JPH10507919A - Manufacture and use of human and plant methyltransferases - Google Patents

Abstract

  (57) [Summary] The isolated recombinant human L-isoaspartyl / D-aspartyl protein methyltransferase was obtained by overexpression of a cDNA encoding isozyme II in an E. coli strain, a cDNA clone of wheat enzyme and a purified enzyme of wheat. Is obtained. These enzymes are useful for treating medical conditions and diagnosing diseases associated with increased L-isoaspartyl / D-aspartyl residues of polypeptides in tissues.

Description

DETAILED DESCRIPTION OF THE INVENTION           Manufacture and use of human and plant methyltransferases                                   background Field of the invention   The present invention relates to the production and use of methyltransferase. Further Specifically, the present invention relates to recombinant human L-isoaspartyl / D-aspartyl protein. Methyltransferase, plant L-isoaspartyl protein methyltra And a purified plant L-isoaspa Related to the production, purification and use of rutile protein methyltransferase It is.Background of the Invention   Proteins gradually lose function due to spontaneous thermodynamic changes (Hardin g, JJ (1985) Adv. Protein Chem.37: 247-334). Normal of such damage Forms of non-enzymatic deamidation, isomerization of asparaginyl and aspartyl residues (Clarke et al. (1992) "Stability of Protein Drugs, Part A: Chemical and physical pathways of proteolysis ") p. 1-29, Plenum Press, New York). In most cells, L-isoaspartyl and D-aspa Proteins containing rutile residues catalyze the methyl esterification of the protein Protein-L-isoaspartate (D-aspartate) O-methyltrans Recognized by Ferase (EC 2.1.1.77) (McFadden et al. (1982) Pr oc. Natl. Acad. Sci. USA79: 2460-2464, Murray et al. Bio l. Chem.25910722-10732, and Aswad (1984) J. Biol. Chem.259: 1071 4-10721). Enzymatic methylation corrects these altered aspartyl residues. This corresponds to the first step in the process of conversion to a normal L-aspartyl residue (McFadden et al. ( 1987) Proc. Natl. Acad. Sci. USA84: 2595-2599, Johnson et al. (1987) J. Bio1. Chem.262: 5622-5629, Lowenson et al. Biol. Chem.26 6 : 19396-19406, Lowenson et al. (1992) J. Biol. Chem.267: 5985-5995). This reaction isomerization and Prevents the accumulation of proteins containing racemized aspartyl residues, and It appears to be an important component that limits the negative effects of the aging process. In bacteria, Because the deletion mutation reduces survival by a factor of 100, this enzyme is Has been shown to be crucial for the survival of mice (Li et al. (1992) Proc. Natl. Acad. Sci USA89: 9885-9889).   L-isoaspartyl / D-aspartyl methyltransferase is a mammal Extensively studied in organizations. Two similar activators were found in bovine brain (Aswad et al. (1983 Year) J. Neurochem.40: 1718-1728) and human erythrocytes (Gilbert et al. (1988) Bi). ochemistry27: 5227-5233, Ota et al. (1988) Biochem. Biophys. Res. Co mmun.151: 1136-1143). These isozymes have about 25,000 Da monomeric polypeptide and have similar catalytic properties, but with isoelectric point Differ by about 1 pH unit (Ota et al. (1988) Biochem. Biophys. Res. . Commun.151: 1136-1143). A third with an isoelectric point between isozymes I and II Active, but the corresponding isozyme has not been isolated or characterized (Ingrosso et al. (1991) Adv. Exper. Med. Biol.307: 263-276). Bovine brain ( (1989) J. Henzel et al. Biol. Chem.264: 15905-15911) and human erythrocytes ( Ingrosso et al. (1989) J. Biol. Chem.264: 20131-21039) Complete amino acid sequence of Zyme I and isozyme II from human erythrocytes Most of the amino acid sequence (Ingrosso et al. (1991) Biochem. Biophys. Res. Commu n.175: 351-358) have been determined. The sequence of human isozyme I consists of bovine enzyme and 96 % Are identical. The amino acid sequences of the two human isozymes are the two amino acids at the C-terminus. Identical except for the acid, and these two amino acids explain the difference in isoelectric point (Ingrosso et al. (1991) Biochem. Biophys. Res. Commun.175 : 351-358).   Southern blot analysis shows that both isozymes are products of a single gene (Ingrosso et al. (1991) Biochem. Biophys. Res. Commun.175: 351〜 358). Encodes an enzyme similar to isozyme I in rat brain and mouse testis The nucleotide sequence has recently been determined (Sato et al. (1989) Biochem. Biophys). . Res. Commun.161: 342-347, Romanik et al. (1992) Gene118: 217-222).   Human L-isoaspartyl / D-aspartyl protein carboxyl methyl ester Corresponding to mRNA of two isozymes of transferase (EC 2.1.1.77) Two cDNA clones (MacLaren et al. (1992) Biochem. Biophys. Res. . Commun.185, 277-283) have been sequenced. One of these (p The DNA sequence of RK1) is the amino acid sequence of the C-terminal half of human erythrocyte isozyme I. Code the column. Other cDNA clones (pDM2) are more acidic Contains the complete coding region for ImII. Potential polymorphisms at amino acid residues 119 and 205 With the exception of the site, the deduced amino acid sequence differs only at the C-terminus, The -RWK sequence is replaced by the -RDEL sequence in Isozyme II. The latter arrangement The columns are identical to mammalian ER retention signals. Deletion of coding region of pRK1 The minutes appear to be comparable to the coding region of pDM2. The existence of another splicing The presence indicates the presence of a third isozyme having a -RC terminal. Finally Evidence was presented that there are three genomic polymorphisms in the human gene for this enzyme. ing. Residue 22 is either isoleucine (I) or leucine (L). Residue 119 can be either isoleucine or valine (V) And residue 205 is either lysine (K) or arginine (R). (Tsai and Clarke (1994) Biochem. Biophys. Res. Commun.203: 491-497). Thus, each isozyme described above has at least eight forms (I_{twenty two} I₁₁₉K₂₀₅, I_{twenty two}I₁₁₉R₂₀₅, I_{twenty two}V₁₁₉K₂₀₅, I_{twenty two}V₁₁₉R₂₀₅, L_{twenty two}I₁₁₉K_{Two 05} , L_{twenty two}I₁₁₉R₂₀₅, L_{twenty two}V₁₁₉K₂₀₅, L_{twenty two}V₁₁₉R₂₀₅) Can exist You.   However, in the above study, the coding for human methyltransferase was DNA was not efficiently expressed due to the lack of a suitable recombinant expression system. Scratch Thus, large quantities of homogeneous enzymes have not been isolated so far. Methyl trans Ferase is purified from bovine brain, where 0.58 kg of cerebral cortex 3.7 mg was obtained in 5 steps (Aswad et al. (1983) J. Neurochem.40: 1718-1726). Purification of the human enzyme was more difficult. That is, from 0.22 liters of blood in 5 steps Only about 0.1 mg of protein was obtained (Gilbert et al. (1988) Biochemistr). y 27 : 5227-5233). Therefore, large amounts of isoaspartyl methyltransferase No efficient purification system has ever been produced on an industrial scale.   In plants, protein L-isoaspartyl methyltransferase Activity is in both monocotyledonous and dicotyledonous plants and green algae, Chlamydomonas reinhard Identified in Chi (Chlamydomonas reinhardtii) (Mudgett et al. (1993) Biochemistry32: 11100-11111). Most plant organs have some enzyme activity Although present, their values can vary considerably. Interestingly, L-a The highest values of soaspartyl methyltransferase activity are found in seeds (Mu dgett et al. (1993) Biochemistry32: 11100-11111). In addition, foreign peptide groups Carboxymethylated protein in the seed soluble fraction in the absence of What is formed in vitro is that the methyl acceptor substrate (Trivedi et al. (1982) Eur. J. Bioch). em.128: 349-354 and Mudgett et al. (1993) Biochemistry32: 11100-11111) .   Isoaspartate value in protein is a measure of the damage value of this protein in use. Using isoaspartyl methyltransferase enzyme The method for measuring the isoaspartyl content of protein is given to Aswad. No. 5,273,886, the disclosure of which is incorporated by reference. And incorporated herein.                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the human L-isoaspartyl / D-aspartyl methyltrans of the present invention. 1 shows the construction of a ferase expression plasmid, pDM2x. 107 bp Kpnl-Narl flag Is removed from plasmid pDM2 and the ribosome binding site and Replace with a synthetic polylinker that has a citator site.   FIG. 2 shows the novel polylinker fragment used in the present invention. This port The relinker is a multiple cloning site (KpnI, KhoI, KbaI, BamHI, N heI, NcoI) and contains a strong prokaryotic ribosome binding site (Shine et al.) (1974) Proc. Natl. Acad. Sci. USA71: 1342-1346).   FIG. 3 shows the human L-isoaspartyl / D-aspartyl methyl trans of the present invention. The nucleotide and deduced amino acid sequences of the ferase expression plasmid, pDM2x are shown (SEQ ID NO: 8).   FIG. 4 shows human L-isoaspartyl / D-aspartyl in E. coli. Isopropyl β-D-thiogalactate on tyl methyltransferase production 9 shows the effect of pyranoside (IPTG).   FIG. 5 shows human methyltransferase in the pDM2x expression system of the present invention. E. coli growth percentage of total soluble protein obtained Spherase yield is shown as a function of time after IPTG induction.   FIG. 6 shows the optimization of the protamine sulfate precipitation step according to the invention.   FIG. 7 shows human L-isoprotein obtained from the clarified supernatant of protamine sulfate according to the present invention. Ammonium sulfate of aspartyl / D-aspartyl methyltransferase 4 shows the optimization of the precipitation fraction.   FIG. 8 shows human L-isoaspartyl / D-aspartyl methyl tra according to the present invention. Figure 4 shows the anion exchange column purification step of the transferase.   FIG. 9 shows purified human L-isoaspartyl / D-aspartylmethyl according to the present invention. Figure 4 shows the pH dependence of transferase concentration.   FIG. 10 shows the electrophoresis of the purified recombinant human methyltransferase of the present invention. Figure 2 shows an electrospray mass spectral analysis. (A) Dithiothreitol Part of spectrum of material purified using (B) 0.1 μM buffer in buffer A When using 15 mM β-mercaptoethanol instead of thiothreitol Part of spectrum of purified material.   FIG. 11 shows the purified human L-isoaspartyl / D-aspartyl methylt of the present invention. 3 shows an ultraviolet absorbance spectrum of transferrinase.   FIG. 12 shows wheat germ L-isoaspartyl methyltransfer according to the present invention. 2 shows purification of the enzyme. (A) DEAE-52 anion exchange cellulose treatment. (B) Reverse Ammonium sulfate gradient solubilization treatment. (C) Sephacryl S-200 gel filtration.   FIG. 13 shows L-isoaspartyl methyl sulfate obtained from wheat germ according to the present invention. Figure 5 shows polypeptide analysis of transferase purification.   FIG. 14 shows the insertion of the wheat germ methyltransferase cDNA used in the present invention. 1 shows the DNA sequencing strategy of the product.   FIG. 15 shows L-isoaspartyl methyltransfer obtained from wheat germ. Sequenced Peptide Fragment of Glycine and Its Predictions Obtained from pMBM1 The alignment of amino acid sequences is shown.                                 Summary of the Invention   One particular object of the present invention is to isolate by expression of a cDNA encoding the enzyme. Recombinant human L-isoaspartyl / D-aspartyl protein methyltransfer To provide a cellulase. Surprisingly, this expression system allows pure recombination It enables very efficient large-scale production of enzymes. The structure of the recombinase is N-terminal alanine residue not modified by post-translational modification by acetylation in enzymes Injury differs from the structure of the purified enzyme obtained from human erythrocytes. Thus, this yeast The element is suitable for use in human studies because there is no potential antigenic problem.   Another object of the present invention is to provide wheat L-isoaspartyl protein methyltrans. Identification of cDNA encoding ferase and purified plant L-I obtained from wheat The provision of soaspartyl protein methyltransferase. High level Methyltransferase is found in wheat, especially in seeds.   Still another object of the present invention is to provide an enzyme for treating a disease caused by protein degradation. To provide pharmaceutical preparations containing Analytical tools to control quality and diagnose disease states associated with protein degradation To provide. For other objects of the invention, reference is made to the following detailed description of the invention. This will be apparent to those of ordinary skill in the art.   