KR19990081420A - Peptide library using ubiquitin that can be searched by yeast 2 hybrid system and preparation method thereof - Google Patents

Abstract

The present invention relates to a peptide library using ubiquitin (UB), which can be searched by a yeast 2 hybrid system, and a method for preparing the same. More specifically, the present invention relates to a carboxy terminal portion protruding from the surface of ubiquitin to bind to a specific enzyme. The present invention relates to a peptide library into which a small peptide consisting of 20 amino acid residues is inserted, and a method for preparing the same, wherein the peptide library can be used to easily select a peptide that binds to a specific enzyme. And so on.

Description

Peptide library using ubiquitin that can be searched by yeast 2 hybrid system and preparation method thereof

The present invention relates to a peptide library using ubiquitin (Ub) which can be searched by the yeast two hybrid system, and a method for preparing the same.

More specifically, the present invention relates to a peptide library in which a small peptide consisting of 20 amino acid residues capable of binding a specific enzyme is inserted into a carboxy terminal portion protruding from the surface of ubiquitin, and a method for preparing the peptide library. Using can easily select a peptide that binds to a specific enzyme and similar compounds can be developed as an effective cancer therapy.

Cellular phenomena are known to be maintained by the interaction of various protein enzymes. These interactions are regulated by transient or stable binding between proteins, and specific processes are involved in these processes. That is, the amino acid sequence, which is a unique primary structure of a protein, and its secondary, tertiary, and quaternary structures determine the function and nature of the protein, and proteins that bind and interact with the protein are also specific to itself. By having a structure, selective and specific binding occurs between two proteins.

The binding between these proteins can be divided into two types. First, the factors necessary for binding are spread on the surface of the protein, and the type of binding between the surface of the protein and the factors necessary for binding to a specific peptide domain of the protein are biased. Binding between the surface of the protein and certain parts of other proteins that bind to it.

The yeast two hybrid system is a new genetic method that analyzes the interaction between proteins using the transcriptional activity of reporter genes in living organisms (yeast) rather than in artificial systems. And as a method of searching for a genome fusion library (Fields and Song, 1989, Nature, 340: 245-246).

The yeast 2 hybrid system distinguishes it from other existing in vitro (in vitro) methods because of its ability to confirm protein interaction in vivo. Indeed, this method is a major contributor to understanding the interactions between proteins in higher animal cells.

This is a method to confirm the binding between proteins by quantitatively analyzing the transcriptional activity of reporter genes in yeast cells. Basically, transcriptional regulatory proteins are divided into domains that involve DNA binding and transcriptional activation. In addition, these areas can function independently of each other, and even if an exchange occurs between these areas, the function of a hybrid of the functions of each area appears. For example, in the case of using the yeast transcriptional regulator GAL4, the DNA binding domains of the target proteins X and GAL4 and the transcriptional activation domains of the proteins Y and GAL4 are fused to the same yeast cells. Express within. In this case, when the protein X and the protein Y present in the fusion protein in the yeast cells are mutually coupled, the two hybrid proteins may perform transcriptional regulation as in the normal GAL 4. Specifically, proteins that bind to each other by expressing auxotrophy marker genes such as β-galactosidase, HIS3, Leu2, URA3, etc. in yeast cells using a promoter whose transcription is regulated by GAL 4 Can be confirmed by the transcriptional activity of these reporter genes. Of course, in this case, the yeast used as a host should remove the endogenous GAL4 gene.

In addition, the yeast 2 hybrid system may also be used for the purpose of separating new proteins that bind to protein X when using the cDNA library instead of the protein Y. According to this principle, in addition to the GAL4 system, the DNA binding region of the human estrogen receptor (ER) or LexA, which is a DNA binding protein of E. coli, instead of the DNA binding region of GAL4, or the transcriptional activation region of GAL4 A system using the herpes virus VP1 has been developed instead.

The reason why the yeast two hybrid system can be widely applied to various proteins is that there is a relatively high structural flexibility in transferring DNA bonds to transcriptional activity, which can be mediated by mutual binding between proteins of different structures than the original protein. Not only that, but also proteins that function in extracellular nucleus, such as cytoplasm and cell membrane, can also enter the nucleus when the fusion protein is expressed in yeast and cause mutual binding between the two fusion proteins. The advantage of the yeast 2 hybrid system is that the conventional in vitro system determines whether the two proteins are bound only by the mutual binding force of the two proteins, whereas in the yeast 2 hybrid system, the fused DNA binding region binds DNA and the transcriptional activation region is a general transcription factor. By binding to each of the transcription factors), even if the binding strength between the two proteins can be stabilized. For this reason, yeast 2 hybrid systems are generally more sensitive than conventional in vitro assays and can be useful for analyzing them even when binding between proteins is weak or temporary occurs.

