JP2002306182A - Method for producing antibacterial protein and fused protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To realize an inexpensive and advantageous large amount expressing system of a basic antibacterial protein on a condition of forming proper disulfide bonds for exhibiting the antibacterial activity. SOLUTION: This method for producing the antibacterial protein is provided by expressing a fused protein not having the antibacterial activity, from the above basic antibacterial protein with a partner protein having <7 isoelectric point and a shaperon function, collecting the fused protein, separating the both and activating the antibacterial protein with a partner protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a system for expressing a large amount of a basic antibacterial protein in which the formation of an intramolecular disulfide bond in a certain mode is an active condition.

[0002]

2. Description of the Related Art Due to the application of gene recombination technology, proteins having various functions are being expressed in various expression systems such as bacteria, fungi, and mammals. In view of efficient mass production of proteins, an expression system using a prokaryotic cell such as Escherichia coli as a host is particularly advantageous. On the other hand, the expression of antibacterial proteins, which are attracting attention in many industrial fields, has specific problems such as toxicity of antibacterial proteins to host cells and degradation of antibacterial proteins having a relatively low molecular weight in host cells. Therefore, for example, in the production of antibacterial proteins in Escherichia coli, the following techniques have been proposed.

[0003] First, there has been proposed a method of masking the toxicity of an antibacterial protein and suppressing its degradation in cells by multimerizing a fusion unit of the antibacterial protein and the acidic protein (Jae H. Lee). , Il Minn, C
han B, et al (1998) ProteinExpression and Purificat
ion 12: 53-60). Others have aimed at the same effect by fusing antibacterial protein production with prochymosin expressed in the insoluble fraction (Chris Ha).
ught, Gregory D, Rajesh Subramanian, et al (1998)
Biotechnology and Bioengineering 57, 1: 55-61).
For the same purpose, a method of fusing an antibacterial protein with glutathione-S-transferase 3 (GST) (Kiri
ll A Martemyanov, Alexander s spirin, Anatory T Gu
dkov (1996) Biotechnology Letters 18, 12: 1357-136
2) and a method of fusing an antimicrobial protein with a cellulose binding domain (CBD) (Kevin L Piers, Mel
issa H Brown, et al (1993) Gene 134, 1: 7-13), a method of fusing an antimicrobial protein with protein A (L. Zhan
g, T. Falla, M. Wu, et al (1998) Biochemical and Bio
physical Res. Com. 247: 674-680).

[0004]

By the way, some important antibacterial proteins include cysteine in the amino acid sequence and have a basic form in which the formation of intramolecular disulfide bonds in a certain mode is an active type condition. Is an antibacterial protein. Representative examples include plant-derived thionin, PR protein, lipid transfer protein, ribosome-inactivating protein, and plant, insect or human-derived defensin.

[0005] These types of basic antibacterial proteins, in addition to the above-mentioned problems of toxicity to host cells and degradation in host cells, furthermore, the activity of antibacterial proteins with correct intramolecular disulfide bonds in a certain mode. There is a problem that it is difficult to obtain by type. In general, eukaryotic cells have the function of refolding proteins in the endoplasmic reticulum such as the endoplasmic reticulum (changing the wrong mode of intramolecular disulfide bonding to the correct certain mode), whereas prokaryotic cells have There is no such function.

[0006] For example, none of the above-mentioned prior arts is directed to basic antibacterial proteins of the type described above, and a method for obtaining these types of basic antibacterial proteins in an active form. No disclosure or suggestion. It is also known to convert a basic antibacterial protein obtained in an inactive form into an active form by separately administering a protein having a chaperone function in vitro, but it is a costly method. Is rarely used.

[0007] On the other hand, in the field of functional proteins other than antibacterial proteins, and in expression systems using prokaryotes as hosts, proteins that have a certain mode of formation of intramolecular disulfide bonds as active conditions are refolded. There have been proposed a few techniques for expressing as a fusion with a certain protein having For example, Japanese Patent Application Laid-Open No. Hei 7-250685 proposes a method for expressing a certain functional protein as a fusion with thioredoxin.
No. 9 proposes a method for expressing a functional protein as a fusion with protein disulfide isomerase. However, even if these techniques can be applied to an antibacterial protein expression system and an active antibacterial protein can be expressed, an effective amount of antibacterial protein is exhibited because the antibacterial protein exerts toxicity on host cells. Unable to get protein.

[0008] In view of the above, in order to obtain a basic antibacterial protein with an active type condition of forming a certain mode of intramolecular disulfide bond using an expression system using a prokaryote as a host, with high efficiency, The basic antibacterial protein is expressed in a host cell in an inactive state without undergoing intracellular degradation,
The problem is that after being collected from a host cell, it can be activated by an easy and low-cost operation. An object of the present invention is to provide a method for producing an antibacterial protein that can solve this problem.

[0009]

Means for Solving the Problems (Structure of the First Invention) The structure of the first invention (the invention described in claim 1) for solving the above-mentioned problem is to form an intramolecular disulfide bond in a certain manner. The basic antimicrobial protein A as an active type condition is expressed in a prokaryotic cell as a protein fusion with a partner protein B having an isoelectric point of less than pH 7 and having a chaperone function, and after collecting this, the partner A method for producing an antibacterial protein, wherein the antibacterial protein A is converted into an active form by an activation step including utilization of the function of protein B.

(Structure of the Second Invention) The structure of the second invention of the present application (the invention according to claim 2) for solving the above problems is
A method for producing an antibacterial protein, wherein the expression of the protein fusion according to the first invention is performed by culturing a prokaryotic cell into which a DNA encoding the protein fusion has been introduced so as to be expressible.

(Structure of Third Invention) The structure of the third invention of the present application (the invention according to claim 3) for solving the above problems is as follows.
The antibacterial protein A according to the first or second invention, wherein the antibacterial protein A is any one of plant-derived thionin, PR protein, lipid transfer protein, and ribosome-inactivating protein, or a plant, insect or human-derived defensin, respectively. This is a method for producing a protein.

