KR100195372B1 - Enzymes which articipate in c-terminal amidation, and production and use thereof - Google Patents

Abstract

The present invention relates to enzymes that act on peptide C terminal α-hydroxyglycine adducts of formula (II) to produce C terminal amidated peptides of formula (III).

Formula II

Formula III

In the above formula,

A represents a residue other than an α-amino group or an imino group and an α-carboxyl group derived from a natural α-amino acid,

X represents the residue of the amino acid derivative couple | bonded with N atom through a hydrogen atom or a carbonyl group.

Description

Enzyme involved in C-terminal amidation

Conventionally, enzymes involved in C-terminal amidation of peptide C-terminal glycine adducts (compounds in which glycine is peptide-bound to C-terminal residues) according to in vivo enzymatic reactions are peptidylglycine-α-amiditin monooxygenase ( Peptide C terminal amidase) (EC. 1, 14, 17, 3) (Bradbury et al., Nature, 298, 686, 1982: Glembotski et al., J. Biol. Chem., 259, 6385, 1984) It is thought to catalyze a reaction such as

-CHCONHCH ₂ COOH → -CHCONH ₂ + Glyoxylic acid Peptides produced according to the elucidation of the amidation mechanism in vivo and recombinant DNA technology, the peptides that are the first physiological activity by amidating the C-terminal, for example calcitonin, gastrin Attempts have been made to purify the enzyme, which can be used as a method of ex vivo conversion. Examples of such enzymes include mesenchymal pituitary (Murthy et al., J. Biol. Chem., 261, 1815, 1986), porcine pituitary (Kizer et al., Endocrinology. 118, 2262, 1986: Bradbury et al., Eur. J. Biochem., 169, 579, 1987), swine atria (Kojima et al., J. Biochem., 105, 440, 1989), African claw frog body epidermis (Mizuno et al., Biochem. Biophys. Res. Commun., 137, 984, 1984), Reported from rat thyroid tumors (Mehta et al., Arch. Biochem. Biophys., 261, 44, 1988).

In addition, since it is difficult to obtain such purified enzymes in large quantities, it has recently been attempted to isolate the corresponding cDNAs required for expression of these enzymes and to prepare the enzymes using them using recombinant DNA techniques which are generally performed. For example, Eipper B. A. et al., Mol. Endocrinol 1, 777-790, 1987, Ohsuye, K et al., Biochem. Biophys. Res. Commun. 150, 1275-1281, 1988, Stoffers, D. A. et al., Proc. Natl. Acad. Sci. VSA, 86, 735-739, 1989 and Glauder, J. et al., Biochem. Biophys. Res. Commun, 169, 551 to 558 1990, discloses peptide C terminal amidation enzyme cDNA from bovine pituitary, frog skin, atrium of rats and human thyroid cells, and is not necessarily satisfactory for its productivity. Examples of production of peptide C terminal amidation enzymes using recombinant DNA technology using frog-derived and bovine-derived cDNA are also known (e.g., Japanese Patent Application Laid-Open No. 89-104168, WO89 / 02460 and Perkins). Et al., Mol. Endocrinol. 4, 132-139, 1990).

On the other hand, these proteins have been reported to have a molecular weight of 38, 42 or 54 KDa from cows, a molecular weight of 39 KDa from frogs, and a molecular weight of 41, 50 or 75 KDa from rats. See, eg, Bradbury et al., Supra, Ramer et al., J. Am. Chem. Soc., 110, 8526-8532 (1988) and by Young et al., J. Am. Chem. Soc., 111, 1933-1934 (1989) also suggest the presence of reaction intermediates. However, there is currently no clear example of the isolation of this intermediate and the relationship between its intermediate and amidation enzymes.

As mentioned above, peptide C terminal amidation enzymes have a very interesting action in vivo and compositions having a certain purity derived from a specific biological organ are also known. However, the use of such a composition in vitro for the production of peptide C terminal amidide is not sufficient in terms of production cost and its purity and stability. In order to solve this problem, the present inventors need to clarify the mechanism of collecting basic views on the enzyme, that is, conducting the C-terminal amidation reaction, and have attempted to isolate the intermediate product. I succeeded in making the decision. As a result, it was found that the peptide C terminal amidation reaction is not a one step reaction as previously considered, but a two step reaction via an intermediate (corresponding C terminal α-hydroxyglycine adduct). It is therefore desirable to provide such enzymes because it is expected that the individual, or combination of enzymes catalyzed in the respective reactions under appropriate conditions can be efficiently converted to the corresponding amidates of the peptide C terminal glycine adducts. . It is also desirable to provide such an efficient production method when the presence of these enzymes can be confirmed.

The present invention relates to novel enzymes involved in C-terminal amidation of peptide C-terminal glycine adducts, methods for their preparation and uses. C terminal amidation refers to the action of promoting any step of converting a peptide C terminal glycine adduct to its peptide C terminal amidate.

1 is an HPLC pattern for preparing FGFhyG using FGFG as a substrate and the enzyme-I of the present invention (in the specification and the drawings, each symbol is conventional in the art). HyG is α-hydroxyglycine),

2 is a result of FAB-MS spectral analysis performed to confirm the molecular structure of the prepared FGFhyG,

Figure 3 is a chromatographic pattern depicting the separation of Enzyme-I and Enzyme-II of the present invention according to Mono Q column chromatography (a full quadrant float shows the activity of Enzyme-II and a black circle float shows Enzyme- The activity of I and the dashed line is the linear concentration gradient of sodium chloride),

4 is a HPLC pattern over time when using FGFhyG as a substrate to prepare FGF-NH ₂ using the enzyme-II of the present invention.

FIG. 5 shows, in one letter representation, the amino acid sequence estimated from peptide C terminal amidation enzyme cDNA cloned from human, horse, bovine, rat frog.

Figure 6 shows the nucleotide sequence of the C-terminal amidase enzyme cDNA cloned from rat pituitary mRNA and the amino acid sequence thus estimated,

FIG. 7 schematically shows the five C-terminal amidase enzyme cDNA cloned from rat pituitary mRNA [indicated by the box where the putative enzyme is encoded and the number is the base number (bp) with translation initiation point 1 TM represents the portion corresponding to the transmembrane region, KK represents the lysine-lysine sequence, and restriction enzymes are the following abbreviations, respectively.

B (BamH I), N (Nsi I), RI (EcoR I), RV (EcoR V),

S (Sph I), X (Xma I)],

8, 9 and 10 show the Sephacly S-200 column chromatography patterns of enzymes expressed according to plasmids SV-a, SV-b and SV-203, respectively.

11 and 12 show changes over time when the α-hydroxyglycine body and the C-terminal amidated body are produced when PheGlyPheGly is used as a substrate.

Figure 13 shows, in one letter representation, the nucleotide sequence of the longest cDNA fragment and the amino acid sequence encoded therein in a cDNA encoding a polypeptide having peptide C terminal amidase enzyme activity from an isolated horse,

Fig. 14 and Fig. 15 are base sequences of a portion of cDNA encoding peptide C terminal amidation enzymes derived from rats which were digested with different restriction enzymes and used as probes, respectively.