That is, one gist of the present invention is that SEQ ID NO: 1 (isozyme I) and SEQ ID NO: 2 (Isozyme II) of a polynucleotide having a sequence selected from the group consisting of Isolated recombinant human L-isoaspartyl / D-aspartylta obtained by expression Protein methyltransferases, each of which is isozyme I Or the eight amino acid sequence of II,_{twenty two}I₁₁₉K₂₀₅, I_{twenty two}I₁₁₉R₂₀₅, I_{twenty two}V₁₁₉K₂₀₅, I_{twenty two}V₁₁₉R₂₀₅, L_{twenty two}I₁₁₉K₂₀₅, L_{twenty two}I₁₁₉R₂₀₅, L_{twenty two}V₁₁ 9K₂₀₅And L_{twenty two}V₁₁₉R₂₀₅Contains a total of 17 nucleotide sequences encoding You.   Another aspect of the present invention provides that SEQ ID NO: 3 (isozyme I) and SEQ ID NO: 4 ( Isolated recombinant human having an amino acid sequence selected from the group consisting of L-isoaspartyl / D-aspartyl protein methyltransferase And each contains the eight amino acid sequences described above.   Another aspect of the present invention has the coding sequence of the sequence shown in SEQ ID NO: 5. An isolated polynucleotide, which is a plant L-isoaspartyl protein Encodes a methyltransferase. ATG code of base numbers 117 to 199 This starts with an AGC codon and ends at the AGC codon before the TGA codon at positions 807-809. 690 base pairs in the sequence indicate the coding sequence for this polynucleotide.   Another aspect of the present invention relates to a polynucleotide having the sequence shown in SEQ ID NO: 5. Recombinant plant L-isoaspartyl protein obtained by expression of Transferase.   Another aspect of the present invention has the amino acid sequence shown in SEQ ID NO: 6 or 7. Purified plant L-isoaspartyl protein methyltolan obtained from wheat germ Spherase.   Another gist of the present invention is to provide human L-isoaspartyl / D-aspartyltan A method for producing a protein methyltransferase by a recombinant method,   Using oligonucleotides, human L-isoaspartyl protein methyl Plasmid pDM2 containing the entire coding region for transferase (Genebank By modifying a plasmid such as S37495). Works in efficient ribosome binding sites and bacterial and / or eukaryotic expression systems Providing a powerful translation initiator area that is designed to   The constructed vector was converted to isopropyl β-D-thiogalactopyranoside (IPT G) transfecting into a host having an inducible T7 polymerase gene, And   IPTG induces methyltransferase overexpression to induce methyltransferase To produce cellulase, It is a method including that.   This expression method should be advantageously applied to any variant of methyltransferase Can be. The enzyme is preferably BL21 (DE3) grown in LB broth. Obtained from overexpressed human cDNA in E. coli. More efficient and efficient An efficient expression is achieved in a more nutrient medium, such as a strong broth (Sambrook et al. (1989) "Molecular Cloning: Laboratory Manual", Cold Spring Harbor Laboratory) Which results in higher final cell densities as well as longer exponential A growth and methyltransferase production phase is obtained. In the present invention, finally, Human methyltransferase makes up about 10-30% of total bacterial soluble protein Resulting preparation.   Another aspect of the present invention is a lytic bacterium in which methyltransferase expression has occurred. Recombinantly produced human L-isoaspartyl / D-aspa present in the extract A method for purifying rutile protein methyltransferase, and The method is   Nucleotide precipitation, such as protamine sulfate or polyethyleneimine To remove the DNA present in the extract after removal of cell debris. ,   Precipitate methyltransferase with ammonium sulfate,   Dialysed to remove ammonium sulfate, and   Use anion exchange chromatography to convert methyl dialysate under new conditions. Purifying the transferase, Including.   Obtain about 50 mg of enzyme from 4 liters of bacterial culture based on this new purification method be able to. Successful purification of this enzyme on a single column means that the enzyme can be Weakly bind and isocratically with starting buffer without applying a salt gradient This is probably due to the finding of conditions that can elute. Complete with phosphate / EDTA buffer Best fractionation on a non-equilibrated DEAE-cellulose column Have been found.   Another aspect of the present invention is a plant L-isoaspartyltan obtained from wheat. A method for purifying protein methyltransferase, and the method comprises:   Obtain crude (crude) cytosol from raw wheat (raw wheat) germ,   The crude cytosol is fractionated by DEAE-cellulose chromatography,   Add ammonium sulfate to the collected active fractions in the presence of a protein carrier. ,   The resulting material was reverse ammonium sulfate gradient solubilized (reverse ammonium sulfate gr). adient solubilization), and   The collected active fractions were purified by gel filtration chromatography and Purify the transferase as a 28,000 Da monomeric species, Including.   Another aspect of the present invention relates to L-isoaspartyl / polypeptides in tissues. A method of treating a medical condition associated with an increase in D-aspartyl residues, and Can be used in tissues with or without S-adenosylmethionine. Converting the L-isoaspartyl / D-aspartyl residue to an L-aspartyl residue Administering to the tissue a sufficient amount of methyltransferase to obtain the tissue.   According to another feature of this preferred embodiment, L-isoaspartyl / D- A method of diagnosing a disease state in which aspartyl residues are accumulated is provided, and The method uses methyltransferase as a probe to detect disease-related proteins. Measures the content of L-isoaspartyl / D-aspartyl residues accumulated in carbonaceous materials Including doing.   Another aspect of the present invention is a method for measuring degradation of a pharmaceutical polypeptide, This method uses methyltransferase as a probe and Measure the content of L-isoaspartyl and D-aspartyl residues in the peptide Including.   Yet another embodiment of the present invention provides a method for treating L-isoaspa of a polypeptide in a tissue. Pharmaceutical formulations for the treatment of medical conditions associated with increased rutile / D-aspartyl residues And the pharmaceutical formulation preferably has the substrate S-adenosylmethionine Human L-isoaspartyl / D-aspartyl protein methyltransfer And a pharmaceutically acceptable carrier.   The commercial utility of this enzyme, as large quantities of the enzyme can be obtained by the present invention. Will be appropriate. Methyltransferase is a protein and peptide drug product Highly sensitive analysis of L-isoaspartyl and D-aspartyl residues in quality control Can be used as a probe. β-amyloid product (Roher, A.E. et al. (1993 Year) J. Biol. Chem.268: 3072-3083) Modified tan in biological fluids It can be used in medical diagnostics to assay proteins and peptides. Intrinsic Sexual human methyltransferase is restricted to the cytosol (Clarke, S. (198 5 years) Annu. Rev. Biochem.54: 479-506), so the damaged proteins in the extracellular environment It cannot undergo repair catalyzed by this enzyme. Thus, methyltransfer Formulation and topical use of chelase and its substrate S-adenosylmethionine Therapeutic formulations are useful. In particular, it may deteriorate and contribute to skin tissue aging In the repair of damaged proteins in the skin such as collagen and elastin. Advantageously, enzymes are useful.                       Detailed Description of the Preferred Embodiment Human L-isoaspartyl / D-aspartyl methyltransfer in Escherichia coli Overexpression of chelase   Plasmid pBluescript SK (-) (pDM2: Genebank accession number S37495) Human L-isoaspartyl / D-aspartyl methyltransferase The complete coding region for acidic isozyme II (SEQ ID NO: 8) in SEQ ID NO: 9) (Ma cLeren, et al. (1992) Biochem, Biophys. Res Commun. 185: 277/283). This Is derived from human brain tissue using mouse cDNA as a probe From a cDNA library (Stratagene, La Jolla, CA). Expression Isozyme I (SEQ ID NO: 9) having a sequence other than pDM2 (SEQ ID NO: 9) No. 1) or a plasmid containing a cDNA encoding isozyme II (SEQ ID NO: 2). Is isozyme I having the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4. Can be used to generate isozyme II with the sequence Sozyme has at least eight forms (I_{twenty two}I₁₁₉K₂₀₅, I_{twenty two} I₁₁₉R₂₀₅, I_{twenty two}V₁₁₉K₂₀₅, I_{twenty two}V₁₁₉R₂₀₅, I_{twenty two}V₁₁₉R₂₀₅, L_{twenty two}I₁₁₉K₂₀₅ , L_{twenty two}I₁₁₉R₁₀₅, L_{twenty two}V₁₁₉K₂₀₅, L_{twenty two}V₁₁₉R₂₀₅). Or , Isozyme III having a -RC end (226 residues long) can be generated in the same manner. You. Such plasmids include pBK phage Vector (Strategene) and pTrc99A expression plasmid (Pharmacia, Piscatwa) y, NJ). E. coli is preferred for expression of human methyltransferase. New Because the characteristics of isozyme II purified from human erythrocytes were revealed Was found to be unmodified after translation (Ingrosso, e t al. (1989) J. Biol. Chem. 264: 20131/20139). Therefore, the E. coli expression system is human Can produce almost the same recombinant methyltransferase as this enzyme. pDM2 The plasmid already has a T7 promoter site suitable for transcription of the inserted cDNA. In position and orientation but without the ribosome binding site (MacLaren, et a t. (1992) Biochem. Biophpus. Res. Commun. 185: 277-283). Therefore, the pDM2 Rasmid is the region between the T7 promoter site of the enzyme and the start codon. By replacing the region with a synthetic fragment containing a strong ribosome binding site To produce the overexpression vector pDM2x (Hine, et al (1974) Proc. Natl. Acad. Sci. USA 71: 1342-1346.) (FIG. 1). In FIG. 1, pDM2 The KpnI-NarI fragment of these multiple cloning sites, eukaryotic initiator And a synthetic linker containing a strong ribosome binding site (FIG. 2). The fragment shown in FIG. 2 has Kpnl and NarI restriction for proper insertion. Polymerase chain reaction using oligonucleotide primers containing PCR). Region from Kpnl restriction site including T7 promoter site Zone 5 'was originally part of the pBluescript SK (-) plasmid. Ncol start Sequence 5 'is not present in the original methyltransferase cDNA insert. p DM2 is a human methyltransferase eye inserted at its EcoRI site. PBluescript SK (-) (Stratagene) plasmid containing Sozyme II cDNA . The sizes of the given fragments were 4 and 2 salts of Kpnl and Narl, respectively. Does not include base pair overhangs. This replacement fragment can be used in various expression And insertion of a methyltransferase cDNA (FIG. 3) into an organism. It was processed to have multiple cloning sites. In FIG. The EcoRI site at the 3 'end is one of the pBluescript SK (-) cloning plasmids. 7 bases are shown. The region 5 'from the Kpnl restriction site is the T7 promoter And is also part of the pBluescript SK (-) plasmid. This flag The comment has the sequence of SEQ ID NO: 8. T7 promoter site PDM2x expression plasmid from the 3 'EcoRI linker on the end of the human cDNA insert to the The nucleotide sequence of the sumid and the translated amino acid sequence are shown in FIG. . The structure of the modified region of pDM2x and the overall plasmid Confirmed by NA sequence analysis and restriction endonuclease digestion analysis. Methyl A ribosome binding site that can be inserted into a transferase-encoding vector and Any synthetic polylinker that contains an initiator and an initiator site is within the scope of the present invention. The T7 RNA polymerase-inducible expression plasmid is then human L-isoasparatyl / D For expression of -aspartyl methyltransferase, E. coli strain BL21 (D E3) (Studier, et al. (1986) J. Mol. Biol. 18) 9: 113-130). This bacterial strain is isopropyl β-D-thiogalactopyranoside (IP TG) Phage T7 is added to the chromosome under the control of the inducible lacUV5 promoter.  Contains RNA polymerase gene. IPTG-induced T7 polymerase inheritance Other bacterial host strains, including offspring, are also contemplated. For example, T7 polymerase is heat-induced Plus encoded on plasmid pGP1-2 behind a sexual promoter A mid system can be used. A variety of bacterial strains are transformed with the plasmid. Purification of methyltransferase   Used here to increase methyltransferase in lysed bacterial extracts The first batch purification process used was by Fu, et al. (Fu, et al, (1991) J. Biol. Chem. 266: 14562, 14572.). Overexpressing bacteria Is lysed by ultrasonication, and cell debris is removed by centrifugation. To the supernatant The remaining nucleic acid is added with the precipitant protamine sulfate or polyethyleneimine And removed by centrifugation. The required amount of protamine sulfate is optimized, The addition of 0.1 volume of a 4% solution of protamine in essentially quantitative yield It was found that the transferase was purified. Next, this methyl transfer The enzyme is concentrated and further purified by precipitation with ammonium sulfate. Again, reserve The conditions of this step were optimized in the experiment, with the highest purification at 50% -55% saturation Is achieved, but the best compromise between yield and purification occurs at 60% saturation, These conditions are also used in large-scale purification. Small amounts of pelleted protein Buffer A And dialyzed against buffer A to remove ammonium sulfate.   