(Structure of the Fourth Invention) The structure of the fourth invention of the present application (the invention according to claim 4) for solving the above problems is as follows.
A protein fusion wherein the partner protein B according to any one of the first to third inventions is one of the following 1) or 2). 1) Protein disulfide isomerase (hereinafter referred to as "P
DI)) or an acidic protein encoded by DNA present downstream of the base sequence relative to plant-derived thionin. 2) Thioredoxin (hereinafter referred to as “Tx”) or chaperonin.

(Structure of the Fifth Invention) The structure of the fifth invention (the invention described in claim 5) for solving the above problems is as follows.
Production of an antibacterial protein, wherein the partner protein B according to any of the first to third inventions comprises at least an acidic partner protein B1 having an isoelectric point of less than pH 7 and a chaperone partner protein B2 having at least a chaperone function. Is the way.

(Structure of Sixth Invention) The structure of the sixth invention of the present application (the invention according to claim 6) for solving the above problems is as follows.
A fusion of a basic antibacterial protein A, which has a certain mode of formation of an intramolecular disulfide bond as an active condition, and a partner protein B having an isoelectric point of less than pH 7 and a chaperone function, wherein the antibacterial protein A And partner protein B
Are protein fusions composed of a series of polypeptide chains via an oligopeptide moiety cleavable by an enzyme.

(Structure of Seventh Invention) The structure of the seventh invention (the invention according to claim 7) for solving the above problems is as follows.
A basic antibacterial protein A whose active condition is the formation of an intramolecular disulfide bond in a certain mode, an acidic partner protein B1 having at least an isoelectric point of less than pH 7, and a chaperone partner protein B2 having at least a chaperone function. A fusion, a protein fusion.

(Structure of Eighth Invention) The structure of an eighth invention (an invention according to claim 8) for solving the above problems is as follows.
The protein fusion wherein the acidic partner protein B1 according to the seventh invention is a carboxy terminal region of protein disulfide isomerase derived from Fumicola insolens, and the chaperone partner protein B2 is peptidylprolyl-cis-trans-isomerase. Body.

(Structure of the ninth invention) The structure of the ninth invention (the invention according to claim 9) for solving the above problems is as follows.
An acidic partner protein used for forming a protein fusion with a basic antibacterial protein A whose active condition is the formation of an intramolecular disulfide bond in a certain mode, wherein the acidic partner protein has at least an isoelectric point of less than pH7. B1 and
A partner protein comprising at least a chaperone partner protein B2 having a chaperone function.

(Structure of the Tenth Invention) The structure of the tenth invention of the present application (the invention of the tenth invention) for solving the above-mentioned problem is that the protein fusion according to any of the sixth invention to the eighth invention is used. It encodes DNA.

(Structure of the eleventh invention) The structure of the eleventh invention (the invention according to the eleventh invention) for solving the above-mentioned problem is based on a prokaryotic cell into which the DNA according to the tenth invention has been introduced so that it can be expressed. There is a host cell.

[0020]

According to the method for producing the antibacterial protein of the first invention, the basic antibacterial protein A is compared with the partner protein B having an isoelectric point of less than pH7. It is not degraded in host cells because it is expressed as a high molecular weight protein fusion. In addition, antibacterial protein A
In prokaryotic hosts, it is normally expressed as an inactive form with incorrect intramolecular disulfide bonds, but even when expressed as an active form, toxicity is masked by fusion with partner protein B. From the above points, the antimicrobial protein A can be expressed in large quantities in prokaryotic cells with fast cell division.

Next, after the protein fusion is collected from the host cell, the antimicrobial protein A is converted into an active form by an activation step. At this time, the antimicrobial protein A is used by utilizing the function of the partner protein B having a chaperone function. Since refolding of A is performed, activation can be performed at low cost. In this activation step, usually, an operation of separating the antibacterial protein A and the partner protein B is also performed. However, the separation operation is a simple operation selected according to the fusion state of the protein fusion, and the antimicrobial protein A can be easily activated.

(Action / Effect of Second Invention) The second invention provides a typical embodiment of the method for producing an antibacterial protein according to the first invention.

(Action / Effect of the Third Invention) According to the third invention, the basic antibacterial protein A according to the first invention or the second invention, wherein the formation of a certain mode of intramolecular disulfide bond is an active condition, A representative example embodiment is provided.

(Action and Effect of the Fourth Invention) The fourth invention provides a representative example of partner protein B having an isoelectric point of less than pH 7 and having a chaperone function. Among them, PDI or acidic proteins encoded downstream of plant-derived thionine are particularly excellent in the refolding function of antibacterial protein A for disulfide bonds.

(Function / Effect of the Fifth Invention) The partner protein B in the protein fusion is composed of the acidic partner protein B1 and the chaperone partner protein B2 as in the fifth invention.
It may consist of In this case, the protein fusion is
Antibacterial protein A, acidic partner protein B1, and chaperone partner protein B2 are fused with three types of proteins.

(Function / Effect of the Sixth Invention) The protein fusion of the antibacterial protein A and the partner protein B may be fused by the electric affinity of the two, but as in the sixth invention, both are a series of polymorphs. When configured as a peptide chain, both expressed in the host cell will reliably form a protein fusion. In addition, since an oligopeptide portion cleavable by an enzyme is interposed between the two proteins, both proteins can be easily separated by enzymatic cleavage of this portion in the activation step.

(Function / Effect of Seventh Invention) According to the seventh invention, an example of a protein fusion different from that of the sixth invention is provided. In this protein fusion, the acidic partner protein B1 and the chaperone partner protein B2 share the role of partner protein B.

(Effect of the Eighth Invention) According to the eighth invention, a typical example of the acidic partner protein B1 having at least an isoelectric point of less than pH 7 and a typical example of the chaperone partner protein B2 having at least a chaperone function And a protein fusion comprising:

(Function / Effect of the Ninth Invention) The ninth invention provides a partner protein to be a partner of the antibacterial protein A in the protein fusion of the seventh or eighth invention.

(Effect of the Tenth Invention) The tenth invention provides a specific means for genetic engineering production of the protein fusion according to the sixth to eighth inventions.

(Operation and Effect of Eleventh Invention) According to the eleventh invention, a specific site for expression of a protein fusion using the DNA of the tenth invention is provided.