Best Mode for Carrying Out the Invention

In the present invention, as the peptide C terminal glycine adduct of the formula (I), that is, the enzyme substrate of the present invention, Amino acid derivatives whose portions are natural or synthetic, in particular those compounds derived from peptides or proteins and whose glycine is peptide-bound to their C-terminal amino acid residues (represented by -H (H) -A-CO-) may be enumerated. . Such C-terminal amino acid residues include natural α-amino acids, in particular aliphatic amino acids such as protein constituting amino acids such as glycine and alanine; Side chain amino acids such as valine, leucine, isoleucine; Hydroxyamino acids such as serine and threonine; Acidic amino acids such as aspartic acid and glutamic acid; Amides such as asparagine, glutamine; Basic amino acids such as lysine, hydroxylysine and arginine; Sulfur containing amino acids such as cysteine, cystine, methionine; Aromatic amino acids such as phenylalanine and tyrosine; Heterocyclic amino acids such as tryptophan and histidine; The residues derived from imino acid, such as proline and 4-hydroxyproline, can be enumerated. In addition, as the residue [represented by X-] of a hydrogen atom or an amino acid derivative bound to an amino acid group or an α-amino group, which is an amino acid residue, peptides capable of peptide bonds via a single amino acid or α-amino group may be used. There is no restriction on the kind of the constituent amino acid residues and the chain length of the peptide, and a phosphoric acid, a sugar or other substituent may be covalently bonded to the constituent amino acid residues thereof, and may be complexed with fat. Specific examples of the substituents include groups substituted with an arginine residue guanidine group corresponding to each amino acid residue, for example, an alkyl group such as a methyl group or an ethyl group or a residue derived from adenosine diphosphate ribose, citrulline or ornithine; Groups substituted in the lysine residues ε-amino group, for example, glucosyl group, pyridoxyl group, biotinyl group, lipoyl group or compound having acetyl group or phosphoric acid, δ-hydroxy group, compound having δ-glucosyl group, glue Residues derived from aldehydes or citraconic anhydride; Residues derived from groups substituted by imidazole groups that are histidine residues, such as methyl groups, phosphate groups or iodine atoms or flavins; Groups substituted at the proline residues, such as hydroxyl, dihydroxy or glycosyloxy groups; Groups substituted in the benzene ring which is a phenylalanine residue, for example, a hydroxyl group or a glycosyloxy group; Derived from a group substituted by a hydroxyl group which is a tyrosine residue, for example, a glycosyloxy group, a sulfonic acid group, an iodine atom, a bromine atom or a compound having a chlorine atom or a hydroxyl group, bisether, adenine, urine or RNA (ribonucleic acid) Residues; Groups substituted in the hydroxyl group which is a serine residue, for example, a methyl group, a glycosyl group, a phosphopantein group, an adenosine diphosphoric acid group or a phosphoric acid group, and the group substituted in the hydroxyl group which is a threonine residue, for example, a glycosyl group, a methyl group, or a phosphate group ; Groups substituted in the SH group which is a cysteine residue, for example, glycosyl group, cystynyl group, dehydroalanyl group or selenium atom or group substituted in carboxyl group which is glutamic acid, aspartic acid or glutamic acid residue, for example, methyl group, glyco The actual group is a γ-carboxyl group, and examples thereof include groups substituted with an amide group of an asparagine or glutamine group, for example, a glycosyl group, a pyrrolidinyl group or an imino group.

The above-mentioned peptide C terminal glycine adduct as the substrate, that is, a peptide or a derivative thereof in which the glycine is peptide-bound to the C terminal residue, may be produced either by natural synthesis or by chemical synthesis, or may be produced using recombinant DNA technology. Thus, as the compound of the present invention, the compound of formula (I) includes peptides, for example, phosphate peptides represented by peptides having a number of amino acid residues of about 2 to 100, casein, protein kinase, adenovirus EIA protein, RAS protein and the like, and hydrolysates thereof. , Riboproteins represented by thromboplastin, α ₁ riboprotein, ribobiterine and the like, and hydrolysates thereof, metal proteins represented by hemoglobin, myoglobin, hemocyanin, chlorophyll, phycocyanin, flavin, rhodopsin, and the like. Glycoproteins represented by their hydrolysates, collagen, laminin, interferon α, serogloids, avidins and the like, along with other hydrolysates thereof, are amidated with other C-terminal carboxyl groups to form mature bioactive peptides such as calcitonin, secretin , Gastrin, vascular functional small intestine peptide (VIP), cholecystoginine, cellulose, pancreatic polypeptide, C-terminal glycine adducts (i.e., amide-binding compounds of C-terminal carboxyl groups and glycine) of peptides that form growth hormone-releasing factor, adrenal cortical stimulating hormone-releasing factor, calcitonin gene-related peptide, and the like. Among them, preferred substrates for confirming the enzymatic activity of the enzyme of the present invention include, for example, D-tyrosylvalylglycine, D-tyrosyl tryptophanyl glycine, glycylphenylalanyl glycyl, phenylalanyl glycylphenylalanyl Glycine, D-Tylosyloylsil asparaginylglycine, arginylphenylalanylglycine, arginylalanylarginylalloylglycine, leuylmethionylglycine, glycylloylsilmethionylglycine, phenylalanylglyciloylsilionylmethionyl Glycine, Asparaginyl arginylphenylalanylglycine, tryptophanyl asparaginylarginylphenylalanylglycine, alanylphenylalanylglycine, lysylalanylphenylalanylglycine, serylalysylalanylphenylalanylglycine , Arginyl tyrosyl glycine, glycylmethionyl glycine, glycyl tyrosyl glycine, glycyl histidyl glycine, histidyl glycyl glycine, tryptophanyl glycyl glycine and glyce It can be exemplified such as Tay carbonyl glycine (Also, except for glycine, and are not specifically shown as D- represents the L- form). On the other hand, a preferable substrate for effectively utilizing the enzyme of the present invention is a mature physiologically active peptide formed by amidating the above-mentioned C-terminal carboxyl group. Peptides to which glycine is peptide-bound in such a C terminal carboxyl group can be enumerated.

Enzyme-I of the present invention may act on the above-mentioned substrate to produce a C-terminal α-hydroxyglycine adduct of formula II.

Formula II

In the above formula,

Specific examples of moieties have the same meanings as defined for Formula I above.

Compound of Formula II It can be converted to the corresponding C terminal amidide by hydrolysis under conditions which do not adversely affect the part or by treatment with the enzyme-II of the present invention described below.

In addition, each of the enzyme-I is different depending on the origin, and according to the molecular weight determination method using gel filtration, for example, it is about 25 kilodaltons (KDa) in horses, and the rat is about It has a molecular weight of 36KDa. In addition, the molecular weight of the enzyme produced using the cDNA of the present invention has an equivalent or slightly larger molecular weight, as it sometimes involves an amino acid sequence derived from the addition base sequence. That is, the molecular weight can be determined according to a gel filtration method known per se (e.g., Seikagakujikken Koja 5, Koso Gankyubo, Sangku, 283-298, Tokyo Kagakudojin (1975)). Specifically, 50mM tris-hydrochloric acid (pH 7.4) containing 100mM potassium chloride is used as an equilibration and eluent, gel filtration is performed in TOYOPAL HW-55S (manufactured by Tosoh Corporation), β-amylase (MW 200,000), alcohol Hydrogenase (MW 150,000), BSA (MW 66,000), carbonic anhydrase (MW 29,000) and cytochrome C (MW 15,400) are determined as indicators.

Enzyme-I of the present invention does not differ by origin and is characterized according to the following physicochemical properties. In other words,

(a) a suitable pH of about 5-7 and a stable pH of 4-9,

(b) the optimum operating temperature is in the range of 25 to 40 ° C;

(c) Metal ions and ascorbic acid are used as carriers.

The properties of (a) and (b) described above are commonly used buffers and are specifically measured using tris-hydrochloric acid, mes-potassium hydroxide, tes-sodium hydroxide, hepes-potassium hydroxide buffer. In addition, the enzyme composition of the present invention acts as a catalyst for the above reaction within a temperature range of 1 ° C to 55 ° C, and it is shown that at 56 ° C, the activity is lost for about 10 minutes and slightly lost at around 40 ° C.