Next, the transparency is determined by using DEAE cellulose anion exchange chromatography. A rapid one-step column method for purifying methyltransferase from effluents Will be implemented. Traditional binding of enzyme to column in non-interacting cation buffer The method was applied but found to be incomplete purification. However, under non-equilibrium conditions It was found that the use of interacting anionic buffers eluted a homogeneous enzyme. Was. If chromatography is performed with the column fully equilibrated before packing Methyltransferase is slightly delayed with similar amounts of contaminating polypeptides. At close to the void volume (data not shown). Identification of L-isoaspartyl methyltransferase in plants   Peptide-dependent L-isoaspartyl methyltransferase is a green algal C . found in plant cells of reinhardtii, which is present in at least one species of the plant kingdom Have demonstrated. In the plant kingdom, methyltransferase activity is Species in both monocots and dicots You. Activity levels in various tissues vary considerably. Of the plant kingdom analyzed Among them, the highest specific activity of methyltransferase was found in wheat embryos (malt). You. In contrast, lettuce leaves or tomato fruits have L-isoaspartyl pea. Almost no peptide / specific methyltransferase activity can be detected. Malt The specific activity of the enzyme (14.0 micromol / min / mg) was determined for E. coli (1-2.5 micromol / min / mg; F u, et al. 1991) and human erythrocytes (1.9-9.4 micromol / min / mg; Ota, et al. , 1988; Gilbert, et al, 1988). Follow Malt, an inexpensive and abundant by-product of flour production, is an excellent source of enzyme purification material. You. Wheat seed and seedling L-isoaspartyl methyltransferase   Peptide-dependent L-isoaspartyl methyltransferase activity matures Wheat seeds, the activity of which is significantly higher after water swelling and germination Decrease. Northern analysis showed that methyltransferase mRNA was Only expressed in seeds as otide species, not in whole seedlings, leaves or roots It is shown that. Enzyme levels fluctuate with the stage of husk development, Highest levels of spherase mRNA are detected in stage IV seeds where embryos have grown to their maximum However, no methyltransferase mRNA can be detected at stage II. No Zan analysis was performed for 10 hours with water deprivation, 50 μM (+)-cis, transabscisic acid (ABA) Exposure to salt stress (0.25 M NaCl) resulted in dramatic methyltransfection. This shows that the expression of the mRNA for E. coli is expressed. In contrast, methyl transfer Lase gene expression is induced in seedlings exposed to low (4 ° C) or high (37 ° C) stress. Not guided. These results indicate that isoaspartyl methyltransferase mR NA expression and enzyme activity are not only induced in seed development and germination It also shows that seedling water deficit and salt stress are increased. Special In addition, when seedlings are treated with both ABA and NaCl, methyltransferase Offspring expression is increased approximately two-fold over alone. ABA-NaCl combination treatment phase The additive effect is that the methyltransferase gene has a salt reaction residue in addition to the ABA response gene. It is a gene. Causes the expression of methyltransferase The necessary hormonal and environmental stresses are preferably ABA concentrations of 10-100 μM, salt The concentration is 0.1-1 M or the dehydration time is 5-24 hours (dehydration is 7 for other plants). Days). Purification of L-isoaspartyl methyltransferase from wheat germ   Wheat was chosen because of its high methyltransferase activity. Purification of the present invention The strategy is based on the protein carboxyl methyl tiger reported by Tribedi, et al. Based on partial purification of the phosphatase (Trivedi, et al. (1982) Eur. J. Biochem. .128,349-354). Methyltransferase is purified from the cytosolic fraction of raw malt. Made. This material is first purified by DEAE cellulose chromatography at pH 7-10. , Preferably at 9.3 (FIG. 12A). Second, the active fraction is 60-100%, preferably up to 80% ammonium sulfate in the presence of protein carrier Saturate, inject into column and fractionate by reverse ammonium sulfate gradient solubilization at room temperature (FIG. 12B). About 20-50%, preferably 26-31%, containing saturated ammonium sulfate The active fraction having is more preferably Sephacryl S-200 gel filtration chromatograph. Using , But other gel filtration materials are also contemplated. Surprisingly, L-isoaspa Rutile methyltransferase is present in fractions roughly equivalent to the total volume of the column. And elute in a highly purified state. In this step, methyltransferase It is unique in that it is not fractionated based on its size. Rather, methyl tran Spherases have a solvent effect which can be obtained by a very high concentration of ammonium sulfate in the fraction. Interaction with Sephacryl Sphacryl S-200 resin through hydrophobic interaction for fruit (Belew, et al. (1978) J. Chromatogr. 147, 205-212). In the absence of ammonium sulfate, methyltransferase is From the Sephacryl S-200 column at a position that matches the molecular weight of the monomer. Elution with the lipopeptide. Therefore, highly purified enzymes from gel filtration columns Continuous isolation of the formulation is due to this unusual adsorption phenomenon.   This polypeptide was obtained from Clarke (Clarke (1981) Biochim, Biophys. Acta 670, 1). Gel slices in the presence of Triton X-100 as described by Was analyzed by regenerating L-isoaspartyl methyltransfection. Corresponding to the enzyme. The purity of this drug product can be estimated by measuring the concentration of Coomassie colored gel. It is about 80 to 100% (Fig. 13). L-isoaspartyl / D-aspartyl methyltransferase derived from wheat DNA sequence to encode   The DNA sequence of the 952-bp cDNA insert in plasmid pMBM1 is the sequence shown in FIG. Determined using a decision strategy. DNA sequence of methyltransferase cDNA and The predicted amino acid sequence is shown in SEQ ID NOs: 5 and 6, respectively. 690-bp open Of the 230 amino acid polypeptide predicted from the reading frame The molecular weight is 24,710. In contrast, purified methyltransferase As determined by gel electrophoresis (SDS-PAGE) of SDS-polyacrylamide , 28,000 Da polypeptide. Sequence of L-isoaspartyl methyltransferase from malt Comparison of peptide fragments with predicted amino acid sequences from pMBM1   Interestingly, the predicted amino acid sequence of wheat cDNA and malt L-isoaspartyl 12 sites between the sequence of the peptide fragment of methyltransferase There are contradictions (Figure 15). In six of these locations, experimentally determined Of amino acids whose amino acid sequence data is not clearly encoded by cDNA Is shown. At the other six positions, other residues are added in addition to the encoded residue. Identified by Doman decomposition. These results are consistent with the hexaploid nature of this type of wheat. Match. In that case, the three diploid genomes (AABBDD) have different sequences with different sequences. Gene and can result in the production of various gene products (Peumans, et a l., (1982) Planta 154, 562-567 and Wright, et al. (1989) J.C. Mol. Evol. 28 327-336). Most amino acid changes are shared between methyltransferases Located outside the three highly conserved regions. Differences between these amino acids Results in an enzyme with a slightly different methyl receptor specificity, which It would provide the ability to recognize and potentially repair a wide range of damaged proteins. It is interesting to measure. Many human methyltransferase genes Shapes were also identified (Ingrosso, et al. (1989) J. Biol. Chem. 264, 20131/20139) And Maclaren, et al. (1992) Biochem. Biophys. Res Commun, 185, 277-283) . Isolated recombinant malt L-isoaspartyl protein methyltransferase Manufacturing of   Methyltransferase cDNA inserts as described for the human enzyme Inserted into a well-known prokaryotic expression vector and transformed into competent E. coli, It is then used to induce the T7 polymerase gene using IPTG. plant Enzyme expression can be performed in exactly the same way as human enzymes. Bacterial expression systems Designed to express cDNA sequences regardless of their phylogenetic origin You. The expressed recombinant protein is purified as described above. Application of L-isoaspartyl methyltransferase   Methyltransferase is an atypical L-isoas of peptides and proteins For S-adenosylmethionine-dependent methylation of partyl and D-aspartyl residues Has a catalytic effect. This reaction is an aging and stress-induced denaturation of proteins. In addition to being used as an analytical tool to detect the presence of groups, these residues Initiating a non-enzymatic pathway that can convert to normal L-aspartyl residues it can.   The methyltransferase enzyme of the present invention is incorporated herein by reference. As disclosed in U.S. Pat.No. 5,273,886, L-isoas Can be used in connection with measurement of partate and D-aspartyl residues. in short, This method breaks down a polypeptide into fragments using proteolytic enzymes. Then, the isoaspartyl residue in the fragment is replaced with a methyltransferase enzyme. Consists of quantitative methylation using elemental. Methyl introduced into the fragment The total amount of the hydroxyl groups indicates the amount of isoaspartyl residues in the polypeptide. Polypeptide The amount of isoaspartyl residues in Can be used as an indicator of quantity.   Interestingly, the structure of the recombinant enzyme is derived from human erythrocytes at the N-terminal alanine residue. Is different from the purified enzyme structure and is measured by electrospray mass spectrometry As such, the recombinase does not include covalent post-translational modifications. Therefore, this yeast Is suitable for use in human studies without causing potential antigenic problems ing. Endogenous human methyltransferase is restricted to the cytosol (Clarke, S. (1985) Annu Rev Biochem 54: 479-506), causing damage in the extracellular environment. This protein cannot be repaired catalyzed by this enzyme. Therefore, Injection using luciferase and its substrate, S-adenosylmethionine Possible topical therapeutic formulations are useful. Recombinant human enzymes pose potential antigenic problems Since it does not have it, it can be injected directly into the brain, eyes, blood, etc. Further purified Plant Enzymes Are Peptide and Protein Damaged Isoaspartyl Residues Can be used in skin care products as a topical formulation.   In the tissue, the L-isoaspartyl / D-aspartyl residue is replaced with an L-aspartyl residue Sufficient amount of methyltransferase to convert to By administering together with S-adenosylmethionine, L- Treatment for medical conditions associated with increased isoaspartyl / D-aspartyl residues Is performed. Such medical conditions include cross-linking of matrix proteins. And a decrease in the softness of the skin tissue, for example, cataracts, Alzheimer's disease, etc. This is the case. For this purpose, human or plant enzymes can be used, The dosage of the enzyme is adjusted so that the concentration of the enzyme in the preparation is preferably in the range of 0.4 to 40 μM. I can decide. This enzyme uses S-adenosylmethionine and a pharmaceutically acceptable carrier. Can be easily formulated into an ointment. Typical ointments contain 0.001-10% by weight of enzyme And S-adenosylmethionine in an amount of 0.00004 to 0.4% by weight.   