[0032]

Next, embodiments of the first invention to the eleventh invention will be described. In the following, the term "the present invention" simply refers to the first to eleventh inventions collectively.

[Protein fusion] The protein fusion according to the present invention is a fusion between the antibacterial protein A and the partner protein B. The term "fusion" as used herein refers to a state where some or all of two or more heterologous proteins are integrated by some force. Examples of the “fusion” include a fusion form in which the antibacterial protein A and the partner protein B are chemically bonded. This fusion form includes the case where the antibacterial protein A and the partner protein B are configured as a series of polypeptide chains via a cleavable oligopeptide portion. A fusion form in which a part or all of the antibacterial protein A and the partner protein B are associated with each other by hydrophobic affinity, electrical properties, or the like can also be exemplified. In each case, the antibacterial protein A is a basic protein having cysteine on the amino acid sequence, and the partner protein B has an isoelectric point of pH7.
, The two are surely fused.

[Antimicrobial Protein A] The antimicrobial protein A according to the present invention is a basic antimicrobial protein having cysteine in the amino acid sequence and having a certain mode of formation of intramolecular disulfide bonds as an antimicrobial active condition. . The state of the antimicrobial protein A constituting the protein fusion according to the present invention includes:
An example is the case of expression as an inactive form, with the formation of disulfide bonds in the wrong manner originally. There is also exemplified a case where the active conformation is obtained by forming an intramolecular disulfide bond in a correct manner, but toxicity is masked due to neutralization or insolubilization of charge by fusion with partner protein B. You. A case is also exemplified in which a disulfide bond, which is a condition of an antibacterial activity type, cannot be formed by fusion with partner protein B, so that the compound is detoxified.

The type of the antibacterial protein A is not limited as long as the formation of an intramolecular disulfide bond in a certain mode is a condition of the antibacterial activity type and the basic antibacterial protein is used.
However, any of plant-derived thionin, PR protein, lipid transfer protein, and ribosome-inactivating protein, or plant, insect or human-derived defensin can be particularly preferably exemplified. Preferred examples of the plant-derived thionin include those derived from barley, and more specifically, those comprising amino acids at positions 10 to 54 in the amino acid sequence shown in SEQ ID NO: 1.

[Partner protein B] The partner protein B according to the present invention is a protein having an isoelectric point of less than pH 7 and having a chaperone function. As used herein, the term "chaperone function" refers to a function capable of correctly changing the tertiary structure of a target protein. In the present invention, in particular, a protein capable of changing the formation site of an intramolecular disulfide bond of a protein to the correct state as an active form. A folding function is preferred.
As the above isoelectric point, those having a pH of 6 or less, especially pH
Those having 5.5 or less are preferred. Also, partner protein B
Are those consisting of a single partner protein, the acidic partner protein B1 and the chaperone partner protein B2
May consist of

The type of the partner protein B is not limited as long as it meets the above rules. However, as a single partner protein B, PDI (isoelectric point pH 4.6
8) or an acidic protein (isoelectric point pH 3.61) encoded by DNA present on the base sequence downstream of plant-derived thionin can be particularly preferably exemplified.

The above-mentioned PDI is, for example, one derived from the aforementioned Humicola insolens, and more specifically, SEQ ID NO: 2
And those consisting of the amino acids at positions 59 to 543 in the amino acid sequence shown in the above. As the acidic protein, an acidic protein encoded by DNA present on the base sequence downstream of thionin derived from barley,
More specifically, 61 in the amino acid sequence shown in SEQ ID NO: 1
Those consisting of the amino acids at positions 124 to 124 are preferably exemplified.

As a single partner protein B, Tx (isoelectric point pH 5.14) can be preferably exemplified. Furthermore, GroEL (isoelectric point pH 5.08), Gro
ES (isoelectric point pH 4.51), HSP90 (isoelectric point pH
4.67) and the like can also be preferably exemplified.

The acidic partner protein B1 includes:
A region of glutamic acid at position 514 to leucine at position 543 (isoelectric point pH 3.95), which is the carboxyl terminal of PDI derived from Humicola insolens, may be exemplified, but any other protein having an isoelectric point in an acidic region may be used. Absent.
PPI can be preferably exemplified as the chaperone partner protein B2. Chaperone partner protein B2
Although its isoelectric point is not limited, if its isoelectric point is not in the acidic range, any means for fusing with antimicrobial protein A during expression in host cells (for example, enzymatically cleavable oligos) Antimicrobial protein A and a series of polypeptide chains via a peptide moiety).

[DNA Encoding Protein Fusion] In the method for producing an antibacterial protein of the present invention, DNA encoding any of the aforementioned protein fusions is introduced into a prokaryotic host cell so as to be expressible. A protein fusion can be efficiently mass-produced. The specific base sequence of these DNAs is not limited as long as they encode the antibacterial protein A and the partner protein B.

As an embodiment of the above-mentioned coding, there may be a case where the antibacterial protein A and the partner protein B (or the acidic partner protein B1 and the chaperone partner protein B2) are continuously encoded as a single structural gene. When the antibacterial protein A, the intermediate oligopeptide portion cleavable by any means, and the partner protein B (or the acidic partner protein B1 and the chaperone partner protein B2) are continuously encoded as a single structural gene. It is possible. When the antimicrobial protein A and the partner protein B, or two or more of the antimicrobial protein A, the acidic partner protein B1, and the chaperone partner protein B2 are encoded as different structural genes through appropriate intervening sequences. It is possible. As such a DNA, for example, those each having the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 can be particularly preferably exemplified.

[Host cell into which DNA has been introduced so that it can be expressed]
Generally, the DNA can be introduced so as to be expressible into any appropriate host cell, and the type of the host cell is not limited. For example, prokaryotic cells such as Escherichia coli, eukaryotic cells such as yeast, etc., as well as any kind of plant cells or non-human animal cells can be used. However, in the present invention,
Prokaryotic cells are used as hosts.

In order to introduce the DNA into the host cell so that it can be expressed, any appropriate expression vector can be used. Particularly preferred expression vectors include, for example, plasmids such as "pET Expression system" manufactured by Novagen for Escherichia coli. Insertion of DNA into an expression vector and introduction of an expression vector into a host cell can be performed by any known method.