The high sensitivity of this system is an advantage in most cases, but when applied to research, in addition to specific binding between target proteins, the protein itself fused to the transcriptional activation region itself binds to DNA to activate transcription of the reporter gene. The disadvantage is that so-called false positives are obtained by binding. Therefore, even if a new binding protein is identified through this system or a binding region and amino acid residue of a specific protein is found, it is desirable to reconfirm it through genetic or biochemical methods. In addition, it is possible to reduce the incidence of false positives by simultaneously using two reporters whose transcription is controlled by different promoters.

As such, the yeast 2 hybrid system has high sensitivity and fluidity and may be used to search for bioactive peptides and medicinal peptides or to isolate drugs that inhibit inhibitory proteins. For example, in order to isolate a peptide having a binding ability to a reporter protein that acts on a signal transduction system, a peptide library having various amino acid sequences is introduced into an active region to prepare a peptide library, and then isolate and treat a peptide that can bind to a target receptor. You can search for drugs that prevent the interaction between proteins that develop as peptides or cause pathologies.

In cellular phenomena, maintenance of homeostasis by protein interaction is very important, and if the function of the protein is altered due to genetic mutation and the homeostasis is broken, the cell stops proliferating or dies, and cancer cellization ( tumorigenesis). As a suitable example, overexpression or over-activation of protein enzymes involved in growth promoting regulatory mechanisms leads to cell arrest, cell death or cancer cellization. Many data to date indicate that many diseases (especially cancer) caused by overexpression and overactivation of growth promoters can be treated by blocking a series of chain reactions of proteins that interact with and interact with the growth promoter. It is thought. As such, most protein enzymes involved in intracellular chain reactions perform their functions by the interaction between proteins. Thus, when a chemical compound having a small molecular weight that can inhibit the binding between proteins is produced, it may be used as a therapeutic agent for cancer. Can be used.

In order to search for a small peptide that binds to a specific enzyme causing the disease by the yeast 2 hybrid system, the present inventors use a ubiquitin, whose structure is well known, and 20 amino acid residues at a carboxy terminal portion protruding from the surface thereof. The present invention has been completed by preparing a peptide library that is easy to select by inserting a gene coupled to a peptide consisting of a target protein effectively accessing the peptide library.

It is an object of the present invention to provide a peptide library using ubiquitin and a method for preparing the same, which can be searched by a yeast 2 hybrid system to develop a peptide compound that binds a specific enzyme as a cancer therapeutic agent.

1 schematically shows the mechanism by which the yeast two hybrid system works,

Figure 2 shows the amino acid sequence and gene base sequence of the ubiquitin used in the present invention,

Figure 3 shows the manufacturing process of the peptide library of the present invention.

In order to achieve the above object, the present invention provides a peptide library using ubiquitin that can be searched by the yeast 2 hybrid system.

Specifically, the present invention provides a peptide library p4-5 UbCLib in which a peptide consisting of 20 amino acid residues is inserted at the carboxy terminal portion of ubiquitin.

The present invention also provides a yeast expression vector and an E. coli transformant comprising the ubiquitin gene.

In addition, the present invention is to prepare a peptide library consisting of the step of inserting a small peptide gene in the carboxy terminal region of the ubiquitin gene of the plasmid vector using a primer having a nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4 and transforming into E. coli Provide a method.

Hereinafter, the present invention will be described in detail.

The present invention provides a peptide library using ubiquitin that can be searched by the yeast two hybrid system.

In this case, the yeast 2 hybrid system is capable of self-nourishment when the DNA binding domain (DB) and transcription activation domain (AD) are physically strongly bound in the yeast cell, so that the yeast becomes blue. Identifying peptides by selecting and fusing cells can easily search for peptides that bind specific proteins from the library.

The present invention clones the ubiquitin gene by performing a polymerase chain reaction (PCR) using the existing expression vector pETUB A545 (Accession Number: KCTC 0151 BP) as a template. The primers include the amino terminal gene of ubiquitin, the oligonucleotide of SEQ ID NO: 1 including the restriction enzyme Eco RI recognition site, and the restriction enzyme Rsr II recognition site, the carboxy terminal gene of ubiquitin, the termination codon and the restriction enzyme Xho I recognition site. The oligonucleotide of SEQ ID NO: 2 is used.