Other medical conditions include plaque formation in brain tissue and For these purposes, human enzymes are preferably used. It is used in an amount such that the concentration of the enzyme in the extracellular space is in the range of 0.4 to 40 μM. In the brain For administration, the enzyme is present in a pharmaceutically acceptable carrier at 0.001 to 10% by weight of the enzyme. Injection solution typically containing 0.00004-0.4% by weight of S-adenosylmethionine. Provided as a liquid. Preliminary evidence suggests that L-isoaspartyl and D-aspartyl Suggests that residues can accumulate in amyloid protein of Alzheimer's disease I have. Since the fraction of β-amyloid protein is found in cerebrospinal fluid (CSF), Treatment of Alzheimer's disease by injecting certain enzymes into the cerebrospinal fluid It might work.   Another medical condition is reduced vascular flexibility, which makes human enzymes preferred. Or red blood cells, endothelial tissue, heart arterial tissue, immune cells, all cells or lungs It is used in such an amount that the concentration of the enzyme in the body ranges from 0.4 to 40 μM. Vascular system For the enzyme, 0.001 to 10% by weight of the enzyme and 0% An injection solution typically containing 0.0004-0.4% by weight of S-adenosylmethionine; Provided. Solutions can be administered by catheter or direct infusion.   Other medical conditions include infertility for eggs and / or semen and tissue fibrosis. For these purposes, the human enzyme is preferably used in egg or sperm cells. It is used in an amount such that the concentration of the enzyme ranges from 0.4 to 40 μM. For the vascular system , The enzyme is present in a pharmaceutically acceptable carrier at 0.001 to 10% by weight of the enzyme and Supplied as an injection solution typically containing 4% by weight of S-adenosylmethionine Re You.   L-isoaspartyl and D-aspartyl residues are accurately methyltransferred The presence of these damaged residues in pharmaceutical polypeptides It is possible to determine the presence and thus the purity and purity of such protein products. And shelf life can be confirmed. These analyzes indicate that pharmaceuticals are¹⁴C-methyl) Incubate with methionine in the presence of purified methyltransferase, It is performed by measuring the radioactivity transferred to the pharmaceutical. This removes the reaction product Incubate with alkaline solution to remove bound methyl ester And then released as (Lowenson, et al. (1991) J. Biol Chem. 26 6: 19396-19406) is collected in the scintillation fluid.   Diagnosis of pathological conditions in which L-isoaspartyl and D-aspartyl residues accumulate Uses methyltransferase as a probe to detect disease-related proteins. Measure the content of L-isoaspartyl and D-aspartyl residues accumulated in the protein It is implemented by setting. β-amyloid protein fraction is converted to bone marrow fluid (CSF) L-isoaspartyl and D-aspartyl residues in the sample of bone marrow fluid To develop a diagnostic test for Alzheimer's disease by measuring the content of groups Is possible. An accurate diagnosis of this disease that has rendered millions of Americans incapable It was not possible before. Diagnostic tests control pharmaceutical quality of proteins This is achieved using the same analytical method described above forExperiment 1: Nucleotide sequence of human methyltransferase coding region cDNA library synthesis and clone screening   A cDNA library constructed from the temporal cortex of the human brain of a two-year-old woman is Stra Purchased from tagene (# 935205). This cDNA was isolated from the mR isolated with oligo-dT. EcoRI site of lambda ZAP bacteriophage vector synthesized from NA (Stratagene). The library was grown in E. coli BB4 and 5x10 each^Five22 plates (1.1 x 10⁷(Plaque total) is 15 Radioisotope from coding region of 80 bp murine methyltransferase cDNA Screened using a 768 bp HaeIII fragment labeled with (Romanik, et al. (1992) Gene, 118: 217-222). Fragment is PRIME-I 10 with T Random Priming Kit⁹cpm / μg specific activity (α³²Use P) -dCTP And labeled (Stratagene). Standard plaque lift and southern block Method (Sambrook, et al (1989) Molecular cloning: A Laboratory Manual, 2nd  Ed., Cold Spring Harbor Laboratory) yielded three positives. These programs Lark clones were isolated by subsequent screening. The clone is λZ In vivo excision by AP protocol to XL1-blue cell plasmid Reinserted. Successful excision was indicated by ampicillin resistance. Insert problem Cells containing the plasmid carrying the product are grown in LB / ampicillin medium, The plasmid was isolated and purified using a Qiagen plasmid isolation column. Nucleotide sequencing and analysis   The nucleotide sequence of the clone was determined by the dideoxy chain-termination method (Sanger, et al. (1977) P roc. Natl. Acad. Sci. USA 74: 5463-5467), Sequenase 2.0 kit (USB), M1 With 3 and T7 universal primers, and synthesized 22mer primers Determined on both strands. Sequence data from Macintosh computer Was analyzed with the DNAStar program using.   1.1x10⁷Three clones of the plaque gave positive and were isolated. Claw The two sequences (pDM2 (SEQ ID NO: 9) and pRK1 (SEQ ID NO: 10)) Was determined from. Plasmid pDM2 is available from GeneBank under accession number S37495 it can. In SEQ ID NOs: 3, 4, and 9, the encoded methionine is numbered 0. And the next acid, alanine, is numbered 1. This is the first methionine excision This is done to better fit the final protein numbering. Both clones The numbers start at these positions. Clone pRK1 starts at position 358. The division The number to the left of the eye number represents pDM2. The number to the right of that number is for pRK1 is there. The nucleotide sequence and encoded amino acid sequence of pDM2 are shown in (a). , (B). (B) represents the 47 base insert found in clone pRK1.Experiment 2: Human methyltransferase expression in E. coli Construction of expression vector pDM2x   The pDM2 plasmid enhances the region between the T7 promoter site and the start codon of the enzyme Modified by replacement with a synthetic fragment containing a unique ribosome binding site. To produce the overexpressed vector pDM2x (FIGS. 1-3). Bacterial growth   The E. coli strain DH5α (Gibco-BRL, Gaithersburg, MD) is a plasmid construct. Used for cloning and propagation. Transformation of E. coli is performed by Cjhung, et al. . (Proc. Natl. Acad. Sci. USA (1989) 86: 2172-519). Achieved by the method described above. For protein expression, E. coli strain BL21 (DE3 ) (Studier, et al. J. Mol. Biol. (1986) 189: 113-130) Transformed with Sumid. 100 μg of BL21 (DE3) bacteria containing the pDM2x plasmid Luria Bertrani (LB) broth containing 1 / ml ampicillin (Sambrook, et. al. "Molecular cloning: a laboratory manual," (1989) Cold Spring Harbo r Laboratory, Cold Spring Harbor, NY) medium at 37 ° C. Protein concentration measurement   The protein concentration of the crude extract was determined using bovine serum albumin as a reference. According to the chloroacetic acid lowry method (Chang, YC (1992) Anal Biochem. 205: 22-26). Measured. The protein density of the column fraction was measured at 280 nm and the optical density was measured. A concentration of 1.0 mg / ml of the mixture of proteins (Sambrook, et al. (1989) "Molecular cloning : a laboratory manual "Cold Spring Harbor Laboratory, Cold Spring H arbor, NY) or homogeneous methyltransferase (Mach, et al, Anal. iochem. (1992) 200: 74-80) was measured as the absorbance of 1 was equal to the concentration of 1.12 mg / ml. Was done. Methyltransferase analysis   The concentration of active methyltransferase is determined by vapor diffusion as base) on methyl receptor ovalbumin Determined by measuring steal formation (Gilbert, et al. Biochemistry). (1988) 27: 5227-5233). The final concentration of the 50 μL reaction mixture is 10 μM S-adenosyl -L- (methyl-¹⁴C) Methionine (53 mCi / mmol, 100 cpm / pmol, ICN Biomedicals, Irvine, CA), 40 mg / ml chicken egg white (fraction V, Sigma, St. Luouls, MO), and 0.2 M sodium citrate, pH 6.0. Incu Is carried out at 37 ° C. for 30 minutes and an equal volume (50 μL) of 0.2 N The reaction was terminated by adding 1.0% (w / v) SDS of NaOH. This mixture Is on a piece of 1cm x 9cm thick filter paper prefolded like accordion pleats Include 6 ml Safariy-Solve counting flow with spots on spots 20ml plastic scintillation vial placed in the neck (No. 111177, Research Products International). The vial is capped and kept at 23 ° C for 2 hours. After incubation, the filter paper insert was removed and the vial was closed again. . wide¹⁴Radioactivity was measured in the C channel. Measured by SDS-PAGE Once purified to homogeneity, methyltransferase Was measured (10,000 pmoles / min / mg at 37 ° C). This value is a measure of the enzyme activity. It was used to determine the mass of methyltransferase from the fixed value. Methyltransferase expression   Ten 10-liter cultures of E. coli strain BL21 (DE3) containing the expression plasmid pDM2x Luria Bertrani (LB) broth containing 0 μg / ml ampicillin at 37 ° C Culture at 250 rpm with shaking to an optical density of 0.5 at 600 nm. Next, the enzyme The current was induced by adding IPTG to 50 μM. Cell growth is optical at 600nm At a density of 1.6, growth continued for more than 4 hours until saturation was reached. Cells are 15 minutes at 4 ° C , Harvested by centrifugation at 5,000 g to give a 14 g wet weight pellet. Here or All operations from column fractionation to column fractionation were performed at 4 ° C.   To determine the optimal concentration of IPTG for expression, log phase expression media should be A range of IPTG was induced and analyzed for methyltransferase concentration. Figure 4 is a very small amount (0.03m) to induce methyltransferase expression. M) indicates that only IPTG is required. In FIG. 4, 100 μg / ml Add pDM2 to a 1 liter solution of Luria Beltrani (LB) broth at 37 ° C containing phosphorus. x BL21 (DE3) E. coli harboring the methyltransferase expression plasmid And cultured to an optical density of 0.2 at 600 nm. The culture medium has four equal volumes (250m each) l), final concentrations of 0, 0.03 mM, 0.5 mM, 048.00 mM isopropyl β-D- Thiogalactopyranoside was used. Continue shaking vigorously at 37 ° C., sample over 3 hours Collected. Each sample is placed on ice and cells are pelleted by low speed centrifugation. Was The pelleted cells are resuspended in buffer A, sonicated, And analyzed for methyltransferase activity. Soluble protein Concentrations were determined by modified Lowry analysis. 10,000 pmol / min / mg specific activity The methyltransferase concentration was used to calculate. Importantly, methyl Transferase reaches 20% of total soluble protein in full active form It was found that methyltransferase was measured as determined by SDS-PAGE. Polypeptide was not found in the insoluble fraction where inclusion bodies were normally found.   Next, a concentration of 50 μM IPTG was used to further increase methyltransferase production. Extensive analysis has been performed. The data in FIG. 5 show that enzyme production rapidly increases during log phase growth. Occur, resulting in increased methyltransferase accumulation Is shown. In FIG. 5, 1 liter of BL21 (DE3) cells containing pDM2x Cultured at 37 ° C with shaking at 250 rpm in Luria Bertrani (LB) broth Was. Expression was started at time 0 by adding IPTG to 50 μM. Soluble protein Quality and methyltransferase were analyzed for the times shown in FIG. Only when the rate of cell growth begins to slow, does methylation in the soluble protein fraction occur. The concentration of the transferase fraction decreased.Experiment 3: Purification of human methyltransferase Methyltransferase purification   Buffer A is used for all preparations and contains 5 mM sodium phosphate, pH 8.0 Yes, 5 mM EDTA, 25 μM phenylmethanesulfonyl fluoride (PMSF), 0.1 mM It contained dithiothreitol (DTT), and 10% V / V glycerol.   The cell pellet (14 g) was suspended in 200 ml of buffer A at 4 ° C. Bacterial cells are my Continuous output power using a Branson W-350 sonifier with a black tip probe Lysed by sonication at a power level of 5 for 2 minutes. Sonication is ice water Dissipate heat and mix thoroughly in a 500 ml beaker cooled in a slurry bath. Made to be combined. These sonication settings are based on the sample at various power levels. At various times, and enzyme content by methyltransferase activity Optimized in preliminary experiments analyzed for Cell destruction starts at power level 3. As a result, the activity of the enzyme began to decrease at level 7, probably due to heating. Dissolved The cells were centrifuged at 13,000 g for 15 minutes and the supernatant was saved. 200ml loose Impulse A is added to the pelletized debris, and the material is again sonicated and The supernatant was stored. The supernatants of the two extracts are mixed and the final A volume of 390 ml was obtained.   