[Method for Producing Antibacterial Protein] The method for producing the antibacterial protein includes a process for producing a protein fusion. This process involves, for example, culturing a host cell into which the predetermined DNA has been introduced so that it can be expressed. The method and conditions for culturing the host cell are not limited.

As to the method of collecting and purifying the produced protein fusion from the host cells, any method may be used as necessary. The protein fusion according to the present invention may have better storage properties than antibacterial protein A depending on the storage method and storage conditions. Therefore, there may be a case where the method for producing an antibacterial protein is not carried out until completion, and only the process up to production and collection of the protein fusion is carried out for the purpose of preserving and distributing the protein fusion. The “collection” of the protein fusion may be a collection in a crude state or a purification state.

The method for producing an antibacterial protein includes at least the step of converting the antibacterial protein A into an antibacterial active form utilizing at least the function of the partner protein B. Usually, the antibacterial protein A in the protein fusion is separated from the partner protein B. Including the process of doing. Both of the above processes may proceed at the same time, or may proceed sequentially over time.

In the process of separating the antimicrobial protein A from the partner protein B, when both are constituted by a series of polypeptide chains, the boundary (or the oligopeptide portion for cleavage interposed at the boundary) is used. With the operation of cleaving the peptide bond in When both are composed of separate polypeptide chains and are associated by electrical properties or the like, an appropriate treatment for separating them is involved.

The process of converting antimicrobial protein A to an antimicrobial active form can be expected to be automatically advanced and completed by the function of partner protein B. However, any other disulfide bond refolding operation may be performed in parallel to promote this process.

[0050]

The present invention can be preferably carried out in the following embodiments. In this section, the phrase “above” indicates that all of the embodiments with the preceding numbers of the corresponding contents can be alternatively selected. (1) A basic antibacterial protein A whose active condition is the formation of an intramolecular disulfide bond in a certain mode,
After expression in a prokaryotic cell as a protein fusion with a partner protein B having a chaperone function of less than 7 and collecting it, the antimicrobial protein A is activated by an activation step including utilization of the function of the partner protein B. A method for producing an antibacterial protein that can be converted into a mold. (2) In the above protein fusion, antibacterial protein A and partner protein B are chemically bonded. (3) In the above protein fusion, the antibacterial protein A and the partner protein B are constituted as a series of polypeptide chains via an oligopeptide portion that can be cleaved by an enzyme. (4) In the protein fusion, part or all of the antibacterial protein A and the partner protein B are associated with each other by hydrophobic affinity or electrical properties. (5) The antimicrobial protein A is any one of plant-derived thionin, PR protein, lipid transfer protein, and ribosome-inactivating protein, or plant, insect, or human defensin. (6) The plant-derived thionin is derived from barley. (7) The plant-derived thionin comprises the amino acids at positions 10 to 54 in the amino acid sequence shown in SEQ ID NO: 1. (8) the isoelectric point of the partner protein B is pH 6 or less;
Especially pH 5.5 or less. (9) The chaperone function of the partner protein B is a refolding function that changes the intramolecular disulfide bond formation site of the protein to a correct state as an active form. (10) DNA in which the partner protein B is present on the base sequence downstream of PDI or plant-derived thionin
Is an acidic protein encoded by (11) The PDI is a PDI derived from Humicola insolens, more specifically a PDI consisting of amino acids 59 to 543 in the amino acid sequence shown in SEQ ID NO: 2. (12) The acidic protein is an acidic protein derived from barley, more specifically, the amino acid sequence of positions 61 to 61 in the amino acid sequence shown in SEQ ID NO: 1.
It is an acidic protein consisting of the amino acid at position 124. (13) The partner protein B is Tx or chaperonin. (14) The partner protein B comprises at least an acidic partner protein B1 having an isoelectric point of less than pH 7 and a chaperone partner protein B2 having at least a chaperone function. (15) The acidic partner protein B1 is a predetermined region at the carboxyl terminus of PDI derived from Humicola insolens. (16) The chaperone partner protein B2 is a PPI
It is. (17) A protein used for forming a protein fusion with the antimicrobial protein A, wherein the partner protein comprises the acidic partner protein B1 and the chaperone partner protein B2. (18) DN encoding any of the above protein fusions
A. (19) The DNA continuously encodes antibacterial protein A and partner protein B (or acidic partner protein B1 and chaperone partner protein B2) as a single structural gene. (20) The DNA is a single structural gene comprising an antimicrobial protein A, an intermediate oligopeptide portion cleavable by any means, a partner protein B (or an acidic partner protein B1 and a chaperone partner protein B).
2) are coded continuously. (21) The above-mentioned DNA is mediated by any intervening sequence between the antibacterial protein A and the partner protein B, or two or more of the antibacterial protein A, the acidic partner protein B1 and the chaperone partner protein B2, including the antibacterial protein A. Thus, they encode different structural genes. (22) The DNA has a base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. (23) the DNA has a nucleotide sequence that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and has an antibacterial protein A, a partner protein B, an acidic It encodes at least two of the partner protein B1 and the chaperone partner protein B2, including the antibacterial protein A. (24) A host cell into which the above DNA has been introduced so that it can be expressed. (25) The host cell is a prokaryotic cell. (26) A process for producing a protein fusion, wherein the host cell is cultured to collect any of the protein fusions. (27) The protein fusion is stored or distributed. (28) Production of an antimicrobial protein comprising a step of separating antimicrobial protein A from partner protein B in any of the above protein fusions and a step of converting antimicrobial protein A to an antimicrobial active form by the function of partner protein B Method. (29) The process of separating the antimicrobial protein A from the partner protein B involves an operation of cleaving a peptide bond at a boundary between the two (or an oligopeptide portion for cleavage interposed at the boundary). (30) The process of converting the antimicrobial protein A into an antimicrobial active form involves a usual disulfide bond refolding operation to accelerate the process.

[0051]

EXAMPLES Example 1 Thionine-acidic protein fusion
Construction of a gene ) A gene encoding a fusion of barley-derived thionin and an acidic protein encoded by DNA present downstream of the base sequence with respect to barley-derived thionin, and a plasmid manufactured by Novagen Cloning was carried out by inserting between restriction sites of Nde1-BamH1 in pET-19b.

