JP4779103B2 - Method for producing oxidized polypeptide in which sulfur-containing amino acid residue is oxidized - Google Patents

Description

The present invention relates to a method for producing an oxidized polypeptide in which a sulfur-containing amino acid residue is oxidized. Specifically, DJ-1 in which a cysteine residue corresponding to position 106 in human DJ-1 is oxidized to cysteine sulfonic acid or The present invention relates to a method for producing the modified product.

  DJ-1 was discovered as an oncogene that transforms NIH3T3 cells (Non-patent Document 1).

Moreover, PARK7 was identified as a causative gene of Parkinson's disease, and it was found that the gene was identical to a DJ-1 point mutation (the 166th leucine was mutated to proline) (Non-patent Document 2).

  Furthermore, it is suggested that DJ-1 is caused by oxidation of cysteine from the change in spot position due to the shift of the spot on two-dimensional electrophoresis due to oxidative stress caused by hydrogen peroxide and paraquat (Non-patent Document 3).

Furthermore, Non-Patent Document 4 is a paper on X-ray crystal structure analysis of DJ-1, which shows that a change in the dimer structure occurs due to the introduction of the L166P mutation related to Parkinson's disease, and that the 106th cysteine is caused by X-ray irradiation. Is oxidized to sulfinic acid (SO ₂ H). Patent Document 1 discloses oxidized human DJ-1 in which the cysteine residue at the 106th position is oxidized to finic acid or cysteine sulfonic acid that is cysteine.
WO2005 / 75513 Nagakubo D, TairaT, Kitaura H, Ikeda M, TamaiK, Iguchi-Ariga SM, ArigaH.J-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras.Biochem Biophys Res Commun. (1997) 231, 509-13 Bonifati V, RizzuP, van Baren MJ, Schaap O, Breedveld GJ, Krieger E, DekkerMC, Squitieri F, Ibanez P, JoosseM, van Dongen JW, VanacoreN, van Swieten JC, Brice A, MecoG, van Duijn CM, Oostra BA, Heutink P. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism.Science. (2003) 299, 256-9. Mitsumoto A, Nakagawa Y, Takeuchi A, Okawa K, Iwamatsu A, Takanezawa Y. Oxidized forms of peroxiredoxinsand DJ-1 on two-dimensional gels increased in response to sublethallevels of paraquat.Free RadicRes. (2001) 35, 301-10. Wilson MA, Collins JL, Hod Y, Ringe D, Petsko GA.The 1.1-A resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease.Proc Natl Acad SciU S A. (2003) 100, 9256-61.

  An object of the present invention is to provide a method for producing an oxidized polypeptide in which a sulfur-containing amino acid residue is oxidized.

In order to obtain oxidized DJ-1 as an example of an oxidized polypeptide, the present inventor first purifies human recombinant DJ-1 and adds hydrogen peroxide thereto to oxidize the cysteine residue at position 106. Tried method. However, this method hardly yielded DJ-1 in which the cysteine residue at position 106 was oxidized.

Therefore, as a result of various investigations on the oxidation conditions of the cysteine residue at position 106 of DJ-1 to cysteine sulfonic acid, surprisingly, by treating a culture in which a host having recombinant DJ-1 was cultured with an oxidizing agent, It was found that DJ-1 in which the cysteine residue at position 106 was oxidized was easily obtained. This method is not limited to DJ-1, but can be similarly applied to polypeptides having cysteine or methionine, and it has been found that an oxidized polypeptide can be obtained in good yield.