The present invention prepares a plasmid vector including the ubiquitin gene by using an existing plasmid vector p4-5. Specifically, the present invention produces the yeast expression vector p4-5 Ub by cutting the ubiquitin gene with restriction enzymes Eco RI and Xho I and inserting it into the plasmid vector p4-5.

The present invention prepares a peptide library by transforming Escherichia coli by inserting a small peptide gene into the ubiquitin gene region of the yeast expression vector.

Specifically, the present invention provides a peptide library p4-5 UbCLib by inserting a peptide consisting of 20 amino acid residues into the carboxy terminal region of ubiquitin using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 and then transforming E. coli MC1061 strain. do.

The transformant obtained by transforming the yeast expression vector p4-5 Ub into the E. coli HB101 strain was deposited on March 8, 1998, at the Korea Institute of Science and Technology Biotechnology Research Institute Gene Bank (Accession Number: KCTC 0451 BP).

Hereinafter, the present invention will be described in detail by examples.

The following examples illustrate the invention, but the scope of the invention is not limited by the examples.

Example 1 Isolation of the Ubiquitin Gene

In order to clone the ubiquitin (hereinafter abbreviated as "Ub") gene, the present invention uses the expression vector pETUB A545 (Accession No .: KCTC 0151 BP) deposited in the Genetic Bank of Korea Institute of Science and Technology as a template to polymerize the enzyme. Chain reaction (PCR) was performed.

Oligonucleotides used to perform the PCR were designed in the following procedure and synthesized with a nucleic acid synthesizer (DNA synthesizer, Applied Biosystems, USA). In this case, the primer having the nucleotide sequence of SEQ ID NO: 1 includes a portion of the nucleotide sequence corresponding to the amino terminus of Ub and the restriction enzyme Eco RI recognition site, and the primer having the nucleotide sequence of SEQ ID NO: 2 is the restriction enzyme Rsr II recognition site and the Ub. Carboxy terminus, termination codon, and restriction enzyme Xho I recognition site.

2 μg of each of the primers of SEQ ID NO: 1 and SEQ ID NO: 2 was used as a template, using 1 ng of the expression vector pETUB A545 as a template, and then 10 μl of 10-fold polymerization buffer (500 mM potassium chloride, 100 mM tris-hydrochloric acid, pH 9.0, 1). % Triton X-100, 2.5 mM magnesium chloride), 10 μl of 2 mM dNTPs (2 mM dGTP, 2 mM dATP, 2 mM dCTP, 2 mM dTTP), 2.5 monomer Taq polymerase and distilled water were added and the total volume was 100 μl. Then, PCR was repeated 25 times under conditions of 1 minute (modification step) at 95 ° C, 40 seconds (immersion step) at 55 ° C, and 2 minutes (polymerization step) at 72 ° C. As a result, the Ub gene was obtained (hereinafter, abbreviated as "fragment Ub").

2 μg of the plasmid vector p4-5 was then completely purified with restriction enzymes Eco RI and Xho I under conditions of NEB buffer solution 2 (50 mM sodium chloride, 10 mM tris-hydrochloric acid, 10 mM magnesium chloride, 1 mM dithiothreitol, pH 7.9). The 6.1 Kb nucleic acid fragment was isolated by cleavage and 1% agarose gel electrophoresis (hereinafter abbreviated as “fragment p4-5-RI / X”).

Meanwhile, 2 μg of the fragment Ub obtained above was completely digested with restriction enzymes Eco RI and Xho I under conditions of NEB buffer and subjected to 7% polyacrylamide gel electrophoresis to separate nucleic acid fragments of about 240 bp (hereinafter referred to as “fragment” Abbreviated "Ub-RI / X"). Put 100 ng of the fragment Ub-RI / X and fragment p4-5-RI / X obtained above into the conjugation reaction tube, and then add 2 μl of 10-fold conjugation buffer (50 mM Tris-hydrochloric acid, pH 7.8, 10 mM magnesium chloride). , 10 mM dithiothreitol, 1 mM ATP, 25 μg / ml bovine serum albumin), 10 units of T4 DNA ligase, and distilled water were added to a total volume of 20 μl, followed by reaction at 16 ° C. for 12 hours. The reaction was terminated and transformed into E. coli HB101 (ATCC 33694) to obtain a yeast expression vector p4-5 Ub containing the Ub gene.

The nucleotide sequence of the Ub gene cloned into the recombinant expression vector p4-5 Ub obtained above was confirmed by a die cycle sequencing system (Dye Cycle Sequencing System, Applied Biosystems, USA) according to Sanger et al. This was analyzed by sequencing.