The barley-derived thionin gene has the nucleotide sequence at positions 28 to 162 in SEQ ID NO: 1, and the acid protein gene has the nucleotide sequence at positions 181 to 372 in SEQ ID NO: 1. In SEQ ID NO: 1, the nucleotide sequence of CATATG at positions 7 to 12 is a region cleaved by the restriction enzyme Nde1, and GGATCC at positions 378 to 383.
Is a region cut by the restriction enzyme BamH1. In the amino acid sequence described together, positions 5 to 8 and 5
The amino acid sequence of Thr-Glu-Gly-Arg from position 6 to position 59 is:
This is the recognition site for Factor Xa, a protease.

Example 2 Inheritance of Thionine-PDI Fusion
Construction of offspring ) A gene encoding a protein fusion between barley-derived thionin and Phumicola insolens-derived PDI was created and inserted between the restriction sites Ndel-BamH1 in the Novagen plasmid pET-19b. To perform cloning.

The barley-derived thionine gene has the nucleotide sequence at positions 25 to 159 in SEQ ID NO: 2. The PDI gene is located at position 175 to position 1 in SEQ ID NO: 2.
It has the nucleotide sequence at position 629. In SEQ ID NO: 2, the nucleotide sequence of CATATG at positions 7 to 12 is a cleavage region by the restriction enzyme Ndel, and GGAT at positions 1635 to 1640.
The base sequence of CC is a region cut by the restriction enzyme BamH1.
On the other hand, in the amino acid sequences described together, the amino acid sequences of Thr-Glu-Gly-Arg at positions 5 to 8 and positions 55 to 58 are recognition sites for Factor Xa.

Comparative Example 1: Construction of a Thionine Gene Cloning was performed by inserting a gene encoding thionin derived from barley between the cleavage sites of the restriction enzyme Ndel-BamH1 in plasmid pET-19b manufactured by Novagen.

The barley-derived thionine gene has the nucleotide sequence at positions 28 to 162 in SEQ ID NO: 3. In SEQ ID NO: 3, the nucleotide sequence of CATATG at positions 7 to 12 is a cleavage region by restriction enzyme Ndel, and the nucleotide sequence of GGATCC at positions 168 to 173 is a cleavage region by restriction enzyme BamH1. On the other hand, in the amino acid sequence
The amino acid sequence of Thr-Glu-Gly-Arg at positions 5 to 8 is F
It is the recognition site of actor Xa.

( Comparative Example 2: Thionine-PDI acidic region melting)
Construction of union gene ) PD derived from Humicola insolens
At the carboxyl terminus of I, there is an acidic region rich in acidic amino acids. A gene encoding a protein fusion between barley-derived thionin and the acidic region was created, and
Restriction enzyme Ndel in ovagen plasmid pET-19b
-Cloning was performed by inserting between the BamH1 cleavage sites.

Since the acidic region does not contain an active site of PDI, it is considered that the acidic region does not have a catalytic action of refolding a disulfide bond.

The barley-derived thionine gene has the nucleotide sequence at positions 25 to 159 in SEQ ID NO: 4, and the gene in the acidic region has the nucleotide sequence at positions 175 to 264. In SEQ ID NO: 4, CATA from position 7 to position 12
The base sequence of TG is a cleavage region by restriction enzyme Ndel, and the base sequence of GGATCC at positions 270 to 275 is restriction enzyme Ba.
This is a region cut by mH1. In the amino acid sequence shown, Thr-G at positions 5 to 8 and positions 35 to 38
The amino acid sequence of lu-Gly-Arg is the recognition site for Factor Xa.

Example 1 ( Expression of Gene in Escherichia coli )
Escherichia coli BL21 (DE3) pLysS was transformed using the vectors constructed in Example 2, Comparative Example 1 and Comparative Example 2, respectively. Each of the obtained transformants was placed in an LB medium (bact
o-tryptone 1%, bacto-yeast extract 0.5%, NaCl
(1%) overnight at 37 ° C.
% Inoculated. Then, the cells were cultured at 37 ° C. until OD = 0.5, IPTG (isopropylthio-β-D-galactoside) was added to a final concentration of 1 mM, and induced expression was performed for 6 hours.

Thereafter, the culture solution is collected by centrifugation,
A Sonication buffer 10 times the wet cell weight was added and suspended. After disrupting the cells by ultrasonic waves, 15000
After centrifugation at 30 rpm for 30 minutes, the supernatant was separated into the soluble fraction,
The precipitate was the insoluble fraction.

As a result of subjecting them to SDS-PAGE,
The thionin according to Comparative Example 1 was not expressed, but the protein fusions according to other Examples and Comparative Examples were all expressed in the insoluble fraction.

( Recovery of Protein Fusion) The insoluble fractions of the protein fusions according to Examples 1, 2 and Comparative Example 2 in which the above expression was observed, and the absence of the vector as a control when measuring the antibacterial activity. The insoluble fraction of the E. coli BL21 (DE3) pLysS culture was combined with 0.5% TritonX-100 / 1 mMED
After washing twice with a TA solution, a urea solution (8 M urea, 50 mM
Tris-HCl (pH 8.0, 1 mM DTT, 1 mM EDTA) was added for solubilization. After centrifugation, the supernatant was transferred to a dialysis tube, and dialyzed against a 4 M urea solution at 4 ° C for 1 hour. It is considered that the disulfide bond of thionine was broken during dialysis with the urea solution containing DTT.

Thereafter, the dialysate solution was subjected to a 2M urea solution and a buffer (50 mM Tris-HCl pH
8.0, 100 mM NaCl and 1 mM calcium chloride), and dialyzed overnight against the latter buffer. At the time of this dialysis, it is considered that the disulfide bond of thionin has been refolded. After completion of the dialysis, the mixture was centrifuged and the supernatant was used for cutting by Factor Xa described later.

During the above operation, in the protein fusion of thionin and PDI or the protein fusion of thionin and acidic protein (Example 1 or Example 2), in addition to the refolding effect of thionin by dialysis itself, Improvement of refolding efficiency by PDI or acidic protein can be expected.