As the metal ion, Cu ²⁺ , Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Fe ³⁺ and the like are suitable, and Cu ²⁺ and Zn ²⁺ are particularly preferable.

The present invention also provides another one of the following enzymes. That is, enzyme-II is provided which acts on the peptide C terminal α-hydroxyglycine adduct of formula II to produce peptide C terminal amidide and glyoxylic acid of formula III.

Formula III

In the above formula,

A and X are as described above.

These enzymes also have molecular weights of about 40 KDa in horses and a molecular weight of about 43 KDa in rats, for example, according to the molecular weight determination method using gel filtration, when separating and purifying from the following enzymatically active contents, which differ in molecular weight depending on their origin. It is an enzyme involved in C-terminal amidation of peptide C-terminal glycine adducts. In addition, the molecular weight of the enzyme produced using cDNA may be slightly larger than that of enzyme-I. This enzyme is specifically used The meaning of the moieties is the same as that used specifically for Enzyme-I. In order to confirm the enzymatic activity of enzyme-II, preferred substrates include C-terminal α-hydroxyglycines corresponding to the substrates specifically listed for enzyme-I. For example, specific examples thereof include D-tyrosylvalyl α-hydroxyglycine, D-tyrosyl tryptophanyl α-hydroxyglycine, glycylphenylalanyl α-hydroxyglycine, phenylalanyl glycylphenylalanyl α-hydroxyglycine, D-tyrosylosilyl asparaginyl α-hydroxyglycine, arginylalanylarginyloxyl α-hydroxyglycine, leuylmethionyl α-hydroxyglycine, glycylosilmethionyl α -Hydroxyglycine, phenylalanylglycyloylsiliononyl α-hydroxyglycine, asparaginylarginylphenylalanyl α-hydroxyglycine, tryptophanylasparaginylarginylphenylalanyl α-hydroxyglycine, Alanylphenylalanyl α-hydroxyglycine, lysylalanylphenylalanyl α-hydroxyglycine, cerylsilylalanylphenylalanyl α-hydroxyglycine, arginyltyrosyl α-hydroxyglycine Shin, glycylmethionyl α-hydroxyglycine, glycyltyrosyl α-hydroxyglycine, glycyl histidyl α-hydroxyglycine, histidyl glycyl α-hydroxyglycine, tryptophanyl glycyl α-hydro Hydroxyglycine, glycesteininyl α-hydroxyglycine, and the like. Enzyme-II is also specified by having the following properties as other physicochemical properties. In other words,

(a) a suitable pH of about 5 to 6 and a stable pH of 4 to 9,

(b) The optimum temperature for action is in the range of 15 to 35 ° C.

The properties of (a) and (b) described above are commonly used buffers, and are specifically measured using tris-hydrochloric acid, mes-potassium hydroxide, tes-sodium hydroxide and hepes-potassium hydroxide buffer. In addition, it can be seen that the enzyme composition of the present invention acts as a catalyst for the above reaction within a temperature range of 1 ° C to 55 ° C or loses activity at about 56 ° C for about 10 minutes and slightly loses activity at around 40 ° C.

Preparation of the enzyme

The enzyme-I and enzyme-II of the present invention described above can be prepared according to the separation and purification method of enzymes known per se from the enzymatic activity-containing content, and preferably in the production method of the present invention described below. Can be obtained accordingly. That is, enzyme-I or enzyme characterized in that the enzymatic activity of enzyme-I or enzyme-II is subjected to substrate affinity chromatography and anion exchange chromatography with the peptide C terminal glycine adduct of formula (I) as a ligand. The preparation method of -II can be used.

The enzymatic activity content used in the present method can be used to control all those containing the enzyme of the present invention, and any naturally found or artificially provided one is good. Generally found in nature are organisms having such enzymatic activity, for example, mammals such as humans, cattle, horses, pigs, sheep, rabbits, goats, rats and mice, birds such as chickens, wild chickens and kawara pigeons. Reptiles such as dolphins, salsas, rattlesnakes and cobras, amphibians such as eternity, african claws, introductory copper and toads, seven-growing eel, blind eel, oil shark, hibiscus eel, butterfly shark, herring, salmon, eel, bumbok And products derived from insects such as fish such as sea bream, circular pattern cockroaches, silkworm moths, fruit flies, and bees. Specific examples of such preferable products include biological fluids containing homogenates derived from organs such as mammalian brain, pituitary gland, stomach, heart and liver, and body fluids such as blood and lymph fluid.

That is, biological fluids having the present enzymes as described above are commonly used for purification of enzymes as needed using substrate analog affinity chromatography with ligands of peptide C terminal glycine adducts of formula (I)

Formula I

In the above formula,

A and X are the same as described above.

(1) fractions from precipitation

(2) heparin affinity chromatography

(3) molecular weight fractionation method and / or according to dialysis, gel filtration, etc.

(4) By using a combination of ion exchange chromatography, the enzymes (enzyme-I and enzyme-II) of the present invention can be obtained.

As the ligand, any of the peptide C terminal glycine adducts of the above formula (I) may be used, but peptides consisting of 2 to 6 amino acid residues including glycine, which are specifically listed as preferable to confirm the enzyme activity, may be listed. Can be. Among them, D-Tyr-Trp-Gly, Phe-Gly-Phe-Gly and Gly-Phe-Gly are more preferred, and Phe-Gly-Phe-Gly is a ligand or in the enzyme of the present invention (called this enzyme). It is especially preferable because it has a strong affinity.

Such ligands are usually used in combination with a water-insoluble carrier, but the carboxyl group of the C-terminal glycine residue of the peptide used as the ligand is important in the binding to the present enzyme, and the N-terminal amino acid residue is linked to the carrier via the amino group. Need to be combined. In other words, any carrier can be used as long as it can bind to the amino group of the peptide, and a commercially available carrier which has introduced the active group reacting with the amino group chemically from the carrier or has already been introduced may be used. The method of chemical introduction is good as a commonly used method, for example, as described in Seigakakujikkenbo Vol. 5, 257-281 Kasaiiser Tokyo Kagakudojin (1975). For example, cyanide bromide is introduced into agarose to introduce an imide carboxyl group. Commercially available activating carriers include, for example, agarose, cellulose, hydrophilic polyvinyl, and the like, and any of these may be used. As the agarose carrier, CNBr-activated Sepharose 4B (manufactured by Pharmacia) using CNBr method in the method of binding a ligand to an amino group, CH-Sephalos 4B, ECH-Sephalos 4B (manufactured by Pharmacia company) using carbodiimide method ), Apigel 10, Apigel 15 (above, manufactured by Biorad, Inc.), tresyl activated cephalos 4B (manufactured by Pharmacia) using the tresyl chloride method, and the like. As the hydrophilic polyvinyl-based carrier, AF-carboxyyopal 650 using carbodiimide method, AF-formal density Yopal 650 using formyl method, AF-tresylyopal 650 using tresyl chloride method and epoxy activation method are used. We can enumerate AF-Epoxy Toy arm 650 (above, Tosoh Corporation) to say. The binding reaction with the ligand is good if the reaction according to the instructions of each carrier.

Among these, the manufacturing method regarding Apigel 10 is described. The reaction of Apigel 10 with the peptide is 0.001 to 1 M and is preferably carried out in a buffer such as 0.1 M morphs-potassium hydroxide. The reaction conditions may be 0 to 20 ° C., 10 minutes to 24 hours, and pH 3 to 11, but preferably 4 ° C., 4 to 24 hours, and pH 5 to 9 conditions. The mixing ratio of the peptide used in the binding to Apigel 10 is as much as the peptide is added to 1ml of Apigel up to about 25μmol, so it may be any number within the range, but from 1 to 20μmol is convenient in terms of binding efficiency. . After the reaction, the reaction solution was sufficiently washed with the buffer used in the reaction, and then tris-hydrochloric acid (pH 8.0) was added so that the final concentration was 50 mM, followed by shaking at 4 ° C for 1 hour. Blockade. Substrate analog affinity gels are prepared above.