Example 2 Synthesis of DNA for Library Preparation

In order to obtain the insert DNA for the preparation of the peptide library, the primers of SEQ ID NO: 3 and SEQ ID NO: 4 are prepared by cleno buffer solution (100 mM ATP, 10 mM tris-hydrochloric acid, pH 7.5, 5 mM magnesium chloride, 7.5 by 4 nmole). Incubated in mM dithiothreitol, 200 μmole dNTPs) at 65 ° C. for 5 minutes, cooled to room temperature, and reacted for 25 minutes using a cleno enzyme to synthesize double-stranded DNA for library preparation.

Example 3 Preparation of Peptide Library p4-5 UbCLib

In the present invention, to prepare a peptide library, the plasmid vector p4-5 Ub obtained in Example 1 was completely digested with restriction enzyme Rsr II, and the reaction buffer (50 mM sodium chloride, 10 mM tris-hydrochloric acid, pH 7.9, 1 mM) was prepared. Dithiothreitol) was dephosphorylated by using alkaline phosphatase for 1 hour at 37 ° C. This was separated by 1% agarose gel electrophoresis to obtain a 6.4 Kb DNA fragment (hereinafter abbreviated as "fragment p4-5 Ub-Rsr II").

The library preparation DNA obtained in Example 2 was 37 ° C. in a reaction buffer solution (10 mM tris-hydrochloric acid, pH 7.9, 10 mM magnesium chloride, 1 mM dithiothreitol, 50 mM sodium chloride) using restriction enzyme Ava II. After cutting for 1 hour at 7% polyacrylamide gel electrophoresis, about 90 bp of nucleic acid fragments were isolated (hereinafter abbreviated as "fragment library-Ava II"). 100 ng of the fragment library-Ava II and fragment p4-5 Ub-Rsr II obtained above were added to the conjugation reaction tube, followed by 2 μl of 10-fold conjugation buffer solution (50 mM tris-hydrochloric acid, pH 7.8, 10 mM magnesium chloride, 10 mM dithiothreitol, 1 mM ATP, 25 μg / ml bovine serum albumin), 10 units of T4 DNA ligase, and distilled water were added to a total volume of 20 μl, followed by reaction at 16 ° C. for 12 hours. Next, the E. coli MC1061 strain was transformed using an electroporation method to obtain a peptide library p4-5 UbCLib having 20 amino acid residues at the carboxy terminus of ubiquitin (Ub). At this time, the number of transformed E. coli constituting the peptide library is about 0.6 × 10 ⁷ , which is the size of the library to be searched soon.

As described above, the peptide library of the present invention inserts a small peptide capable of binding to a specific enzyme causing a disease to a carboxy terminal portion protruding from the surface of ubiquitin, thereby allowing the target protein to be spatially and easily. Access and strong binding allow for easy and accurate retrieval of peptides.

Accordingly, the peptide library is developed using a yeast two hybrid system to obtain information on the amino acid sequence and structure of the lead compound, a peptide that binds to a specific enzyme, and develop a compound having a similar structure as a new cancer therapeutic agent. It is useful to

(Sequence list)

SEQ ID NO: 1

Sequence length: 39

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

5'- GCGAGAATTC GGTGGTCATA TGCAAATTTT CGTCAAAAC -3 '

(Sequence list)

SEQ ID NO: 2

Sequence length: 39

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

5'- CGACTCGAGC TAACGGACCG CACCGCGGAG TCTCAACAC -3 '

(Sequence list)

SEQ ID NO: 3

Sequence length: 88

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

5'- GCTAGCGGTCCAA (NNT / G) ₂₀ TAGGTCCAGTCAGTC -3 '

(Sequence list)

SEQ ID NO: 4

Sequence length: 15

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

5'- GACTGACTGG ACCTA -3 '

Claims (4)

Peptide library using ubiquitin which can be searched by yeast 2 hybrid system. The peptide library p4-5 UbCLib according to claim 1, wherein a peptide consisting of 20 amino acid residues is inserted into the carboxy terminal region of ubiquitin. The transformant E. coli HB101 / p4-5 UbCLib obtained by transforming Escherichia coli with the yeast expression vector of claim 2 (Accession No .: KCTC 0451 BP). Method for producing a peptide library comprising the step of inserting a small peptide gene into the carboxy terminal region of the ubiquitin gene of the yeast expression vector p4-5 Ub using a primer having the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 and transforming E. coli .