( Cleavage of Protein Fusion by Protease
The protein fusions according to Example 1, Example 2 and Comparative Example 2 subjected to dialysis as described above were, as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, respectively, thionine and its partner. Between the protein and the protease
Holds Factor Xa recognition sequence. Therefore, 1 mg of each of the above protein fusions was subjected to Factor Xa cleavage at 30 ° C. overnight using 30 μg of Factor Xa.

( Measurement of Antibacterial Activity ) 30 μL of the protein solution obtained by the above-mentioned cleavage operation was applied using Microcon-3 (MWCO: 3000) which is a concentration unit using an ultrafiltration membrane.
Concentrated. 30 μL of such many concentrated sample solutions
Was injected into each well of a 96-well microplate, and 200 mM MES (2- (N
-morpholino) ethanesulfonic acid) buffer 10μL,
Sweet potato black spot fungus (Ceratocystis fimbriata: IFO3050
1) Conidiospore suspension (100,000: 8 per mL of spores)
60% of 0% potato dextrose broth)
The cells were cultured at 6 ° C. Separately, as a control, the same operation was performed for the insoluble fraction of the above-described vector-free E. coli culture.

Then, absorbance (415 nm) after 30 minutes and 48 hours using a Model 3550 microplate reader.
Was measured. The growth inhibition rate was determined by the following equation 1. In addition, in Formula 1, A represents a value obtained by subtracting the measured value of the absorbance after 30 minutes from the measured value of the absorbance after 48 hours in the sample solution, and B represents the value after 48 hours in the solution containing no sample. It represents a value obtained by subtracting the measured value of the absorbance after 30 minutes from the measured value of the absorbance.

Growth Inhibition Rate (%) = (BA) × 100 / B Equation 1 As a result, the control growth inhibition rate was 9.6%. Comparative Example 2 has a growth inhibition rate of 11.5%,
Substantially no antibacterial activity was observed. Example 1 has a growth inhibition rate of 99.8%, and Example 2 has a growth inhibition rate of 99.1%.
%, Indicating high antibacterial activity. The above difference between Comparative Example 2 and Examples 1 and 2 can be attributed to the presence or absence of the effect of improving the refolding efficiency by the partner protein.

[0070]