That is, the present invention relates to the following method for producing an oxidized polypeptide.
1. A polypeptide containing a sulfur-containing amino acid selected from the group consisting of cysteine and methionine is oxidized to contain at least one oxidized sulfur-containing amino acid selected from the group consisting of cysteine sulfonic acid or a salt thereof and methionine sulfoxide A method for producing an oxidized polypeptide, characterized by treating a culture of a transformed host into which a gene encoding the polypeptide has been introduced with an oxidizing agent, and recovering the oxidized polypeptide. A method for producing a polypeptide.
2. Oxidized DJ-1 in which the polypeptide containing sulfur-containing amino acid is DJ-1 or a variant thereof, and the oxidized polypeptide has a cysteine residue corresponding to position 106 of DJ-1 oxidized to a cysteine sulfonic acid residue Item 2. The method according to Item 1, which is a variant thereof.
3. Item 3. The method according to Item 1 or 2, wherein the oxidizing agent is hydrogen peroxide.
4). Item 3. The method according to Item 2, wherein DJ-1 is human DJ-1.
5. Item 5. The method according to any one of Items 1 to 4, wherein the transformed host is Escherichia coli transformed with an expression vector having a DNA encoding the polypeptide.
6). Item 6. The method according to any one of Items 1 to 5, wherein the concentration of the oxidizing agent is 0.5 to 500 mM.

  DJ-1 and peroxiredoxin are known as proteins that are oxidized under oxidative stress, but no monoclonal antibodies against oxidized proteins have been obtained so far. For peroxiredoxins, polyclonal antibodies that react with oxidized proteins have been obtained, but have low specificity and are not very reactive. Conventionally, it is a conventional method to synthesize a peptide at a site to be oxidized and use it as an immunogen. However, antibodies that react with peptides can be obtained, but in most cases, the reactivity of intact proteins that actually exist is reduced. In fact, when an antibody against oxidized DJ-1 was prepared, an oxidized peptide was synthesized and examined, but an antibody that reacts with the peptide was obtained, but an antibody that reacts with an intact protein was not obtained.

  There is a possibility that the oxidized polypeptide is produced in vivo, and a preparation of the oxidized polypeptide or an antibody against the oxidized polypeptide can be easily prepared by the method of the present invention. . Oxidized polypeptide can be an indicator of oxidative stress in vivo.

  For example, oxidized DJ-1 is known to be a marker for diseases associated with oxidative stress such as Parkinson's disease, Alzheimer's disease, and arteriosclerosis, and is useful as an antigen to produce antibodies against oxidized DJ-1 It is.

  By using the oxidized polypeptide obtained by the method of the present invention, an oxidized protein produced in vivo can be identified.

In the present invention, the polypeptide containing a sulfur-containing amino acid is widely exemplified by a polypeptide containing one or more cysteine residues and methionine residues having a free SH group, and is not particularly limited. Examples include peroxiredoxins such as peroxiredoxin 6 and various physiologically active polypeptides such as enzymes, hormones, receptors, and ligands. The polypeptide may be a natural type or a variant in which one or a plurality of amino acids other than a sulfur-containing amino acid that may be oxidized are substituted, added, deleted, or inserted.

  A polypeptide derived from a mammal is used. Examples of mammals include humans, monkeys, cows, horses, sheep, dogs, rats, mice, rabbits, and the like, preferably humans.

  The structures of oxidized methionine sulfoxide and cysteine sulfonic acid are shown below.

  Examples of salts of cysteine sulfonic acid residues include, but are not limited to, alkali metal (Na, K, Li, etc.) salts of sulfonic acid, alkaline earth metal (Ca, Mg, Ba, etc.) salts, and the like.

When a polypeptide contains two or more sulfur-containing amino acids, it can be oxidized from sulfur-containing amino acid residues that are easily oxidized, or only one residue can be oxidized, and two or more residues can be oxidized. The number of residues to be oxidized can be changed depending on the oxidation conditions (type of oxidizing agent, amount used, reaction time, pH, etc.).

  Examples of the oxidizing agent used in the present invention include peroxides such as hydrogen peroxide and cumene hydroperoxide, azo radical initiators, paraquat, and chloramine. Hydrogen peroxide is preferably used.