Substrate affinity chromatography may be carried out batchwise or continuously by charging the column. The time for contacting the sample with the gel is good enough that the enzyme is sufficiently adsorbed, and is usually 20 minutes to 24 hours. At low ionic strengths of the same composition used to equilibrate the gel, the nonadsorbed components are washed out with a buffer of pH 6.0 to 11.0, preferably 7.0 to 9.0, for example 10 mM Hepes-potassium hydroxide (pH 7.0). . Among these, the fraction in which this enzyme activity exists is eluted. The eluate may be any composition so long as the enzyme is efficiently obtained, but preferred is a buffer between pH 7.0 and 9.0 containing 0.1 to 10 M sodium chloride with about 1 to 40% acetonitrile, for example 20% acetonitrile, 10 mM Hepes-Sodium Hydroxide (pH 7.0) containing 0.4 M sodium chloride may be enumerated. In addition, elution may be carried out over the whole concentration gradient, when a column is filled.

Further, in some cases, fractions obtained by pre-drying before or after or after performing the substrate analog affinity chromatography process (hereinafter, referred to as (5)) (hereinafter, referred to as (1)), heparinchin Chemical chromatography [hereinafter referred to as (2)] The molecular weight fraction [hereinafter referred to as (3)] and / or ion exchange chromatography [hereinafter referred to as (4)] according to dialysis, gel filtration, etc. may be performed. Do. In this way, the enzymes (enzyme-I and enzyme-II) can be separated from other miscellaneous products, but it is effective to perform the steps (3) and / or (4) to separate the enzyme-I and enzyme-II. Do. In general, it is preferable to carry out the above steps 1 to 6 and then to carry out the step (5) or (3) in the final step. As a specific example which combined each process, only in (5),

It is more preferable to advance the process in the order of.

The above-mentioned steps (1) to (4) will also be described in detail. In addition, they are all performed at 0 ° C to 10 ° C, preferably 4 ° C.

Substances used in the fraction according to precipitation in (1) may include salts such as ammonium sulfate, organic solvents such as ethanol and acetone, and polymers such as polyethylene glycol. The concentration to be added is not particularly limited, and conditions are preferred in which the enzyme can be recovered in good yield and can be separated from other protein components. For example, the addition of 30-50% saturated ammonium sulfate and 10-15% (W / V) polyethylene glycol 6000 results in an efficient purification since the enzyme is a precipitate fraction and most of the protein is in the upper portion. Can be. Moreover, when adding, it is preferable to carry out gradually, stirring with a stirrer. After completion of the addition, the mixture is allowed to stand for at least 1 hour and then centrifuged to recover a fraction present in the enzyme. When recovering the precipitated fraction, it is dissolved in a suitable buffer. The buffer may have any composition if the pH is 6.0 to 11.0 and preferably the pH is 7.0 to 9.0, and examples thereof include tris-hydrochloric acid, hepes-potassium hydroxide, tes-sodium hydroxide and the like. In addition, the concentration is not particularly limited as long as it maintains the buffering capacity, and 5 to 50 mM is preferable.

The active fraction obtained according to (1) is then good to proceed to any one of (2) to (5) even if (1) is again performed, but using salts such as ammonium sulfate in the fraction of (1) ( When proceeding to 2) or (4) or (5), it is necessary to add the appropriate buffer solution in step (3) or lower the salt concentration to which the enzyme can bind in the gel used in the next step. Insoluble matter may be generated when the precipitate is dissolved and purified for 1 hour or more, or when dialysis is performed, and thus, it is removed by centrifugation or filtration.

The heparin affinity chromatography of (2) may be carried out in a batchwise manner by gel filling the column in a continuous manner. Heparin-based ligand gels are commercially available and include Heparin Sepharose CL-6B (manufactured by Pharmacia), Apigelheparin (manufactured by Biorad), Heparin-Agarose (manufactured by Sigma), AF-heparindopal 650 (manufactured by Tosoh) .

The biological extract is subjected to treatment of the fractions directly or according to the precipitation described in (1), followed by contact with the heparin affinity gel. The contact time is good enough that the enzyme is sufficiently adsorbed, and is usually 20 minutes to 12 hours. A component that has no affinity for heparin is so low in ionic strength that the enzyme is not eluted and has a pH of 6.0 to 11.0, preferably 7.0 to 9.0, for example, 10 mM Hepes-potassium hydroxide (pH 7.0). Remove enough. Among them, the fraction containing this enzyme is eluted. As the eluate, it is preferable that the recovery rate of this enzyme activity is high, and for example, pH 6.0-11.0 containing salts generally used for enzyme purification, such as 0.5 M-2 M sodium chloride, potassium chloride, ammonium sulfate, etc., is preferable. When the elution is charged to the column, the elution may be carried out according to a salt concentration gradient or a one-step elution. For example, it is eluted with 10 mM Hepes-potassium hydroxide buffer (pH 7.0) containing 0.3-2.0 M sodium chloride.

The active fraction obtained in the step (2) may then be provided to any of the steps (1) to (4), but if (2) is carried out again or proceeds to (4) or (5), (3) (2), (4) or by adding a buffer of pH 6.0 to 11.0, preferably 7.0 to 9.0, which has been carried out in advance or in large amounts at a low ion strength of 50 mM or less, for example 5 mM Hepes-sodium hydroxide (pH 7.0) or the like. It is necessary to lower the ionic strength which the enzyme can adsorb to the gel used in (5).

In the case of dialysis in the step of removing low molecular weight substances according to dialysis, gel filtration and the like of (3), the membrane to be used may have a fractional molecular weight such that the enzyme cannot pass, but 1,000 to 10,000 is preferable. The method of dialysis is generally used, for example, as described in Seikagakujikken Koza Vol. 5, No. 5, pages 252 to 253, Saida Low Tokyo Kagakudojin (1975). For several days, the ionic strength is low and is performed with a buffer of pH 6.0 to 11.0, preferably pH 7.0 to 9.0, such as 10 mM Hepes-potassium hydroxide (pH 7.0), 10 mM Tris-hydrochloric acid (pH 7.5), and the like. do. In addition, when an insoluble substance precipitates at the time of dialysis, it is removed by centrifugation, filtration, etc., for example.

As regards gel filtration, anything that can be generally used as a carrier for gel filtration does not matter. For example, Sephadex G-10, G-15, G-25, G-50, G-75, G-100, Sephacryl S-200, S-300 (above, Pharmacia company), Toyota HW-40 , HW-55 (manufactured by Tosoh Corporation), Biogel P-2, P-4, P-6, P-10, P-30, P-60, P-100 (above, manufactured by Biorad Company), and the like are preferable. In addition, the buffers to be used are the same as the composition to be used for dialysis. However, if the ionic strength is considerably low, the concentration of the enzyme is considered to be 5 to 200 mM, preferably 10 to 20 mM. The method of gel filtration may be carried out as described in, for example, Seikagakujikken Koza Vol. 5, No. 283-298, Saida, Tokyo Kagakudojin (1975). Samples are added in an amount such that sufficient separation capacity is obtained for the bed volume of the gel filtration carrier, followed by elution to recover the fraction in which the present enzymatic activity is present.