Protamine sulfate (4% w / v protamine sulfate solution in 0.1 volume (3 9 ml) (grade X, sigma)) and slowly add and mix at 4 ° C. The acid was precipitated. After mixing for 30 minutes, the solution was centrifuged at 13,000 g for 15 minutes. The methyltransferase was concentrated, and the solid ammonium sulfate was further reduced to 60% (167%). g) Slowly add at 4 ° C. until saturated, mix for 30 minutes, and add Purified by centrifugation at 00 g (Scopes, RK (1993) "Proteinpur ification: orinciples and proctice, "Springer-Verlag, New York). To remove ammonium sulfate for anion exchange column purification, the protein The pellet was resuspended in 18 ml of buffer A, and a 3500 molecular weight cut-off dialysis membrane (Spectr apor3, Spectrum) and permeate three times by changing 1 liter of buffer A every 12 hours Was analyzed.   Final purification step using DEAE cellulose chromatography at room temperature under non-equilibrium conditions Was. Height 14.7cm × ID. 2.5 cm DE-52 (Whatman) column measures eluent pH As described above, equilibrium was maintained at a flow rate of 2 ml / min with buffer A. Trial To prepare the column for loading, the column is buffered with NaCl to a final concentration of 1M. A for 1 hour at a flow rate of 2 ml / min, and finally without NaCl. Washed with Impact A for 6 hours. Next, the dialysed ammonium sulfate preparation Was packed into the column and washed with buffer A at 2 ml / min for 2 hours. This The substance bound to the column at the time of is eluted by washing with 1 M NaCl buffer A Was done. Two minute (4 ml) fractions were collected.   Edman Protein Sequencing by Dr. Audree Fowler Is UCLA Prote in Microsequencing Facility using a Porton 2909E sequencer Was. Electron Spray Mass Spectroscopy is used for UCLA Molecular and Medical Dr. Ken Coklin and Kym at Sciences Mass Spectroscopy Facility Performed by Dr. Faul using a Perkin-Elmer Sciex AP13 instrument. ultraviolet X-ray and visible light spectroscopy are performed on a Hawlett Packard 8452A diode analysis spectrometer. It was implemented.   To increase the methyltransferase fraction in eluted bacterial extracts The first batch purification step used here is by Fu, et al. The method used Corrected (Fu, et al. (1991) J. Biol Chem. 266: 14562/14572). Ultrasonic After lysis of the overexpressing bacteria by treatment, cell debris is removed by centrifugation. Was. The nucleic acid remaining in the supernatant is centrifuged by adding the precipitant protamine sulfate. Has been removed. The required amount of protamine sulfate has been optimized and a 4% solution of protamine sulfate 2.4 volumes of purified product with essentially quantitative yield by adding 0.1 volume of (FIG. 6, Table 1). In FIG. 6, various volumes of 4% w / v protamine sulfate were added to buffer A was sonicated by pipette clarified by centrifugation at 1.0 ml in addition to A The supernatant was added and stirred. Nucleic acids and other debris are precipitated at 13,000g for 15 minutes Was. The supernatant contains total protein and methyltransferase as shown in FIG. Was analyzed in search of. Next, methyltransferase is precipitated with ammonium sulfate. The solution was further concentrated and purified. Also, the conditions of this process were Optimized. Protein precipitated by varying proportions of ammonium sulfate saturation The total amount of and methyltransferase is shown in FIG. In FIG. Solid ammonium sulphate is added to a 1.0 ml sample and the indicated percentage of saturation And mixed at 4 ° C. for 30 minutes on a shaking table. Protein for 13 minutes, Pelleted by centrifugation at 000 g. The pelleted protein is the first Resuspended in a volume of buffer A and the protein and methyltrans Analyzed for total spherase. The highest purification is from 50% to 55% Although obtained at saturation, the best compromise between yield and purification was obtained at 60% saturation. Conditions were used in current large-scale purifications (Table 1). Pelletized protein The material is then resuspended in a small amount of buffer A, dialyzed against buffer A and treated with ammonium sulfate. Um was removed.   Dialysate by using DEAE cellulose anion exchange chromatography Rapid one-step column method for purifying methyltransferase from Was. Conventional methods where the enzyme is bound to the column in a non-interacting cation buffer Upon application, purification was found to be incomplete. However, under non-equilibrium conditions, The use of an anion buffer can provide homogeneous, non-gradient elution of the enzyme. It turned out (FIG. 8). In FIG. 8, the optical density is measured at a protein concentration of 280 nm. It was decided by Electrophoresis was performed using 12% Duracryl (Millipore) separation gel. Implemented using the Laemmli buffer system (Laemmli, UK (1970) Nature 227: 680-5) Was done. Polypeptides were visualized by rapid silver staining (Blum, et al. (1987). ) Electrophoresis 8:93:99). Fractions were analyzed on two minigels. That is, For gel # 1, 1 μL of sonicate and load, 10 μL of fractions 10-21; In the turn, 15 μL of LMWS / 100, 10 μL of fractions 22-25, 27, 29, 31, 33, 35, 1 μL Fractions 76-80 were analyzed. Under these conditions, methyltransferase Eluted as a narrow spike of enzyme collected in the center of fraction 18, from fractions 21-40 Elutes as a broader peak. Methyl in these two peaks Transferases look similar. Both peaks are in SDS gel analysis And a major 25 kDa polypeptide (FIG. 9). In FIG. The 1 ml fraction of the enzyme produced was at the indicated pH value against 20 mM sodium citrate. Dialysed using a Centricon-10 microconcentrator (Amicon, Beverly, MA) Concentrated by ultrafiltration. First peak is low level due to other polypeptides (Less than 5% for fraction 18), but the second broad peak is No protein contamination is indicated. Specific activity of methyltransferase is both Are the same in their peaks, and their weight by mass spectrometry is also the same (see below). See). Partial separation of these peaks not only It seems to be due to the non-equilibrium nature of the graph. For example, reducing the volume of dialysate One broad peak of the homogeneous enzyme further eluted from the void volume. When the amount of dialysate is increased, the concentration of the enzyme becomes higher (the amount of contaminating polypeptides is small) Showing a narrow pi Is obtained near fraction 18 (results not shown). If the column is packed If fully equilibrated chromatography has been performed before, methyltrans Ferase is slightly delayed and dissolves near the void volume with small amounts of contaminating polypeptides. Let go.   Table 1 summarizes the purification steps when obtaining a large amount of homogeneous methyltransferase. ing. According to Table 1, the loss of yield is greatest during the ammonium sulfate precipitation step. No. I don't know why. Overall, 1.7 pmol / min / mg of inactive 10,000 pmol / min / mg 17 mg of enzyme was obtained from each of the first broth cultures of Torr, but purified human Comparable to that found for erythrocyte enzymes in G. (Gilbert, et al, (1988) Biochemlstry 27: 5227-5233, Ingrosso, et al. (1989) Biol. Chem . 264: 20130-20139). In this preparation, the column chromatography step (Table 1). DEAE Cellulo The chromatographic cycle is used repeatedly, easily adding additional homogeneous enzyme. Generate.   The purified recombinant enzyme is stable at room temperature for up to 2 months and can be cycled between freezing and thawing. Repeat has no effect on activity. In addition, methyltransferase Can be heated at 50 ° C for up to 30 minutes without loss of activity. Enzyme concentration   The availability of a concentrated form of the enzyme is useful for several applications, including X-ray structure determination. Important to the way. Successful concentrations of methyltransferase are all In most cases, it was found that it was determined depending on the pH. Typical proteolytic properties (Scopes, RK (1993) "Protein purification: Principies and prac tice, "Springer-Verlag, New York." It happened very easily near the point 5.9 pH units (FIG. 8). Characteristics of purified recombinant methyltransferase   The N-terminal sequence of the purified methyltransferase is an automated Edman It was performed by degradation analysis. The sequence determined in the experiment for the first 20 residues is the human enzyme As can be seen from the above, with the removal of the initiator methionine, (Ingrosso, et al. (1989) J. Biol. Chem. 2). 64: 20131-20139) (Table 2). For example, acetylated alanine residues present in human enzymes No evidence for blocked amino termini such as groups (Ingrosso, et al. (1989) J. Biol. Chem. 264: 20131-20139).   The molecular weight of the purified enzyme was 24,551 ± 3D by electron spray mass spectrometry. Measured at a (FIG. 10 (A)). In FIG. 10 (A), a part of the spectrum of the purified substance is shown. Has already been described using dithiothreitol as a reducing agent in buffer A It is shown. No other peaks were observed at other molecular weights. This average is cD Accurately predicts 24,551 Da for the unmodified product encoded by the NA sequence Matched. Β-mercaptoethanol instead of dithiothreitol as a reducing agent Preliminary attempts at purification using Β-mercapto as indicated by two additional minor forms of high molecular weight An enzyme having one or two adducts of ethanol was obtained (FIG. 10 (B)). In FIG. 10 (B) Is a solution of 0.1 mM dithiothreitol in buffer A with 15 mM β-mercaptoethanol. Figure 2 shows a portion of the spectrum of a purified material when used instead. Mass 24,6 The peak at 27 represents the adduct with one β-mercaptoethanol molecule, 24,7 The small peak at 04 represents an adduct with two β-mercaptoethanol molecules .   On-line liquid chromatography of the bromide fragments of trypsin and cyanogen. Of protein products by electrographic electrospray mass spectrometry Was confirmed directly. All but the insoluble hydrophobic nucleus corresponding to residues 37-143 The mass of the detected species corresponded to the expected fragment.   For direct spectrophotometric densitometry of homogeneous methyltransferases, amino acids From composition, 1A_280nm1.12 mg / ml (Mach, et al. (1992) Anal. Biochem. The extinction coefficient of the enzyme was calculated as 200: 74-80). This value is based on bovine serum albumin. This was confirmed by the trichloroacetic acid-Lowry method used as a reference (Chang. 92) Anal. Biochem. 205: 22.26). Homogeneous fraction 29 from the DEAE column (FIG. 8) The ultraviolet spectrum of methyltransferase is shown in FIG. Experiment 4: Identification of methyltransferase in plants Biological material   Fresh carrots, yellow corn, romaine lettuce, green peas, white Topotes, spinach, cherry tomatoes and alfafa are purchased at local stores Was done. Alphafa seeds and raw malt are available from Rainbow Acres, Inc. (Los Soybean seeds were obtained from Arrowhead Mills, Inc, (He reford, TX). Winter Feet (Triticium aestivum cultiva r Augusta) seeds are from Robert Forsberg, University of Wisconsin (Madison, WI). Provided by Dr. Danver Half Long Carrot Seed, Golden Jubile e-corn seeds, romaine lettuce seeds, sugar snappy seeds, new Girand spinach Seeds and seeds of Bonnie Best Tomato were purchased from Chas. H. Lily Co. (Portland, OR). ). The cytosolic fraction of Chlamydomonas reinhardtii (Wt strain 2137) Dr. Gregg Howe and Sabeeh of the University of California, Los Angeles a Provided by Dr. Merchant (Howe & Merchant, 1992). Preparation of plant cytosol   Crude cytosol should be homogenized from plant tissue using a mortar and pestle Extracted by In a chilled mortar, liquid nitrogen is introduced into plant tissue (typically, 20 g of fresh tissue or 5 g of seed) and the tissue was completely frozen. E Undesired polyphenols potentially released from tissues during mogenation To remove oxidase, 3 g of hydrated PVPP (polyvinyl-polyp Loridone) (Loomis, et al. (1966) Phytochemistry 5, 423-438) After being thoroughly mixed with the weave, it was ground with a pestle. Extraction buffer (20 mL of 100 mM HEPE S, pH: 7.5, 10 mM 2-mercaptoethanol, 1 μm leupeptin, 1 mM PMSF , 10 mM sodium hydrosulfite, and 10 mM sodium metabisulfite Was added to the mortar and the slurry was further ground. The crude obtained The homogenate is strained with 4 layers of cheesecloth and then at 2200g for 30 minutes at 4 ° C. Centrifugation removed insoluble PVPP and unmilled plant material. Got The supernatant is further centrifuged at 172,200 xg for 50 minutes at 4 ° C, then two layers of Miracloth M Filtration through