[Sequence list] SEQUENCE LISTING <110> KABUSHIKI KAISHA TOYOTA CHUO KENKYUSHO <120> Antibacterial protein production method, protein fusion <130> POK-01-029 <160> 4 <210> 1 <211> 392 <212> DNA <213> Hordeum vulgare <400> 1 gac aag cat atg att gaa ggt cgt atg aaa agc tgc tgc cgt agc acc 48 Asp Lys His Met Ile Glu Gly Arg Met Lys Ser Cys Cys Arg Ser Thr 5 10 15 ctg ggt cgt aac tgc tat aac ctg tgc cgt gtt cgt ggt gcg cag aaa 96 Leu Gly Arg Asn Cys Tyr Asn Leu Cys Arg Val Arg Gly Ala Gln Lys 20 25 30 ctg tgc gcg ggt gtt tgc cgt tgc aaa ctg acc agc agc gg tgc Cyg Ala Gly Val Cys Arg Cys Lys Leu Thr Ser Ser Gly Lys Cys 35 40 45 ccg acc ggt ttt ccg aaa atg att gaa ggt cgt acg ctg gcg ctg gtt 192 Pro Thr Gly Phe Pro Lys Met Ile Glu Gly Arg Thr Leu Ala Leu Val 50 55 60 agc aac agc gat gaa ccg gat acc gtt aaa tat tgc aac ctg ggt tgc 240 Ser Asn Ser Asp Glu Pro Asp Thr Val Lys Tyr Cys Asn Leu Gly Cys 65 70 75 80 cgt gcg agc atg tgc gat tat atg gtt aac gcg gc g gcg gat gat gaa 288 Arg Ala Ser Met Cys Asp Tyr Met Val Asn Ala Ala Ala Asp Asp Glu 85 90 95 gaa atg aaa ctg tat ctg gaa aac tgc ggt gat gcg tgc gtt aac ttt 336 Glu Met Lys Luu Tyr Cys Gly Asp Ala Cys Val Asn Phe 100 105 110 tgc aac ggt gat gcg ggt ctg acc agc ctg acc gcg tga tag gat ccg 384 Cys Asn Gly Asp Ala Gly Leu Thr Ser Leu Thr Ala *** *** Asp Pro 115 120 125 gct gct aa 392 Ala Ala 130 <210> 2 <211> 1649 <212> DNA <213> Hordeum vulgare <400> 2 gac aag cat atg att gaa ggt cgt aaa agc tgc tgc cgt agc acc ctg 48 Asp Lys His Met Ile Glu Gly Arg Lys Ser Cys Cys Arg Ser Thr Leu 5 10 15 ggt cgt aac tgc tat aac ctg tgc cgt gtt cgt ggt gcg cag aaa ctg 96 Gly Arg Asn Cys Tyr Asn Leu Cys Arg Val Arg Gly Ala Gln Lys Leu 20 25 30 tgc gcg ggt gtt tgc cgt tgc aaa ctg acc agc agc ggt aaa tgc ccg 144 Cys Ala Gly Val Cys Arg Cys Lys Leu Thr Ser Ser Gly Lys Cys Pro 35 40 45 acc ggt ttt ccg aaa atg att gaa ggt cgt tcgat gtc cag ctg 192 Thr Gly Phe Pro Lys Met Ile Glu Gly Arg Ser Asp Val Val Gln Leu 50 55 60 aag aag gac acc ttc gac gac ttc atc aag acg aat gac ctt gtt ctc 240 Lys Lys Asp Thr Phe Asp Asp Phe Ile Lys Thr Asn Asp Leu Val Leu 65 70 75 80 gcc gaa ttc ttc gcg ccg tgg tgc ggt cac tgc aag gct ctc gcc ccc 288 Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala Pro 85 90 95 gag tac gag gag gct gcg acc aca ctg aag gag aag aac atc aag Tyr 336 Glu Glu Ala Ala Thr Thr Leu Lys Glu Lys Asn Ile Lys Leu 100 105 110 gcc aag gtg gac tgc aca gag gag acg gac ctc tgc caa caa cat ggt 384 Ala Lys Val Asp Cys Thr Glu Glu Thr Asp Leu Cys Gln Gln His Gly 115 120 125 gtt gag ggc tac ccg act ctc aag gtc ttc cgc ggc ctt gac aac gtc 432 Val Glu Gly Tyr Pro Thr Leu Lys Val Phe Arg Gly Leu Asp Asn Val 130 135 140 tcc ccc tac aag ggc cag cgc aag gct gct atc acc tcg tac atg 480 Ser Pro Tyr Lys Gly Gln Arg Lys Ala Ala Ala Ile Thr Ser Tyr Met 145 150 155 160 atc aag cag tct ctg ccc gcc gtg tcc gag gtc acg aag gac aac ctg 528 Ile Lys Gln Ser Leu Pro Ala Val Ser G lu Val Thr Lys Asp Asn Leu 165 170 175 gag gag ttc aag aag gcc gac aag gcc gtc ctt gtc gcc tat gtg gat 576 Glu Glu Phe Lys Lys Ala Asp Lys Ala Val Leu Val Ala Tyr Val Asp 180 185 190 gct tcc gac aag gcg tcc agt gag gtt ttc acc cag gtc gcc gag aag 624 Ala Ser Asp Lys Ala Ser Ser Glu Val Phe Thr Gln Val Ala Glu Lys 195 200 205 ctg cgc gac aac tac ccg ttc ggc tcc agc agc gat gct gcg ctg Arg Asp Asn Tyr Pro Phe Gly Ser Ser Ser Asp Ala Ala Leu Ala 210 215 220 gag gct gag ggc gtc aag gct ccc gct atc gtc ctt tac aag gac ttt 720 Glu Ala Glu Gly Val Lys Ala Pro Ala Ile Val Leu Tyr Lys Asp Phe 225 230 235 240 gat gag ggc aag gcg gtc ttc tcc gag aag ttc gag gtg gag gcg atc 768 Asp Glu Gly Lys Ala Val Phe Ser Glu Lys Phe Glu Val Glu Ala Ile 245 250 255 gag aag ttc gcc aag accg gg ccg ctc att ggc gag att ggc 816 Glu Lys Phe Ala Lys Thr Gly Ala Thr Pro Leu Ile Gly Glu Ile Gly 260 265 270 ccc gaa acc tac tcc gac tac atg tcg gcc ggc atc cct ctg gcc tac 864 Pro Gp Thr Ser T yr Met Ser Ala Gly Ile Pro Leu Ala Tyr 275 280 285 att ttc gcc gaa acg gcc gag gag cgg aag gag ctc agc gac aag ctc 912 Ile Phe Ala Glu Thr Ala Glu Glu Arg Lys Glu Leu Ser Asp Lys Leu ag 290 295 ccg atc gcc gag gct cag cgc ggc gtc att aac ttt ggt act att 960 Lys Pro Ile Ala Glu Ala Gln Arg Gly Val Ile Asn Phe Gly Thr Ile 305 310 315 320 gac gcc aag gct ttt ggt gcc cac gcc ggc aac ctg ac aag acc 1008 Asp Ala Lys Ala Phe Gly Ala His Ala Gly Asn Leu Asn Leu Lys Thr 325 330 335 gac aag ttc ccc gcc ttc gcc atc cag gag gtc gcc aag aac cag aag 1056 Asp Lys Phe Pro Ala Phe Ala Ile Gln Glu Ala Lys Asn Gln Lys 340 345 350 ttc ccc ttc gat cag gag aag gag atc acc ttc gag gcg atc aag gct 1104 Phe Pro Phe Asp Gln Glu Lys Glu Ile Thr Phe Glu Ala Ile Lys Ala 355 360 365 ttc gtc gc gac t gcc ggt aag atc gag ccc agc atc aag tcg 1152 Phe Val Asp Asp Phe Val Ala Gly Lys Ile Glu Pro Ser Ile Lys Ser 370 375 380 gag ccg atc cct gag aag cag gag ggc ccc gtc acc gtc gtc gtt gcc 1200 Glu Ile Pro Glu Lys Gln Glu Gly Pro Val Thr Val Val Val Ala 385 390 395 400 aag aac tac aat gag atc gtc ctg gac gac acc aag gat gtg ctg att 1248 Lys Asn Tyr Asn Glu Ile Val Leu Asp Asp Thr Lys Asp Val Leu Ile 405 410 415 gag ttc tac gcc ccg tgg tgc ggc cac tgc aag gcc ctg gct ccc aag 1296 Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala Pro Lys 420 425 430 tac gag gag ctc ggc gcc ag cct agc gag ttc aag gac cgg 1344 Tyr Glu Glu Leu Gly Ala Leu Tyr Ala Lys Ser Glu Phe Lys Asp Arg 435 440 445 gtc gtc atc gcc aag gtt gat gcc acg gcc aac gac gtt ccc gat gag 1392 Val Val Asle Ala Lys Ala Thr Ala Asn Asp Val Pro Asp Glu 450 455 460 atc cag gga ttc ccc acc atc aag ctg tac ccg gcc ggt gcc aag ggt 1440 Ile Gln Gly Phe Pro Thr Ile Lys Leu Tyr Pro Ala Gly Ala Lys Gly 465 470 470 475 480 cag ccc gtc acc tac tct ggc tcg cgc act gtc gag gac ctc atc aag 1488 Gln Pro Val Thr Tyr Ser Gly Ser Arg Thr Val Glu Asp Leu Ile Lys 485 490 495 ttc atc gcc gag aac ggc aag tac aag gcc gcc atc tcg g gat gcc 1536 Phe Ile Ala Glu Asn Gly Lys Tyr Lys Ala Ala Ile Ser Glu Asp Ala 500 505 510 gag gag acg tcg tcc gca acc gag acg acc acc gag acg gcc acc aag 1584 Glu Glu Thr Ser Ser Ala Thr Glu Thr Thr Thr Glu Thr Ala Thr Lys 515 520 525 tcg gag gag gct gcc aag gag acg gcg acg gag cac gac gag ctc tga 1632 Ser Glu Glu Ala Ala Lys Glu Thr Ala Thr Glu His Asp Glu Leu *** 530 535 540 540 543 tag gat ccg gct gct aa 1649 *** Asp Pro Ala Ala 545 549 <210> 3 <211> 182 <212> DNA <213> Hordeum vulgare <400> 3 gac aag cat atg att gaa ggt cgt atg aaa agc tgc tgc cgt agc acc 48 Asp Lys His Met Ile Glu Gly Arg Met Lys Ser Cys Cys Arg Ser Thr 5 10 15 ctg ggt cgt aac tgc tat aac ctg tgc cgt gtt cgt ggt gcg cag aaa 96 Leu Gly Arg Asn Cys Tyr Asn Leu Cys Arg Val Arg Gly Ala Gln Lys 20 25 30 ctg tgc gcg ggt gtt tgc cgt tgc aaa ctg acc agc agc ggt aaa tgc 144 Leu Cys Ala Gly Val Cys Arg Cys Lys Leu Thr Ser Ser Gly Lys Cys 35 40 45 ccg acc ggt ttt ccg aaa t tag gat ccg gct gct aa 182 Pro Thr Gly Phe Pro Lys ** * *** Asp Pro Ala Ala 50 55 60 <210> 4 <211> 284 <212> DNA <213> Hordeum vulgare <400> 4 gac aag cat atg att gaa ggt cgt aaa agc tgc tgc cgt agc acc ctg 48 Asp Lys His Met Ile Glu Gly Arg Lys Ser Cys Cys Arg Ser Thr Leu 5 10 15 ggt cgt aac tgc tat aac ctg tgc cgt gtt cgt ggt gcg cag aaa ctg 96 Gly Arg Asn Cys Tyr Asn Leu Cys Arg Val Arg Gly Ala Gln Lys Leu 20 25 30 tgc gcg ggt gtt tgc cgt tgc aaa ctg acc agc agc ggt aaa tgc ccg 144 Cys Ala Gly Val Cys Arg Cys Lys Leu Thr Ser Ser Gly Lys Cys Pro 35 40 45 acc ggt ttt ccg aaa atg att gaa ggtt gag acg tcg tcc gca acc 192 Thr Gly Phe Pro Lys Met Ile Glu Gly Arg Glu Thr Ser Ser Ala Thr 50 55 60 gag acg acc acc gag acg gcc acc aag tcg gag gag gct gcc aag gag 240 Glu Thr Thr Thr Glu Thr Ala Thr Lys Ser Glu Glu Ala Ala Lys Glu 65 70 75 80 acg gcg acg gag cac gac gag ctc tga tag gat ccg gct gct aa 284 Thr Ala Thr Glu His Asp Glu Leu *** *** Asp Pro Ala Ala 85 90 94