  In the present invention, a host is transformed with an expression vector in which a DNA encoding a polypeptide is incorporated so that it can be expressed, the transformant is cultured in a medium, and an oxidant is allowed to act on the resulting culture. Thus, the target oxidized DJ-1 or a derivative thereof can be obtained. The oxidizing agent may be added at the beginning of the culture of the transformant or during the culture, but preferably the oxidizing agent is allowed to act after the end of the culture. An oxidizing agent may be added directly to the culture, and the culture can be centrifuged, the host can be separated to remove the culture medium, and the oxidizing agent can be applied to the resulting host.

The concentration of the oxidizing agent added to the culture or the host is not particularly limited as long as the sulfur-containing amino acid of the polypeptide is oxidized. For example, when the oxidizing agent is hydrogen peroxide, about 0.5 to 500 mM, preferably Is about 0.5 to 50 mM. The processing time is exemplified by about 1 to 24 hours.

  Hosts that can be used for the preparation of the recombinant polypeptide include microorganisms such as Escherichia coli, Bacillus subtilis and yeast, mammalian cells such as human cells, insect cells, plant cells, etc., preferably Escherichia coli. For example, when Escherichia coli is used as a host, the polypeptide is expressed and accumulated in Escherichia coli.

  Although not wishing to be bound by theory, polypeptide expressed in a host by genetic recombination does not produce an oxide when purified outside the cell and after purification, or by-products and The inventor believes that some factor in the cell is necessary for the oxidation because the decomposition product is generated and the yield is greatly reduced. Thus, the polypeptide is expressed in the cell, not as a secreted protein. Further, an oxidized polypeptide in which the same sulfur-containing amino acid as that of the natural polypeptide is oxidized can be obtained by oxidizing the recombinant protein in the host.

  After treatment with the addition of an oxidizing agent, the desired oxidized polypeptide can be obtained according to conventional methods such as disruption of cells, filtration, gel filtration, centrifugation, chromatography, electrophoresis, affinity chromatography and the like.

  Hereinafter, human DJ-1 will be described as an example of a polypeptide containing a sulfur-containing amino acid, but an oxidized polypeptide can be obtained for a polypeptide other than DJ-1 in the same manner as DJ-1.

  The amino acid sequence of human DJ-1 is shown in SEQ ID NO: 1, and the DNA sequence is shown in SEQ ID NO: 2.

In oxidized human DJ-1, at least the cysteine residue at position 106 is oxidized, and the cysteine residue at position 46 or 53 may be oxidized. In the case of DJ-1 derived from mammals other than humans, the one in which the cysteine residue corresponding to position 106 of human DJ-1 is oxidized to cysteine sulfonic acid is referred to as oxidized DJ-1 or a modified form thereof. As long as the cysteine residue corresponding to position 106 of human DJ-1 is oxidized to cysteine sulfonic acid, other amino acid residues may be further oxidized.

The DJ-1 variant of the present invention has been modified so that the oxidized DJ-1 variant is recognized by an antibody against oxidized DJ-1, and the cysteine at position 106 is not modified, Other amino acids may be substituted, added, deleted or inserted to retain the conformation recognized by the antibody. For example, in the DJ-1 variant, the cysteine residue at position 46 and / or 53 may be substituted with another amino acid (eg, Ser).

DJ-1 is oxidized to a cysteine sulfonic acid by oxidizing the 106-position cysteine residue into an oxidized DJ-1 derivative. This oxidation of DJ-1 occurs under physiological conditions.

The oxidized DJ-1 obtained by the present invention or a variant thereof can be preferably used for the production of, for example, an antibody (monoclonal antibody, polyclonal antibody) that recognizes oxidized DJ-1, particularly a monoclonal antibody. For example, a peptide around the cysteine residue at position 106 is prepared, the cysteine residue corresponding to position 106 of the peptide is oxidized to cysteine sulfonic acid, and this oxidized peptide is administered according to a conventional method such as administration to an animal. Although an antibody that recognizes can be obtained, the antibody cannot sufficiently recognize oxidized DJ-1 produced by oxidative stress in the human body. On the other hand, if oxidized human DJ-1 obtained by the method of the present invention or a variant thereof is used, an antibody that recognizes oxidized human DJ-1 can be easily obtained. In addition, when evaluating the specificity of the obtained antibody for oxidized DJ-1, the oxidized DJ-1 of the present invention or a variant thereof can be preferably used. This makes it possible to develop an ELISA kit using oxidized DJ-1 antibody and a diagnostic agent for diseases associated with oxidative stress such as Parkinson's disease, Alzheimer's disease, and arteriosclerosis.