iracloth (Calbiochem, San Diego, CA) to remove floating lipid layers . This fraction was identified as crude cytosol, stored at 80 ° C, and It was used as a source of ferase. Methylation analysis   Methyltransferase activity was identified using vapor phase diffusion analysis, The analysis was obtained from the hydrolysis of the methyl ester which is unstable towards the base (¹⁴C) By quantifying the release of ethanol, S-adenosyl-L- (methyl Le-¹⁴C) Methyl group labeled with a radioisotope transferred from methionine Quantify the number of In a total reaction volume of 40 μL, 12 μL of enzyme preparation was added to 10 μM S -Adenosyl-L- (methyl-¹⁴C) Methionine (ICN Biomedicals, 50mCi / mmol), 500 Incubated with μM peptide substrate and 0.33 M HEPES, pH 7.5. Pe Peptide substrate, (VYP- (L-isoAsp) -HA (SEQ ID NO: 15), KASA- (L-isoAsp) -LAKY (SEQ ID NO: No. 16), AA- (L-isoAsp) -F-NH2 (SEQ ID NO: 17), VYG- (D-Asp) -PA (SEQ ID NO: 18), KASA -(D-isoAsp) -LAKY (SEQ ID NO: 19)) is a UCLA Peptide Synthesis Facility with Janis Yo synthesized by Dr. ung and characterized as described previously (Lowenson, et al. . (1991b) J. Biol. Chem. 266, 19396-19406 (1992) J. Biol. Chem. 267, 5895-5995) . Otherwise, during purification of malt methyltransferase (see below), sample Was analyzed in a buffer containing a final concentration of 0.2 M sodium citrate, pH 6.0. In each case, the incubation was performed at 25 ° C. for 60 minutes. Each reaction is then Stopped with 40 μL of 0.2 M NaOH and 1% (w / v) SDS, agitated and 60 μL aliquots were 1.5 It is applied on a filter paper (Bio-Rad No. 195-090) folded in a pleated form of × 8 cm in the form of spots, 20 containing 5 mL of Bio-Safe II (RPI, Mount Prospect, IL) counting flow It was placed in the neck of a scintillation vial containing mL. Cover the small bottle,¹⁴C) Methanol was allowed to diffuse from the filter paper through the vapor phase into the flow, but was¹⁴ C radioactivity remained on the filter paper. After 2 hours at room temperature, remove the filter paper from the neck of the vial And counted the vials. Determination of protein   Lawley method (Bailey (1967) Techniques in Protein Chemistry, Else 10% (w / v) of 1 mL using a modified version of vler Publishing Co., New York After precipitation with trichloroacetic acid, the protein concentration was determined. Identification of L-isoaspartyl methyltransferase in plants   L-isoaspartyl is a representative of both angiosperms and green algae -It was investigated whether methyltransferase was present. Crude cytosol Is isolated from various types of plant material and contains L-isoaspartyl-containing peptide, V Methyltransferase activity was determined using YP- (isoAsp) -HA (SEQ ID NO: 15). This was determined to be methyltransferase (kM-0.29) in human erythrocytes. μm; Excellent peptide substrate for OWENSON & cLARKE, 1991) Has been revealed. Endogenous cytosolic oligopeptides also have potential methyl It is a condition. Therefore, parallel experiments were performed in the presence and absence of the peptide substrate. (Table 3). Peptide-dependent L-isoaspartyl methyltransferase Is found in plant cells of the green alga C. reinhardtii and is present in at least one species of the plant kingdom. Has been proven. In the plant kingdom, methyltransferase activity is Detected in two classes, monocotyledonous and dicotyledonous plants. Active in various tissues The bell fluctuated considerably. The best methyltransferase of the types analyzed Specific activity was found in malt. In contrast, lettuce leaves or tomato fruits have L-iso Almost no aspartyl peptide-specific methyltransferase activity can be detected Did not. Significantly all were analyzed for high levels of methyltransferase activity Seen in the seeds of plants, for example, corn, alfalfa, lettuce, Not only on beans, spinach and tomatoes, but also on carrots and potato roots Was. Specific activity of plant seed enzyme (0.66-14.0 p^Mole/ Min / mg) for E. coli (1-2.5 pmol) / Min / mg; Fu, et al., 1991) and human erythrocytes (1.9-9.4 pmol / min / mg; Ota, et  al, 1988; Gilbert, et al. 1988). Experiment 5: Purification of malt-derived methyltransferase Preparation of malt cytosol for enzyme purification   Raw malt (150 g) was added to 750 ml of buffer (20 mM sodium borate (pH 9.3), 5 mM EDTA , 2.4 mM 2-mercaptoethanol and 25 mM NaCl) for 30 minutes at 4 ° C. While stirring. Next, the slurry was squeezed with four layers of cheese cloth, and the resulting crude homogen (585 mL) was centrifuged at 7000 xg for 60 minutes at 4 ° C to remove membranes and cell debris. Was. The supernatant (520 mL) is passed through two layers of Miracloth, and the floating lipid layer is filtered. I have. Purification of malt-derived L-isoaspartyl methyltransferase   The purification strategy of the present invention is based on the protein carboxyl method reported by Trivedi et al. Based on partial purification of chill transferase (Trivedi, et al. (1982) E ur. J. Biochem. 128, 349-354). Referring to FIG. 12, crude malt cytosol (515mL, 30mg protein / mL) is a DE-52 (Whatman) column (diameter 9cm x resin height) 13cm, 827mL), which was previously stored at 4 ° C in buffer (20mL sodium borate). (pH 9.3), 5 mM EDTA, 2.4 mM 2-mercaptoethanol, and 25 mM NaCl). Balanced. Two fractions were collected at an average flow rate of 8-10 mL / min. The packed column is Wash equally with 1 liter of buffer, then add 6 liters of 25-200 mM NaCl to the above buffer. A gradient of the turtle was introduced. Protein profile and NaCl graph Dient measures absorbance and conductivity at 280 nm in the corresponding fractions, respectively. Was monitored by VYP- (L-isoAsp) -HA (sequence No. 15) for L-isoaspartyl methyltransferase Each fraction was analyzed. One peak of methyltransferase activity is pooled (Fractions 80-110, 600 mL, see parenthesis) and further King (Biochemistry 11, 367 -371) to reverse ammonium sulfate gradient solubilization as described by More purified. The pH of the pooled material of DE-52 is 20 mL of 1 M Tris-HCl, pH 7.97. The pH was adjusted to 8.38. Next, 15.62 g of Celite545 (Baker Analyzed Reagent, 1 1 g of Celite / 1 g of protein) with stirring at room temperature for 30 minutes to give 80% After saturation (56.1 g ammonium sulfate / 100 mL starting volume), an additional 45 Stirring continued for minutes. This Celite mixture contains precipitated cytosolic proteins And injected into a 3 cm x 19 cm column and filled with a peristaltic pump at room temperature. This column is 150 mL (about 2 column volumes) of 80 M containing 0.05 M Tris-HCl (pH 7.98). Equally washed with a saturated ammonium sulfate solution, and Eluted with a 550 mL linear gradient decreasing to%. Gradient flow rate Was about 0.6 mL / min, and 7.5 fractions were collected. Ammonium sulfate corresponding fraction Percent protein and protein are determined by conductivity and absorbance at 280 nm, respectively. Was. Every two fractions use VYP (L-isoAsp) -HA (SEQ ID NO: 15) as peptide substrate L-isoaspartyl methyltransferase. Methyl Fractions with the highest specific activity of transferase (65-74) were pooled (95 mL, parentheses). ) And then a Sephacryl S-200 (Sigma) gel filtration column (2 cm diameter x 77.5 cm resin). (Height, 243 mL). 20 mL Tris-acetate (pH 7.0), 0.2 mM EDTA Buffer containing 15 mM 2-mercaptoethanol, and 10 mM NaCl was used. The column was equilibrated and performed at 4 ° C. The column flow rate was maintained at 0.12 mL / min, with 30 fractions. Minutes collected. Every two fractions use VYP- (L-isoAsp) -HA as peptide substrate L-isoaspartyl methyltransferase. 280nm And the protein concentration of these fractions was determined. Refined Malt L-isoaspartyl methyltransferase is present in one or two fractions Eluted at a constant volume, corresponding to a fraction volume of approximately 134-139 mL (see arrow in FIG. 12C). reference).   Surprisingly, L-isoaspartyl methyltransferase is highly purified As eluted, the fraction eluted in a fraction approximately corresponding to the total volume of the column. Sephacryl Highly purified enzyme preparation from S-200 gel filtration column Was due to this abnormal adsorption phenomenon.   Comprehensive purification of L-isoaspartyl methyltransferase from malt This is summarized in Table 4 and shows a typical set of polypeptides corresponding to each purification step. The composition is shown in FIG. Referring to FIG. 13, the methyl tiger from each purification step was Active fractions containing phosphases use the buffer system described by Laemmli And analyzed by SDS-PAGE (Laemmli (1970) Nature 227, 600-685). Tampa The quality fraction was sample buffer (180 mL Tris-HCl (pH 6.8), 6.0% (w / v) SDS, 2.1 M 2-mer Captoethanol, 35.5% (v / v) glycerol and 0.004% (w / v) bromophenol (Le blue) at a ratio of 2: 1 (v / v) and boiled for 3 minutes. These fractions are 12 .5% (w / v) acrylamide / 0.43% (w / v) N, N-methylenebisacrylamide separation gel Was electrophoresed. The gel was stained with Coomassie Brilliant Blue. As molecular mass standards (Bio-Rad), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean bird Psin inhibitor (21.5 kDa) and lysozyme (14 kDa). Analyzed The crude malt homogenate (lane A) and the filtered crude cytosol B), fraction 80-100 from the DEAE-cellulose column (lane C), reverse sulfate Monium gradient solubilization step (lane D) and Sephacryl S-200 color Fraction 39 from the system (lane E). Methyltransferase polypeptide The position of the command (MT) is indicated on the right by an arrow (FIG. 13). MONO Q anion exchange chromatography   L-isoaspartylme from several Sephacryl S-200 gel filtration columns Fractions containing tiltransferase were pooled and dialyzed against buffer A (Spect ropor, cut 3500 Da). Dialysed methyltransferase (0.6 mg Protein) was pre-equilibrated with buffer A to Mono Q HR5 / 5 anion exchange ( (Pharmacia) column (5 mm diameter × 50 mm resin height, 1 mL). One minute Fractions were collected at a flow rate of 0.5 mL / min. The packed column is equally buffered 15 And then a linear gradient of 0-100% buffer B (20 mM tri-acetate). Tate (pH 7.0), 0.2 mM EDTA, 15 mM 2-mercaptoethanol, 10% Serol, and 1M sodium acetate) were introduced over 60 minutes. Column The eluate was monitored at 280 nm. Typically, methyltransferase activity Was detected in fractions 43-44. Fraction containing active methyltransferase Minutes were pooled and used for enzymatic studies.   Major Polypeptides Determined by Electrophoresis of SDS Polyacrylamide Slab Gels The calculated molecular mass of the peptide was 28,000 Da. This polypeptide is Clark ( As described by Clarke (1981) Biochim. Biophys. Acta 670, 195-202). Regeneration of individual gel slices in the presence of Triton X-100 allows L-isomers It was demonstrated to correspond to aspartyl methyltransferase. This key Purity of the product should be 86% by densitometry of Coomassie stained gel Was evaluated. The remaining smaller polypeptide contaminants are It could be removed by an additional step. The dialyzed methyltransferase is MONO A linear gradient of 0.1 M sodium acetate packed in a Q anion exchange column Eluted. Active methyltransferase is about 0.5M sodium acetate Eluted. Experiment 6: Amino acid sequence of purified malt methyltransferase Reverse phase HPLC   Homogeneous methyltransferase suitable for sequence analysis is Sephacryl S-200 process Obtained by reversed-phase HPLC (high performance liquid chromatography) of the enzyme purified by Was. Fractionation was performed on a Vydac C-4 column (1 cm x 25 cm diameter) equilibrated with 65% solvent B. m, 300 ° pores, 5 μm spherical silica support) and 65-80% solvent B Eluted with a linear gradient at a flow rate of 3.0 mL / min for 45 min, but with solvent A 0.1% trifluoroacetic acid aqueous solution (w / v), solvent B was 0.1% in 99% methanol / 0.9% water. % Trifluoroacetic acid solution (w / v / v). The column effluent contains a fraction of 1 minute. Was monitored at 280 nm. Volatile reagents are fractionated from Savanto Speedvac And then these fractions were subjected to SOS-PAGE and silver stained (Jones (199 0) in Current Protocols in Molecular Biology Suppl. 