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Masanori Hirai 41-Yokomichi, Nagakute-cho, Aichi-gun, Aichi Prefecture Inside Toyota Central Research Institute, Inc. No. 41, Yokomichi, Toyota Central Research Institute Co., Ltd. (72) Inventor Katsunori Koda, 41-Cho, Yokomichi, Nagakute-cho, Aichi-gun, Aichi, Japan 1F, 41-Chome, Toyota Central R & D Labs. AC14 BD45 CA41 CA44 4H045 AA10 AA20 BA10 BA41 CA10 CA30 EA01 EA29 FA16 FA74 GA06

Claims (11)

[Claims] The present invention relates to a basic antibacterial protein A whose active condition is the formation of an intramolecular disulfide bond in a certain mode, as a protein fusion with a partner protein B having an isoelectric point of less than pH 7 and having a chaperone function. A method for producing an antibacterial protein, wherein the antibacterial protein is expressed in a prokaryotic cell, collected, and then converted to the active form by an activation step including utilization of the function of the partner protein B. 2. The method for producing an antibacterial protein according to claim 1, wherein the expression of the protein fusion is carried out by culturing a prokaryotic cell into which a DNA encoding the protein fusion has been introduced so as to be expressible. 3. The method according to claim 1, wherein the antibacterial protein A is selected from the group consisting of plant-derived thionin, PR protein, lipid transfer protein, and ribosome-inactivating protein.
Alternatively, the method for producing an antibacterial protein according to claim 1 or 2, wherein the method is a plant, insect or human-derived defensin. 4. The method according to claim 1, wherein the partner protein B is 1)
Or 1) or 2).
A method for producing the antibacterial protein according to claim 3. 1) DNA present on the base sequence downstream of protein disulfide isomerase or plant-derived thionin
Acidic protein encoded by. 2) Thioredoxin or chaperonin. 5. The acidic partner protein B1 wherein the partner protein B has at least an isoelectric point of less than pH7.
And at least a chaperone partner protein B2 having a chaperone function.
A method for producing the antibacterial protein according to any one of claims 1 to 3. 6. A fusion product of a basic antibacterial protein A whose active condition is the formation of an intramolecular disulfide bond in a certain mode and a partner protein B having an isoelectric point of less than pH 7 and having a chaperone function. A protein fusion, wherein the antibacterial protein A and the partner protein B are constituted as a series of polypeptide chains via an oligopeptide portion which can be cleaved by an enzyme. 7. A basic antimicrobial protein A whose active condition is the formation of an intramolecular disulfide bond in a certain mode, an acidic partner protein B1 having at least an isoelectric point of less than pH 7, and a chaperone having at least a chaperone function. A protein fusion, which is a fusion with partner protein B2. 8. The method according to claim 8, wherein the acidic partner protein B1 is a carboxy terminal region of protein disulfide isomerase derived from Fumicola insolens, and the chaperone partner protein B2 is peptidylprolyl-cis-trans-isomerase. The protein fusion according to claim 7, wherein 9. A protein used for forming a protein fusion with a basic antibacterial protein A whose active condition is the formation of an intramolecular disulfide bond in a certain mode, wherein the protein has at least an isoelectric point of less than pH7. A partner protein comprising at least the acidic partner protein B1 and a chaperone partner protein B2 having at least a chaperone function. 10. A DNA encoding the protein fusion according to any one of claims 6 to 8. 11. A host cell, which is a prokaryotic cell into which the DNA according to claim 10 has been introduced so that it can be expressed.