Furthermore, oxidized DJ-1 may have a role of transmitting a signal of oxidative stress in vivo, and oxidized DJ-1 obtained by the present invention or a variant thereof, particularly position 106 of human DJ-1 Oxidized human DJ-1, in which the cysteine residues of the enzyme are selectively oxidized to cysteine sulfonic acid, may be useful as a therapeutic agent for diseases related to oxidative stress, especially Parkinson's disease, Alzheimer's disease, arteriosclerosis, etc. is there.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.
Materials and methods Cells: E. coli transfected with a plasmid encoding human DJ-1 (native with histidine tag or double mutant with Cys46 and Cys53 replaced with Ser) was used. LB medium was used for the culture of E. coli.
Experimental method: Oxidized DJ-1 was prepared according to the following protocol.
To E. coli transfected with human DJ-1 (protein amount 70 μg / ml), 0 to 500 mM hydrogen peroxide was added and reacted at 37 ° C. for 15 minutes. After the reaction, Escherichia coli treated with an oxidizing agent was solubilized and added to a Ni-NTA column. The column was washed and eluted with imidazole, and the proteins contained in the eluted fraction were separated by SDS-PAGE. The separated protein was cut out from the gel and digested in gel with trypsin to obtain a peptide fragment. This peptide mixture was introduced into a nanospray ionization LC-MS / MS system, and peptides containing Cys106 were identified and quantified.
Example 1
Escherichia coli expressing double mutant human DJ-1 in which Cys46 and Cys53 were replaced with Ser was treated with 0 or 500 mM hydrogen peroxide and purified according to the above protocol (FIG. 1). The recognized main band was cut out and the peptide containing Cys106 was identified and quantified (FIG. 2). It was found that Cys106 was highly oxidized by treatment with 500 mM hydrogen peroxide.
Example 2
After reacting by adding 0, 50, or 500 mM hydrogen peroxide to E. coli expressing native DJ-1, purification was performed using the same protocol. A band corresponding to oxidized DJ-1 was cut out and subjected to LC-MS / MS analysis to identify and quantify peptides containing each Cys (FIG. 3). Cys106 was found to be highly oxidized even with 50 mM hydrogen peroxide.

Production and purification of oxidized DJ-1 LC-MS / MS analysis results of oxidized DJ-1 (double mutant) LC-MS / MS analysis results of oxidized DJ-1 (native)

Claims (2)

A polypeptide containing a sulfur-containing amino acid selected from the group consisting of cysteine and methionine is oxidized to contain at least one oxidized sulfur-containing amino acid selected from the group consisting of cysteine sulfonic acid or a salt thereof and methionine sulfoxide A method for producing an oxidized polypeptide, comprising a step of recovering an oxidized polypeptide by treating a culture of a transformed host into which the gene encoding the polypeptide has been introduced with an oxidizing agent, Is a human DJ-1 or a variant thereof, and the oxidized polypeptide is oxidized human DJ-1 in which a cysteine residue corresponding to position 106 of DJ-1 is oxidized to a cysteine sulfonic acid residue or A modified form thereof, wherein the oxidizing agent is hydrogen peroxide, and the transformed host is an expression vector carrying DNA encoding the polypeptide. A method for producing an oxidized polypeptide, which is transformed Escherichia coli. The method according to claim 1, wherein the concentration of the oxidizing agent is 0.5 to 500 mM.

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