11, John Wiley and So ns, New York). Malt methyltransferase has an apparent molecular mass of 28, Eluted as a single polypeptide band with 000 Da. Amines by trypsin and Staphylococcus aureus V8 protease mapping No acid sequencing   Homogeneous methyltransferase suitable for amino acid sequence analysis is reversed as described above. Obtained by phase HPLC analysis. The substance is trypsin and Staphylococcus aureus V8 Protease was digested with protease and the resulting peptide was subjected to reverse phase HP using a Vvdac C-18 column. Collected by LC. N-terminal Edman sequences have been performed on these peptides. Was. Obtained partial peptide sequence data generates oligonucleotide probes And used to confirm the presence of polymorphisms and / or multigenes ( See Experiment 7 below).Experiment 7: cDNA encoding wheat methyltransferase Synthetic oligonucleotide probes   Oligonucleotide probes were used with the Gene Assembler Plus DNA Synthesizer ( Β-cyanoethyl N, N-diisopropane in Pharmacia LKB Biotechnology) It was synthesized using ropyl phosphoramidite chemistry. Oligonucleotides are pBI uescript SK ± phagemid T7 promoter, T7 (DMT-TAATACGACTCACTATAGGG) ( SEQ ID NO: 11) and three types of degenerate oligonucleotides, MB1 [TCTGG (G / A) AT (G / A) TG (C / T) TC (G / A) ATNCCCAT] (SEQ ID NO: 12), EcoRI linker [CTCGAATTCTA (C / T) (C / T) T MB3 containing NAA (G / A) CA (G / A) TA (C / T) GGNGT] (SEQ ID NO: 13), and HindlII linker Represents MB4 containing [TCAAAGCTTTT (G / A) TC (T / G / A) ATNAC (C / T) TGNAG] (SEQ ID NO: 14), As a probe in PCR amplification of a wheat cDNA library (below) Synthesized for use as Primers are Bio-Spin 6 columns (Bio-Rad) Purified by size exclusion chromatography using Isolation of cDNA clone of L-isoaspartyl methyltransferase from wheat   Degenerate oligonucleotides are synthesized based on partial amino acid sequence data ( (See above), then poly (A) isolated from barley seedlings that were yellowed for 48 hours⁺With RNA Methyltransferase cDNA region of constructed wheat cDNA library Used for amplification (Hatfield, et al. (1990) J. Biol. Chem. 265, 15813- 15817). The 850 bp PCR product is a methyltransferase cDNA (peptide 384-fold degenerate oligonucleotide representing the nucleic acid sequence of the 5 'region (corresponding to YLKOYGV) A protocol that encodes the T7 promoter of the leotide, MB3 and pBluescript vectors. Amplified using a primer. Identification of the 850-bp PCR product is methyltransfer Nucleic acid sequence of the middle region of the cDNA of the enzyme (corresponding to peptide GIEHIPE) Demonstrated by Southern hybridization using a 64-fold degenerate oligonucleotide, MB1, representing Was done. Methyltransferase cDNA without pBluescript vector sequence In order to obtain a PCR product containing the 850-bp PCR product, the MB3 primer and 3 ′ region of methyltransferase cDNA (corresponding to peptide LOVIDK) Reaction with the 288-fold degenerate oligonucleotide MB4 representing the nucleic acid sequence of the region Used as a template. PCR amplification by PCR dideoxy chain termination sequencing As determined, a 600-bp fragment containing only the methyltransferase cDNA sequence Created a fragment. Screen the wheat cDNA library with this 600-bp product. As a result, one positive plaque was isolated, which was E. coli XL-1 pU. Return to the C19 pBluescript phagemid to generate the pMBM1 plasmid and then It was used to transform fungal DH5a cells. Gene encoding L-isoaspartyl methyltransferase from wheat DNA sequence   The DNA sequence of the 952-bpc DNA insert of plasmid pMBM1 follows the sequence strategy shown in FIG. Decided to use. Referring to FIG. 14, barley methyltransferase cDNa insertion. Both strands of the pMBM1 clone containing the input (952-bp EcoRI fragment) Sequenced by deoxy chain termination sequencing. Oligonucleotides of 600-bp wheat Synthesized using the sequence of the PCR product containing the methyltransferase cDNA, Prime to sequence the 952-bp fragment as shown in this form of sequencing strategy. Used as a marker. DNA sequence of methyltransferase cDNA and its prediction The measured amino acid sequences are shown in SEQ ID NOs: 5 and 6, respectively. See SEQ ID NOs: 5 and 6 The sequence of the coding strand of the 944 bp insert of plasmid pMBM1 was Shown without terminal EcoRI linker (GGAATTCC) added to the library. 690bp The methyltransferase cDNA begins at the codon for ATG at position 118 and the TGA at position 808 End with a codon. 230 predicted 690 bp open reading frame The calculated molecular weight of the amino acid polypeptide is 24,710. On the contrary, refined The methyltransferase was 28,000 Da po as measured by SDS-PAGE. It migrated as a repeptide (FIG. 13).Experiment 8: Comparison of the sequenced peptide fragment with the predicted sequence Sequence of L-isoaspartyl methyltransferase from malt Amino acid sequence predicted from peptide fragment and its pMBM1   Interestingly, the predicted amino acid sequence of wheat cDNA and malt L-isoaspar 12 parts between the sequence of the peptide fragment of chill methyltransferase A discrepancy in the order was found (FIG. 5). Referring to FIG. 15, trypsin (T) and After digestion with S. aureus V8 protease (V), the peptide was Recovered and sequenced by automated Edman degradation. Number the elution order The peptide sequence of the fragment was compared to the predicted cDNA sequence by a line. It is shown. The existence of the space means that unambiguous identification of amino acid residues is Indicates that it was not done. Other residues identified in a particular cycle are in brackets Shown above. The residue above the parentheses indicates an amino acid substitution at this position, where cD No evidence was found for the NA-encoded residues. As a whole, The amino acid sequence of the produced malt is considered to be shown in SEQ ID NO: 7, except for the following: . At position 41, A was found at T8 and V17; N was found at T9. At position 52 In I, I was observed in T8, V14 and V17. At position 54, only L was found at V17. At position 156, A was found at T6 and V8; V was found at T7 and V9. these The amino acid sequence data determined experimentally at six of the positions Indicates the presence of amino acids not encoded by. In the other six positions, Residues in addition to the loaded residues were identified by the Edman sequence. These results are Match the hexaploid nature of the barley seeds. That is, three diploid genomes (AABBDD) Can have alleles with different sequences and produce different gene products. (Peumans, et al. (1982) Planta 154, 562-567; Wright, et al. (1989) ) J. Mol. Evol. 28, 327-336). Most amino acid changes are methyltransfer Located outside of the highly conserved region shared between the enzymes. These Differences in amino acids can result in enzymes with slightly different methyl receptor specificities. And possibly recognizes a wide range of damaged proteins in the cell It is interesting to speculate that it gives the ability to be repaired. Departure Variants of these enzymes can be similarly purified by the above-described purification methods.Experiment 9: Skin treatment using methyltransferase   Purified methyltransferase (isolated from plant sources or bacteria Either as a recombinant human enzyme purified by S) and S-adenosyl-L-methio Nin and glycerin, respectively, in an aqueous cream containing mineral oil. Final concentrations are 0.01% and 0.00004%. Skin cream or hair cream , Water, glycerin, cetylaryl alcohol, coconut oil glycerides, Ceteareth-20, Mineral oil, petrolatum, sorbitol, avocado oil, DMSO (or other carrier molecule) , Stearic acid, Allantoin, Squalene, Methylparaben, Sodium Carbome r 941, recombinant human L-isoaspartyl / D-aspartyl methyltransfer Lase (or plant methyltransferase), propylparaben, S-adeno Sil-L-methionine, Quaternium-15, fragrance, FD & C Yellow No.5 and FD & C Red Formulated using No. 4. Skin mist ("Methylmist") is water, glycerin, H Allyl alcohol, DMSO (or other carrier molecule), citric acid, recombinant human L-A Soaspartyl / D-aspartyl methyltransferase (or plant methyl Transferase), S-adenosyl-L-methionine, Quaternium-15, and incense Formulated using a fee. Transdermal skin patches use the same ingredients as the skin mist above Formulated using These formulations can be applied directly to the skin. Soap and water on skin After washing with methyl transferase skin cream (a), mist (b) , Patch (c) is used according to the following procedure: (a) a small amount (0.5 ml) of tackiness Lightly apply the formulation to the center point, with or without latex gloves And / or by hand, with or without towels and tissues, for example. Approximately 100cm by rubbing in a small circle^Two(B) Low adhesiveness Using a clean formulation ("MethylMist") and using a spray bottle or atomizer Spray the residue onto the skin as described above; (C) Use the same formulation as "MethylMist", but wrap it in a patch that adheres to the skin, The drug is distributed directly to the skin via the tiny pores of the patch.Experiment 10: Other medical treatments using methyltransferase   Injectable or topical formulation for eye treatment (pH-7.3) (eg, cataract prophylaxis). Brain Injectable (eg, to prevent plaque formation) or blood flow (eg, flexible) 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na_TwoHPO_Four・ 7H_TwoO, 1.4 mM KH_TwoPO_Four, 25 mM recombinant human L-isoaspartyl / D-aspartyl methyltransferer 1-3 μM, 30 μM to match the concentration of S-adenosine (saturated) or S-adenosyl-L-methionine S-adenosyl-methionine, and 0.22 μM of filtered fungicide. Time release For dispensing, the injection is packaged in a carrier liposome or microporous structure. can do. These drugs can be injected directly into the eye, brain or blood stream.Experiment 11: Diagnosis using methyltransferase   Spinal fluid (0.01 mL) sample diluted in 0.2 M sodium citrate buffer (pH 6.0) Is S-adenosyl- [¹⁴[C-methyl] -L-methionine (100 cpm / pmol) Dissolves prepared methyltransferase (from plant source or human recombinant) 0.03 mL of the resulting mixture. The latter mixture is known with 400 pmol of radioisotope. Separated S-adenosylmethionine and 10 μg of purified methyltransferer Contains ze. This mixture is incubated at 37 ° C. for 15 minutes, then 0.04 mL 0.1% sodium dodecyl sulfate solution, 0.2 M sodium hydroxide is added. this The mixture is then applied to a piece of thick filter paper and 7 mL of liquid scintillation cocktail is added. Hang into plastic vials containing. After 2 hours at room temperature, remove the filter paper. Start and count the vials. Injured L-isoaspartyl in radioactive spinal fluid And D-aspartyl residues.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/70 ABN A61K 31/70 ABN ACV ACV 38/45 ADA C12N 9/10 C12N 9/10 C12Q 1/48 Z C12Q 1 / 48 A61K 37/52 ADA // (C12N 15/09 ZNA C12R 1:91) (C12N 9/10 C12R 1:19) (72) Inventor Madget, Mary, Beth United States, 94612-3550 California, Oakland, Lake 300 Side Drive, 21st Floor (72) Inventor Clark, Steven United States, 94612-3550 California, Auckland, 300 Lakeside Drive, 21st Floor

Claims (1)

[Claims]   1. Polynus having a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 Isolated recombinant human L-isoaspartyl / DA obtained by expression of nucleotides Spartyl protein methyltransferase.   2. Has an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4 Isolated recombinant human L-isoaspartyl / D-aspartyl protein methyl tiger Transferase.   3. An isolated polynucleotide having the coding sequence of SEQ ID NO: 5. Thus, plant L-isoaspartyl protein methyltransferase The polynucleotide.   4. Isolated recombinant plant L-isoaspartyl obtained by expression of SEQ ID NO: 5 Protein methyltransferase.   5. Purified plant L-I obtained from wheat germ and having the amino acid sequence of SEQ ID NO: 7 Soaspartyl protein methyltransferase.   6. Human L-isoaspartyl / D-aspartyl protein methyltransfer A method for producing the enzyme by a recombinant method,   Using oligonucleotides, the T7 promoter region and human L-isoaspa All coding regions of rutile / D-aspartyl protein methyltransferase To contain multiple cloning sites, efficient Designed to function in a sosome binding site and in bacterial and / or eukaryotic expression systems Provides a powerful translation initiator area,   This constructed vector is placed in a host having an inducible T7 polymerase gene. Transfection, and   Induces methyltransferase by inducing overexpression of methyltransferase Manufacture, The method comprising:   7. Inducible T7 polymerase gene by heat shock or addition of chemicals 7. The method of claim 6, wherein said inducing and said inducing step comprises the addition of IPTG. .   8. The chemical that induces the T7 polymerase gene is isopropyl β-D-thio. The method according to claim 7, which is ogalactopyranoside (IPTG).   9. A human L-isoform having the sequence shown in SEQ ID NO: 2 Isozyme of Spartyl / D-aspartyl protein methyltransferase The human L-isoaspartyl / D- according to claim 6, which is the entire coding region of im II. A method for producing aspartyl protein methyltransferase by a recombinant method.   