The active fraction obtained according to the process of (3) can be advanced to each process of (1) to (5) without any special treatment.

Regarding ion exchange chromatography, any commercially available carrier for ion exchange chromatography may be used. For example, Aminex, Dowex, Amberlite, SP-Sephacryl M, Asahipak, DEAE-Toyopearl, DEAE-Sephadex, CM-Sepharose, DEAE, Bio-Gel A, CM-Cellulose, DEAE-Cellulofine, Partisil SCY, Mono Q and Mono S and the like are preferred. In addition, the buffer to be used and the method of use are preferably in accordance with the method described in the section of heparin affinity gel. In addition, the basic operation method is satisfactory according to the general method described in Shinkisosei Chemical Co., Ltd. 2, extraction and purification analysis I (Maruzen, 1988).

The active fraction obtained in the step (4) may then be provided to any of the steps (1) to (5), but if (4) is carried out again or proceeds to (2) or (5), (3) (2), (4) or by adding a buffer of pH 5.0-11.0, preferably 6.0-8.0, such as hepes-sodium hydroxide (pH 7.0) or the like, which has been carried out in advance or in a large amount and has a low ion strength of 50 mM or less. It is necessary to lower the ionic strength that the enzyme can adsorb to the gel used in (5). The crude product of the enzyme of the present invention is obtained through the above purification process. The crude product of such an enzyme can also be used as the present enzyme standard by isolating into fractions each having a peak at a molecular weight of about 25,000 and a molecular weight of about 40,000 as a protein separation means using the gel filtration step of (3).

Each of the above-mentioned steps is carried out by using a compound of formula (I) or formula (II) as a substrate and obtaining an active fraction according to the activity of enzyme-I and / or enzyme-II according to another method of measuring enzyme activity of the present invention described below. Is carried out.

Enzyme-I and Enzyme-II of the present invention both of these enzymes from a culture in which the above-described enzyme was produced and accumulated by culturing a host cell transformed with a plasmid capable of expressing and containing cDNA encoding the enzyme. Or it can manufacture by extracting one side.

CDNA encoding the enzyme of the present invention that can be used in this way is mammals such as humans, cattle, horses, pigs, sheep, rabbits, goats, rats, mice, birds such as chickens, turkeys, amphibians such as frogs, snakes, etc. It is derived from DNA in fish such as reptiles, sardines, mackerel, eel, salmon, etc., and the sequence of Lys-Lys exists near the center of the cDNA, and its origin is not a problem. can do. More specifically, the amino acid sequence of the currently known peptide C terminal amidation enzyme is optionally inserted into the deficient part (denoted by-) to increase the homology between types by the one-letter representation of amino acids and as shown in FIG. A cDNA which is a DNA fragment encoding an amino acid sequence and excludes a portion corresponding to a hydrophobic amino acid region near this C terminus may be advantageously used. In addition, each cDNA is described for human, horse, cow, rat, frog I and frog II, respectively. See Biochem, Bioph ys. Res. Commun, 169, 551-558, 1990; Japanese Patent Application No. 90-76331; Mol. Endocrinal. 1, 777-790 pages, 1987; Proc. Natl. Acad. Sci. USA, 86, 735-739, 1989; Biochem. Biophys. Res. Commum., 148, 546 to 552, 1987; And Biochem, Biophys. Res. Commun., 150, pages 1275-1281, 1988. For example, 441 and 442th in the sequence of the end of FIG. 5 correspond to the above-described K (lysine) and K (lysine) sequences. Such sequences are well preserved as cDNA of humans, horses, cattle, and rats. From this sequence the cDNA of the first half (5 'side) encodes a protein having activity to act on peptide C terminal glycine adducts of formula (I) to produce peptide C terminal α-hydroxyglycine adducts of formula (II). The cDNA of the latter half (3 'side) from the KK sequence encodes a protein having an activity that acts on the peptide C-terminal α-hydroxyglycine adduct of formula (II) to produce C-terminal amidide and glyoxylic acid of formula (III). have. CDNA may be separated in the first half and the second half using a restriction enzyme known per se at a site near this KK sequence.

Also, the hydrophobic amino acid region described above corresponds to, for example, the 880th V (valine) to the 901th I (isoleucine) according to the sequence shown in FIG. It is called an area. The use of cDNA fragments other than those corresponding to these regions surprisingly not only secretes the produced enzymes to the outside of the host cell, but also the overall production is remarkably important, so cDNA except for the above-mentioned region is particularly preferable as the cDNA used in the present invention. . In addition, although enzyme-I and enzyme-II of the present invention are encoded in cDNA adjacent to each other as described above, these enzymes are individually released as a process of secretion process in cells, so it is preferable to use cDNA except the above-mentioned membrane transmembrane region. Do. Such cDNA may be prepared by excising its portion from a known corresponding cDNA using a restriction enzyme known per se, and may also be selected from various cDNAs generated by differences in mRNA splicing in the cloning step of the cDNA. . In addition, cDNA isolate | separated as above-mentioned cDNA which encodes enzyme-I and enzyme-II, respectively independently can be used.

Cloning of the cDNA used in the present invention can be carried out using the above-described production of various animals according to a method known per se. Specifically, methods commonly used, such as the +, − method, hybridization method, PCR method, etc. (see Methods in Enzymology, vol. 152; Guide to Molecular Cloning Teehniques, SL Berger et AR Kimmel, 1987, Academic Press. Inc .; Methcds in Molecular Biology, vol. 4; New Nucleic Acid Techniques, JM Walker, 1988, The Humana Press Inc .; Molecular Cloning A Laboratory Manual 2nd Ed.J. Sambrook, EF Fritsch, T. Maniatis, 1989 Cold Spring Harbor Laboratory Press), and determine the cDNA region encoding the protein by determining the base sequence of the obtained cDNA clone, and if necessary select from where the portion corresponding to the membrane penetration region is removed and The desired cDNA can be obtained by cleaving the cDNA near the KK sequence in one central portion.

Using rats as an example, cells are crushed by homogenizing tissues producing large amounts of peptide C terminal amidase, for example, the rat's pituitary with guadiylthiocyanate and cesium chloride equilibrium density gradient ultracentrifugation RNA fractions are obtained. RNA with poly A (poly A ⁺ RNA) is then isolated from the RNA fraction described above by affinity chromatography with carrier oligo dT cellulose.

This poly A ⁺ RNA is used as a template and a cDNA library is obtained according to known methods, preferably Okayama-Berg (Mol. Cell. Biol. 2, 161, 1982). Screen positive cDNA clones using appropriate probes from these libraries, isolate positive cDNA clones obtained by rescreening with appropriate probes from amplified cDNA libraries, and construct the desired cDNA by such restriction enzyme mapping and sequencing. Can be. The plasmid containing the desired cDNA can also be selected by evaluating the productivity of the peptide C terminal amidation enzyme of the host which is inserted into the expression vector and thereby transformed with the cDNA.

As a host expressing such cDNA, cells commonly used, such as cultured cell systems derived from microorganisms such as Escherichia coli, Bacillus subtilis, yeast, insects, animals and the like, can be listed. The expression plasmid is satisfactory as long as it is a plasmid capable of efficiently expressing cDNA in these cells. For example, it can select suitably from the documents described below.

Choku Seika Gakuji Kenken Koza, Idenshi Kankyubo II, -Recombinant DNA Technology-Chapter 7 Expression of the Ring (1986), Nippon Seikagaku, Tokyo Kakukudojin; Recombinant DNA, Part D, Section II, Vectors for expression of Cloned Genes, (1987) RayWu and Lawrence Grossman, Academic Press, INC; Molecular Cloning, A Laboratory Manual 2ne Ed. Book 3, (1989) J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, et al.

