydrazide groups to appear on the liposomes.

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

Before fixation to the β-cyclodextrin QCM surface, an aldehyde group was formed by oxidation to the β-cyclodextrin. Aldehyde groups can react with hydrazide groups to form covalent bonds. 0.2 ml of 50 mM sodium m-periodate was mixed with 0.3 ml of β-cyclodextrin dissolved in distilled water at 1.8 mg / mL and reacted in a dark place for 30 minutes. Unreacted sodium m-periodate was removed by dialysis against distilled water.

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

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

As a result, all three complexes showed higher frequency change than avidin alone without peptide and all showed binding to β-cyclodextrin (FIG. 8). In the QCM biosensor, the frequency change is proportional to the mass change, so that the large frequency change means that the peptide-avidin complex is bound, which means that the peptide has high affinity. Therefore, CD-01, which shows the highest frequency change among the three peptides, is considered to have the highest affinity.

Example 2: On cyclodextrin  Affinity rating

Alternatively, the peptides used in Example 1 were added to the avidin-peroxidase (Av-Prx) complexes to form three peptide-avidin-peroxidase complexes, respectively, to confirm that the three peptides bound to β-cyclodextrin. 10 μg of each complex was mixed with 1.2 mg of β-cyclodextrin polymer beads in 0.4 ml PBS (phosphate-buffered saline) and filtered through a filter having a hole of 0.8 micrometers. In this process, peptide-avidin-peroxidase complexes bound to β-cyclodextrin beads remain in the filter, but unbound complexes exit the filter and are removed. Luminol and hydrogen peroxide were added to the filter to generate light by peroxidase activity. Here, for comparison, samples were prepared containing only β-cyclodextrin beads, samples containing only avidin-peroxidase complex and β-cyclodextrin beads, and samples containing only peptide-avidin-peroxidase complex, and the experiments were simultaneously performed.

As a result, as shown in Figure 9, all three peptides showed a higher value than the control sample, especially CD-01 showed a significantly higher value. This is in line with the experiments with 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 or drug delivery of sensor chips using cyclodextrins.

<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 (4)

A peptide selected from the group consisting of LPVRPWT (CD-01), LPLTPLP (CD-02), LPPQTLI (CD-03) and NQDVPLF (CD-04) sequences that bind to cyclodextrin. The peptide according to claim 1, wherein the peptide binds to the cyclodextrin to introduce a functional group or ligand into the cyclodextrin. The peptide according to claim 1, wherein the peptide is bound to the cyclodextrin by binding to the cyclodextrin in a state in which a fusion protein is formed together with the specific protein. Searching for phage-peptide libraries,
Repeating the biopanning process by adding the phage-peptide library to a cyclodextrin polymer bead,
Identifying the nucleotide sequence of the peptide with high frequency among the peptide recovered in the step
How to identify peptides that bind to cyclodextrins.

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