10. The modified plasmid is a 1580 bp murine methyltransfer Using a 769 bp radiolabeled HaeIII fragment obtained from the coding region of The method according to claim 9, which is obtained from a commercially available cDNA library derived from human brain tissue. L-isoaspartyl / D-aspartyl protein methyltransfer A method for producing an enzyme by a recombinant method.   11. Wherein the plasmid to be modified has the sequence shown in SEQ ID NO: 9. The human L-isoaspartyl / D-aspa according to claim 10, which is mido pDM2. A method for producing rutile protein methyltransferase by a recombinant method.   12. Said modification results in a KpnI between the T7-promoter site and the start codon of the enzyme. The 107 bp region from the site to the NarI site is linked to the ribosome binding site and the initiator. Replaced by a synthetic fragment containing the site and having the sequence shown as SEQ ID NO: 8 7. The human L-isoaspartyl / D- according to claim 6, which is carried out by obtaining. A method for producing aspartyl protein methyltransferase by a recombinant method.   13. 7. The host according to claim 6, wherein the host is E. coli strain BL21 (DE3) or another suitable strain. L-isoaspartyl / D-aspartyl protein methyltrans A method for producing ferase by a recombinant method.   14． The human L according to claim 6, wherein the concentration of the IPTG is 0.03 mM to 8 mM. -Isoaspartyl / D-aspartyl protein methyltransferase Method of manufacturing by replacement method.   15. Methyltransferase expression is present in lysed bacterial extracts L-isoaspartyl / D-aspartyltan recombinantly produced A method for purifying protein methyltransferase,   A nucleotide precipitant was added to the extract to remove cell debris from the extract Later removing the DNA present in the extract,   Precipitate methyltransferase with ammonium sulfate,   Dialysed to remove ammonium sulfate, and   Dialysis using a DEAE-cellulose anion exchange chromatography column Purifying methyltransferase from the product, The method comprising:   16. The nucleotide precipitant is protamine sulfate or polyethylene imine. Human L-isoaspartyl / D-A produced by the recombinant method according to claim 15. A method for purifying spartyl protein methyltransferase.   17． 2. The method of claim 1 wherein said addition of protamine sulfate is 0.1 volume of about a 4% solution thereof. 5. Human L-isoaspartyl / D-aspartyl produced by a recombinant method according to 5 A method for purifying a protein methyltransferase.   18. The method according to claim 15, wherein the saturated state of the ammonium sulfate is 50% to 60%. L-isoaspartyl / D-aspartyl protein produced by the recombinant method described above A method for purifying a methyltransferase.   19. 2. The column is fully equilibrated before loading the dialysate. 5. Human L-isoaspartyl / D-aspartylta produced by the recombinant method according to 5 A method for purifying protein methyltransferase.   20. Plant L-isoaspartyl protein methyltransferer from wheat A method for purifying zeolites, comprising:   Obtain crude cytosol of raw wheat,   Fractionating the crude cytosol by DEAE-cellulose chromatography,   Add ammonium sulfate to the collected active fractions in the presence of a protein carrier. ,   The resulting material was fractionated by reverse ammonium sulfate gradient solubilization, and   The collected active fractions were purified by gel filtration chromatography and Purify the transferase as a 28,000 Da monomeric species, The purification method.   21. 21. Plant L according to claim 20, wherein the crude cytosol is obtained from wheat germ. -A method for purifying isoaspartyl protein methyltransferase.   22. The above fraction by DEAE-cellulose chromatography was run at pH 9.3. 21. The plant L-isoaspartyl protein methyltrans according to claim 20, which is applied. A method for purifying ferase.   23. 21. The plant of claim 20, wherein the ammonium sulfate is added to 80% saturation. A method for purifying L-isoaspartyl protein methyltransferase.   24. 21. The plant L-I according to claim 20, wherein the protein carrier is Celite 545. A method for purifying soaspartyl protein methyltransferase.   25. The reverse ammonium sulfate gradient solubilization is from 80% to 0% of ammonium sulfate 21. The plant L-isoaspa according to claim 20, which is carried out with a linear gradient decreasing to saturation. A method for purifying rutile protein methyltransferase.   26. Active fractions collected after fractionation by reverse ammonium sulfate gradient solubilization Contains 26-31% of saturated ammonium sulphate. A method for purifying aspartyl protein methyltransferase.   27. The gel filtration chromatography was performed on Sephacryl S-200 gel filtration chromatograph. 21. The plant L-isoaspartyl protein medium according to claim 20, which is tomography. A method for purifying chill transferase.   28. Following gel filtration chromatography, purified methyltransferase Dialyzed and dialyzed methyltransferase into Mono Q anion exchange column 21. The plant L-isoaspartyl tampa according to claim 20, further comprising: A method for purifying cytosolic methyltransferase.   29. Using oligonucleotides, human L-isoaspartyl / D-aspa Plasmid containing the entire coding region of rutile protein methyltransferase By modifying multiple cloning sites, efficient ribosome binding sites and Powerful translation initiators designed to function in bacterial and / or eukaryotic expression systems Human L-isoaspartyl / D-aspa constructed to provide a seater region An expression vector for rutile protein methyltransferase.   30. A human L-isoform wherein the entire coding region has the sequence shown in SEQ ID NO: 2; Isozyme of Spartyl / D-aspartyl protein methyltransferase 30. The expression vector according to claim 29, which is the entire coding region of ImII.   31. 31. The method of claim 30, wherein said modified plasmid is plasmid pDM2. The expression vector according to any one of the preceding claims.   32. Said modification results in a KpnI between the T7-promoter site of the enzyme and the start codon. The 107 bp region from the site to the NarI site is linked to the ribosome binding site and the initiator. Replaced with a synthetic fragment containing the site and having the sequence shown in SEQ ID NO: 8 30. The expression vector according to claim 29, wherein the expression vector is performed.   33. Of L-isoaspartyl / D-aspartyl residues of polypeptides in tissues A method of treating a medical condition associated with an increase,   The L-isoaspartyl / D-aspartyl residue in the tissue is replaced with L-aspartyl Administer tissue to the tissue in an amount sufficient to convert it to residues. The treatment method comprising:   34. The condition is cross-linking of matrix proteins and loss of tissue flexibility. 34. A method for treating the condition of claim 33.   35. The method for treating a condition according to claim 33, wherein the condition is cataract.   36. 34. The method for treating a condition according to claim 33, wherein the condition is reduced corneal flexibility. .   37. 34. The condition of claim 33, wherein said condition is plaque formation in brain tissue. Treatment method.   38. The condition according to claim 33, wherein the condition is a decrease in cell function in brain tissue. How to treat the condition.   39. 34. The condition of claim 33, wherein said condition is reduced flexibility in the vasculature. Method of treatment.   40. 34. The method according to claim 33, wherein the condition is infertility associated with eggs and / or sperm. How to treat the listed condition.   41. 34. The method of claim 33, wherein the condition is tissue fibrosis.   42. The methyltransferase is an isolated recombinant human L-isoaspartyl / D-aspartyl protein methyltransferase according to claim 33. How to treat the listed condition.   43. The methyltransferase is injected together with S-adenosylmethionine. 34. The method of treating a condition according to claim 33, wherein the method is provided.   44. The methyltransferase is a purified plant L-isoaspartyl tamper. 34. The method for treating a condition according to claim 33, which is a cytoplasmic methyltransferase.   45. L-isoaspartyl / D-aspartyl residues accumulated in patients Diagnostic method of the condition,   Obtaining a biological sample containing the protein from the patient; and   Accumulates in the protein using methyltransferase as a probe Measuring the L-isoaspartyl / D-aspartyl residue content, The diagnostic method comprising:   46. Of L-isoaspartyl / D-aspartyl residues of polypeptides in tissues A therapeutic pharmaceutical formulation for a condition associated with an increase, wherein   Human L-isoaspartyl / D-aspartyl protein methyltransfer And   A pharmaceutically acceptable carrier, The pharmaceutical preparation for treatment comprising:   47. 47. The composition of claim 46, wherein the formulation is a skin or hair cream. Pharmaceutical preparation for treatment.   48. 47. The pharmaceutical preparation for treating a condition according to claim 46, wherein the preparation is an eye drop.   49. 47. The pharmaceutical preparation for treating a condition according to claim 46, wherein the preparation is an injection.   50. The formulation contains S-adenosylmethionine as a substrate for the enzyme 47. A pharmaceutical preparation for treating the condition of claim 46.   51. Plants associated with increased L-isoaspartyl residues of polypeptides in tissues A method for treating protein degradation,   To convert the L-isoaspartyl residue in the tissue to an L-aspartyl residue Add a sufficient amount of plant L-isoaspartyl protein methyltransferase Administered to the tissue, The treatment method comprising:   52. The degradation of the plant protein is caused by drying, aging and environmental stress 52. The method of treating plant protein degradation according to claim 51.   53. 52. The method according to claim 51, wherein the treatment is applied to seed tissue and / or seedling tissue. Methods for treating plant protein degradation.   54. Inject the methyltransferase together with S-adenosylmethionine 52. The method of treating plant protein degradation according to claim 51.   55. Recombinant plant L-isoaspartyl protein methyltransferase Manufacturing method,   Using oligonucleotides, the T7 promoter region and plant L-isoaspa Plasmid containing the entire coding region of rutile protein methyltransferase The multiple cloning site and efficient ribosome binding site Powerful translation initiators designed to function in bacterial and / or fungal expression systems Provide eater area,   The constructed vector is placed in a host having an inducible T7 polymerase gene. Transfection, and   Induces methyltransferase by inducing overexpression of methyltransferase Manufacture, The method comprising:   56. Methyltransferase expression present in lysed bacterial extracts L-isoaspartyl protein methyltrans produced by recombinant method A method for purifying ferase, comprising:   Nucleotide precipitants are added to the extract to remove the cell debris and remain in the extract. Removing the present DNA from the extract,   Precipitate methyltransferase with ammonium sulfate,   Dialysed to remove ammonium sulfate, and   Using a DEAE-cellulose anion exchange chromatography column Purifying methyltransferase from the dialysate, The method comprising:   57. The nucleotide precipitant contains protamine sulfate or polyethylene imine. 57. The method of claim 56.   58. Of L-isoaspartyl / D-aspartyl residues of polypeptides in tissues Methyltrans for use as a medicament in the treatment of medical conditions associated with an increase Ferase.   59. The condition is cross-linking of matrix proteins and loss of tissue flexibility. A methyltransferase according to claim 58.   60. The condition may be cataract, reduced corneal flexibility, plaque formation in brain tissue, brain Associated with decreased cellular function of tissues, decreased flexibility in the vasculature, eggs and / or sperm 59. The methyltransfer of claim 58 which is infertility or fibrosis in tissue. Rahse.   61. The methyltransferase is an isolated recombinant human L-isoaspartyl / D-aspartyl protein methyltransferase. Methyltransferase.   62. 59. The methyl ester of claim 58 in combination with S-adenosylmethionine. Transferase.   63. The methyltransferase is a purified plant L-isoaspartyl tamper. The methyltransferase according to claim 58, which is a cytoplasmic methyltransferase. -Se.