For example, when CV-1 used as an animal culture cell is used as a host, the promoter of pSV, pL2n, pCol type, and what arrange | positioned selection marker as needed can be used. Also, pGH, pKYP, and pHUB type vectors can be used for E. coli, and YRp and YEp types can be used for yeast. Transformation and transduction of the host cells according to the recombination according to cDNA and the recombinant plasmid of these vectors can be carried out according to their own known levels described in the above-mentioned documents, respectively. The transformed cells thus obtained can be cultured under medium and culture conditions commonly used to propagate derived cells.

Peptide C terminal amidation enzymes produced and accumulated from such cultures can be easily collected from the culture medium after removing the cells, for example, because the production enzymes are secreted outside the cells when animal culture cells are used. If necessary, the sample may be collected from the cell lysate. Such collection and purification may be carried out by combining conventional enzyme purification methods, for example, fractions according to precipitation, heparin affinity chromatography, and dialysis, and the method of extracting the enzyme from the biological fluid described above, peptide C terminus. Preference is given to using a combination of substrate affinity chromatography with glycine adducts as ligands.

The enzyme-I of the present invention thus obtained can be referred to in FIG. 5 with respect to enzyme-I in human, horse, bovine, and rat, respectively, from 42 residues P or S to 442 residues K, or in horses and cattle. The amino acid sequence is from P or S at the 42 residue to K at the 231 residue. On the other hand, it can be obtained that enzyme-II corresponds to the amino acid sequence from the 443th D to the 830th K in humans, horses, cattle and rats, respectively.

Corresponding herein includes those wherein an enzyme of the present invention optionally binds a sugar to an aspartic acid of its amino acid sequence via N-acetylglocosamine.

Use of enzyme

Process for the preparation of the peptide C terminal α-hydroxyglycine adduct of formula (II) characterized in that the use of the enzyme of the present invention, i.e., the peptide C terminal glycine adduct of formula (I) is treated with the above enzyme-I A process for the preparation of the peptide C terminal amidide of formula (III) can be provided which is characterized by treating said adduct with enzyme-II.

These enzyme-I and enzyme-II can also be combined to convert compounds of formula (I) to compounds of formula (III) in a single reaction composition. The use of enzyme-II in the conversion process from the general formula (I) to the compound of the general formula (III) requires the use of chemical hydrolysis conditions in the conversion of the general formula (II) to the general formula (III) in the presence of only the enzyme of the enzyme-I type. It is clear that the conversion can be achieved in comparison with more relaxed enzymatic conditions. In particular, it is suitable for the treatment of unstable substrates under alkaline conditions.

These preparation methods can be used irrespective of their concentration and purity as long as they contain the enzyme of the present invention. However, the present invention is characterized in that the contaminant protein is greatly removed in view of the isolation of the product from the reaction mixture and the compound of formula II. It is advantageous to use enzymes.

The compounds of formula (I) or formula (II) include all of the foregoing, but those suitable for use in their preparation include, in particular, their final products, compounds of formula (III) such as alkynine vasothocin (AVT), luteinizing hormone Releasing hormone (LH-RH), oxytocin, gastrin, gastrin secretion promoting peptide (GGRP), calcitonin (CT), vasoactive intestinal peptide (VIP), thyroid stimulating hormone releasing hormone (TRH), black vesicle stimulating hormone (MSH) , MSH release inhibitory hormone (MIH), cholecystokininoctaheptide (CCK-8), substance P (SP), fat mobilizing hormone, quippeptide (PP), growth hormone releasing factor, secretin, cellulose, mollusk heart excitability Neuropeptides, vasopressin, corticosteroids (ACTH), discoloration hormones, bone besin, acclimatization hormone, motilin, apamin, aridin, eredocin, katsinin, granuliverin R, scothobin, Corresponds to Hiranbate Cellulose, Mast Cell Degranulation Peptides, Pisalmine, Pyrocellulose, Pyrromethezin, Promelitin, Bonvinin, Mastparan, Mastparan-X, Melitin-1, Lanathecin and Ranathecin-R The compound which makes it preferable.

The above treatment may be carried out by adding ascorbic acid and catalase to the reaction solution, especially in a reaction using enzyme-I in a conventional buffer, but it is preferable to consider the conditions of the method for measuring enzyme activity shown below.

Method for measuring enzyme activity and searching for new enzyme using same

The above-described enzyme-I and enzyme-II of the present invention can be traced by the following activity measurement method, and this measurement method is also useful for reasonably carrying out the above-described method for producing the enzyme of the present invention.

Of course, these methods are based on the fact that the peptide C terminal amidation reaction is not a one step reaction as is known in the art, but a two step reaction that mediates an intermediate (peptide C terminal α-hydroxyglycine adduct). will be.

First of all, the activity of enzyme-I is determined by (a) buffering a subject whose pH can be expected to have a pH of 5 to 8, and (b) the peptide C terminus represented by the formula (I) in such a buffer solution. Incubating with the addition of glycine adducts and L-ascorbic acid and catalase, and isolating and measuring the product represented by the above formula (II) from the reaction mixture thus obtained using various known chromatographs described below. Or can be determined by converting the compound of formula (II) to the compound of formula (III) under alkaline conditions and then measuring it isolated. One such preferred isolation measurement method includes the step of detecting the reaction product by HPLC using acetonitrile-containing buffers (pH 6-10).

In addition, the activity of enzyme-II is (a) buffering a subject whose pH can be predicted to have a pH of 4 to 8, and (b) the peptide C terminus represented by the formula (II) in this buffer solution. incubating with addition of the α-hydroxyglycine adduct, whereby the reaction product, formula III or glyoxylic acid, thus obtained, can be determined by a known method known per se. The activity of enzyme-II is also preferably detected by said HPLC.

In these measurement methods, any of the test subjects includes any fluid having an enzyme activity, and in particular, a biological fluid having such activity, i.e., a homogenate of an organ of a living organism, and a body fluid such as blood and lymphatic fluid, Treatment liquids, such as a purification process, are mentioned. In addition, a treatment liquid derived from a microbial cell is also included in the biological fluid.

The buffer used for buffering these subjects is not specifically limited, What is conventionally used can be used. For example, tris-hydrochloric acid, hepes-potassium hydroxide. The concentration of buffer in the buffer may be any concentration as long as buffering can be achieved, but usually 20 to 200 mM is appropriate.

Each buffer is adjusted to pH 6-8, preferably pH 6.5 for the former, and to pH 4-8, preferably pH 6 for the latter. As the peptide C-terminal glycine adduct added in the former to the buffer thus prepared, it is a substrate of the enzyme, and a compound represented by the formula (I) listed as a preferred substrate for confirming the activity of the enzyme-I described above is used. desirable. The concentration of this compound is generally about 0.1 μM to 2 mM. In addition, addition of L-ascorbic acid, which is thought to function as a carrier, and catalase as an activator are required. In general, the concentration of L-ascorbic acid is preferably 0.5 to 2 mM, and the concentration of catalase is suitably 40 to 100 μg / ml. In addition, although metal ions may be added to the buffer solution, such addition is not particularly required for the present activity measurement, but is preferably added because higher activity can be obtained than by addition. As the metal ion to be used, Zn ²⁺ , Cu ²⁺ , Ni ²⁺ , Co ²⁺ , Fe ³⁺ and the like are suitable, and Cu ²⁺ and Zn ²⁺ are particularly preferable. The concentration of the metal ions in the buffer solution is suitably 0 to 1000 µM, preferably 0 to 10 µM. Although not limited, compounds providing such metal ions include CuSO ₄ , CuCl ₂ , ZnCl ₂ , NiCl ₂ , CoCl ₂ , FeCl _3, and the like.

As a specific example of the reaction composition, for example, the reaction composition A of Example 7 to be described later will be referred to. On the other hand, in the latter, the reaction composition is prepared by using the corresponding compound of formula II instead of the compound of formula I. In this case, carriers such as ascorbic acid and catalase are not necessary.

In both measurement methods, the amount of the subject to be used is not particularly limited and can be varied. However, the amount of the substrate (a nanomolar (nmol)) in the reaction system is preferably a pmol / hr or more, more preferably. Is at least 10 X a pmol / hr, more preferably from 10 X a pmol / hr to a mol / hr [unit indicates enzymatic activity and reacts at 37 ° C. for 1 hour (for example, picomol ( pmol))] is appropriately adjusted to contain the enzymatic activity.

The incubation is allowed to react for 2 to 24 hours with shaking at 1 to 55 ° C., especially at the former, preferably at 25 to 40 ° C., particularly preferably at 30 ° C., and in the latter, preferably 15 to 35 ° C., more preferably 25 Allow to stand at < RTI ID = 0.0 > C < / RTI >

In the above processes, the detection of the compounds of formula II and the compounds of formula III, respectively produced, is carried out with these substrates and products, for example in the former compounds of formula I and compounds of formula II, in the latter compounds of formula II and compounds of formula III Any method can be used as long as it can measure separately. In general, the separation measurement can be carried out by separation and purification by the following chromatography. Chromatography that can be used above includes ion exchange chromatography, reverse phase chromatography, gel permeation, affinity chromatography, high performance liquid chromatography (HPLC), thin layer chromatography (TLC) and the like. In the latter reaction system, the substrate represented by the formula (II) and the amidated product represented by the formula (III) each have a carboxyl group and an amide group at the peptide C ends, and differ in charge. Ion exchange chromatography, reverse phase chromatography, etc. using this property are suitable. Affinity chromatography using antibodies of the product is also effective. However, in the former reaction system, the substrate represented by the formula (I) and the product represented by the formula (II) are very difficult to separate by conventional chromatography, but the acetonitrile-containing buffers (pH 6 to 10, Preferably, high-performance liquid chromatography (HPLC) using pH 9) as the eluent can advantageously measure the separation. The eluate is particularly preferably performed by multiplying the linear concentration gradient of acetonitrile concentration. As the column of HPLC, any kind of commercially available column may be used as long as it is suitable for this purpose, but it is particularly advantageous to use capsule pack C18SG, 300 kPa (manufactured by Shiseido).

In this way, each of the separated substrates and products may be measured for the chemical or physical properties of any of them (coupled as necessary). Such measurements may use known properties, known methods, and in general it will be suitable to use UV absorption derived from the amino acids constituting the substrate peptide.

Since the above measuring methods are accurate and simple, the screening of enzymes having enzyme-I or enzyme-II activity can be performed by appropriately using them in the above-described biological fluids.

The biological fluid to be searched can be predicted to have the above-described enzymatic activity, and also includes all living cells, tissues and extracts of other plants and animals. For example, extracts can be found in Experimental Biology 6, Cell Fractionation (Circulation, 1984), Biochemistry Experiment 5, Enzyme Research (Phase) (Tokyo Chemical Drivers, 1975), Basic Biochemistry Experiment 1, Handling of Biomaterials (Circulation) , 1974).

DNA sequence for enzyme-I and enzyme-II derived from horses

According to the present invention, cDNA sequences are provided which encode polypeptides having enzymatic activities of enzyme-I and enzyme-II. Since these enzymes are likely to be superior in activity and stability to peptide C terminal amidation enzymes known in the art (see WO 89/12096), the above DNA sequences are provided in terms of producing them. The meaning can be confirmed. The origin of these enzymes is primarily of the atrial, pituitary, brain or stomach origin, regardless of the type of organ or tissue in which they are present.

Specifically, cDNA encoding a polypeptide having peptide C terminal amidation enzyme activity according to the present invention is shown in FIG. 13. In this figure, the nucleotide sequence of the longest cDNA fragment and the amino acid sequence encoded therein are shown in single letter. In other words, the [] in the figure is a cDNA deletion part believed to be caused by the difference in mRNA splicing revealed by analyzing several cDNAs. Accordingly, cDNAs according to the present invention also exist in several amino acid sequences of polypeptides to be encoded. For example, the amino acid sequence of a polypeptide having the enzymatic activity of the enzyme of the present invention derived from the horse provided in the present invention has a common sequence up to a constant chain length, at least as follows, and further downstream of each of the four sequences There are four species, including which one.

Consensus amino acid sequence

Amino acid sequences of different regions

Since the polypeptides having these amino acid sequences can not only be translated by four cDNA sequences, but also by using DNA combining different codons encoding the same amino acids, the DNA sequences of the present invention include them all. do. Moreover, even if a part of the amino acid sequence is changed by substitution, addition or removal to the extent that the C terminal amidation enzyme activity is not lost, it is interpreted as meeting the original object of the present invention. Specific examples thereof include those having the following common nucleotide sequences and downstream of each having the following different nucleotide sequences:

Consensus base sequence

Different nucleotide sequences

Cloning of the cDNA of the present invention can be carried out by a method known per se using the tissues described above in the same manner as described for the rats described above.

Hereinafter, the cDNA preparation method of the present invention will be described in detail.

In horses, tissues that produce a large number of peptide C terminal amidation enzymes (hereinafter referred to as benign tissues), for example, by homogenizing the atrium of horses with guanidylthiocyanate, cesium chloride equilibrium density gradient RNA is obtained by fractionation by ultracentrifugation. RNA with poly A (poly A RNA) is then isolated from the RNA fraction by affinity chromatography carrying oligo dT cellulose.

Using this poly A ⁺ RNA as a template, cDNA libraries are obtained by known methods, preferably by the method of Okayama-Berg (Mol. Cell. Biol. 2, 161, 1982). The method of Okayama-Berg is carried out as follows. That is, cDNA is synthesized by adsorbing the poly A portion of poly A ⁺ RNA to the poly T portion of the vector of Okayama-Berg and reacting reverse transcriptase. Oligo dC with terminal deoxynucleotidyl transferase is added to the 3 'end of the cDNA, followed by cleavage of the vector DNA with restriction enzyme Hind III. After linking the oligo dG linker and then circularizing the vector, the RNA moiety is replaced with DNA by DNA polymerase to obtain a cDNA containing plasmid. These plasmids are used to transform E. coli by the calcium chloride method (Strik, P. et al., J. Bacteriol. 138, 1033, 1979). Plasmid receptive bacteria are obtained by selecting ampicillin resistant strains from the ampicillin-added plate medium.

On the other hand, the positive tissues, that is, tissues that produce a large amount of C-terminal amidase and tissues that do not produce much C-terminal amidase (hereinafter, referred to as negative tissue), for example horse liver, are prepared. Poly A ⁺ RNA is isolated from the cells in accordance with the method described above. 5 'OH of RNA is labeled with ³² P using polynucleotide kinase and [γ- ³² ] PATP and used as probe.

Next, in the cDNA library described above according to colony-hybridization method (see Hanahan, D. et al., Gene, 10, 63, 1980), the colony was complementary to the probe derived from the benign tissue but not complemented to the probe derived from the negative tissue. Select. The plasmid DNA is obtained from the colonies thus selected, and the nucleotide sequence is determined according to the dideoxynucleotide method (Messing, J. Methods in Enzymology 101, 20, 1983) and the like.

Whether they are cDNA of peptide C terminal amidase, the region encoding its amino acid sequence is introduced into expression vector systems of Escherichia coli, Bacillus subtilis and yeast animal culture cells to produce a protein encoded by cDNA and then its amidase activity. [Reference: PCT / JP 89/00521 specification] can be confirmed by measuring. In addition, the obtained cDNA may be selected by comparing the homology with the above-mentioned known peptide C terminal amidation enzyme cDNA. Furthermore, some amino acid sequences of the enzyme purified using the purification method of the horse C terminal amidation enzyme described in International Publication No. WO 89/12096 are determined by a peptide sequence analyzer or the like, and the amino acid sequence estimated from cDNA. It is also good to check the same with. Moreover, the antibody using the purified enzyme as an antigen may be constructed by rabbits, rats, and the like, and then confirmed by performing an antigen-antibody reaction with a protein expressed in E. coli and the like by the above-described cDNA.

These confirmation means can be used as a method of cDNA cloning using these characteristics. Namely, among the known heterologous C-terminal amidation enzyme cDNAs, regions having high homology among these species are considered to have high homology even with cDNA derived from horses, and a method for screening a cDNA library using DNA of such regions as a probe; a screening method using an antibody as a probe in a cDNA cloning system using λgt 11 phage; The screening method of cDNA library which produces | generates the synthetic DNA (made into several kinds) which linked the corresponding codon by the some amino acid sequence of a purified enzyme with a DNA synthesizer, etc., and this is constructed using a plasmid, phage, etc. as a probe.

The DNA sequence encoding the protein having the peptide C terminal amidation enzyme activity of the present invention thus prepared is expressed by enzyme-I by connecting its DNA to a suitable expression vector to express E. coli, Bacillus subtilis, yeast, animal cells, etc. as a host. And / or a large amount of enzyme-II can be produced.

According to the invention an enzyme which acts on a C-terminal glycine adduct of formula (I) to produce a C-terminal α-hydroxyglycine adduct of formula (II) (hereinafter referred to as enzyme I) and a C-terminal α of formula (II) It acts on a hydroxylglycine adduct to provide an enzyme (hereinafter referred to as enzyme-II) that is involved in the production of C-terminal amidide and glyoxylic acid of formula III.

In the above formula,

A is a residue other than an α-amino group or an imino group and an α-carboxyl group derived from a natural α-amino acid,

X is a residue of an amino acid derivative which bonds to an N atom via a hydrogen atom or a carbonyl group.

In addition, the hydrogen atom (H) in the form of the formulas (I), (II) and (III) means that when A is derived from an α-amino acid having an α-imino group, it does not have a hydrogen atom.

The use of these enzymes individually or in combination allows efficient conversion of the peptide C terminal glycine adducts of formula (I) to the corresponding peptide C terminal amidates of formula (III).

In addition, according to the present invention, a method for preparing such an enzyme from the above-described enzyme content using substrate affinity with a specific ligand as a carrier and a method for efficiently preparing each enzyme using cDNA encoding such an enzyme-activated polypeptide This is provided.

The present invention also provides a method for measuring the enzyme activity described above and a method for screening such enzyme content.

The present invention also provides a cDNA encoding a polypeptide having the above enzymatic activity derived from horses.

Next, the present invention will be described in more detail with reference to Examples.

However, the present invention is not limited by these.

Example 1

Preparation of Substrate-like Affinity Chromatography Gel

5 ml of Apigel 10 is added to a 10 ml Econo column (manufactured by Biorad, Inc.) filled with isopropanol.

After isopropanol was distilled off, it was washed with 50 ml of 10 mM sodium acetate buffer (pH 4.5), followed by 10 ml of 0.1 M morphose-sodium hydroxide buffer (containing pH 80 potassium chloride, pH 7.5). The gel was transferred to a 20 ml bottle, and then mixed with 10 ml of the morphose-sodium hydroxide buffer in which 40 mg (about 100 μmol) of phenylalanyl glycylphenylalanyl glycine (Phe-Gly-Phe-Gly, manufactured by Sigma) was dissolved. The reaction was shaken at 4 ° C. for 18 hours.

Then, 0.5 ml of 1M Tris-base buffer (pH 8.0) was added to shake for 1 hour at 4 ° C, and the unreacted activator was inactivated. The gel is washed with the Mofus-sodium hydroxide buffer, followed by ion exchanged water, then suspended in 0.02% NaN ₃ to fill the column and stored at 4 ° C. From the amount of peptide (Phe-Gly-Phe-Gly) used for the reaction and the amount of peptide in the recovered solution, it was calculated that about 10 μmol per 1 ml of gel was bound.

Example 2

Preparation of Phenylalanylgylsilphenylalanylhydroxyglycine as Substrate

Taken by measuring 3 mg of phenylalanyl glycylphenylalanyl glycine (FGFG) (manufactured by Sigma), 50 mM Hepes-potassium hydroxide buffer (pH 5.5), 3 mM ascorbic acid, 10 mM potassium iodide, 0.25 mg / ml catalase, 0.25 nM Copper sulfate, 7.5% acetonitrile and 200 μl of horse serum amidation enzyme composition as described in Example 2 of International Patent Application No. JP 89-00521 are added to a total amount of 10 ml, and aerobic for 20 hours at 30 ° C. To amidation reaction. The reaction is stopped by addition of 10% formic acid, and phenylalanylglyciylphenylalanylhydroxyglycine (FGFhyG) is obtained separately using high performance liquid chromatography (HPLC). The column of HPLC uses Capsule Park C 18SG, 300 kPa (Sugar Sugar). As the eluting solvent, 1 mM ammonium bicarbonate (pH 9.0) and acetonitrile were used, and the acetonitrile was increased from 0% to 40% for 30 minutes to apply a linear concentration gradient. Peptides are detected at an absorption of 214 nm. The results are shown in FIG. The peak of 10.7 minutes of phenylalanylglysylphenylalanyl glycyl is almost extinguished by the C terminal amidation reaction. Along with this, peaks of the α-hydroxyglycine derivative at 9.9 minutes and the amidated body at 14.5 minutes are found. These materials are structurally identified by FAB-MS spectral analysis and NMR analysis. In FIG. 2, the result of FAB-MS spectrum in a glycerin solution is shown. The parent peak has a molecular weight of 442, where fragmentation results in fragments of 425 and 408 m / z missing one or two -OH groups present at the C-terminus, indicating that they are α-hydroxyglycine adducts. A substrate of enzyme-II of the present invention is prepared by aliquoting a peak of 9.9 minutes and rapidly lyophilizing to 10% formic acid solution. This substrate can be prepared in the same manner using the enzyme-I content of the present invention instead of the amidation enzyme composition. As described above, the α-hydroxyglycine derivative is stable under acidic conditions but unstable under alkaline conditions and decomposes to amidates and glyoxylic acids regardless of the oxygen reaction. Therefore, the C terminal amidation reaction carried out at the beginning of this example is carried out under acidic conditions. Subsequently, the reaction at pH 7.5 or higher no longer confirms the α-hydroxyglycine derivative. It is believed that known C-terminal amidases are converted from peptide C-terminal glycine adducts represented by Formula (I) to peptide C-terminal amidates represented by Formula (III) and glyoxylic acid. Since the conversion is included, the catalytic reaction of oxygen itself is the conversion of the peptide C terminal glycine adduct represented by the formula (I) to the peptide C terminal α-hydroxyglycine adduct represented by the formula (II). Thus, the yield of amidation reactions under acidic conditions with conventional peptide C terminal amidation enzymes is generally low.

Example 3

Preparation of Enzyme-I from Equine Serum

(1) To 100 ml of commercially available serum (produced by Gibuco Co.), 25% aqueous solution of polyethylene glycol 6000 (product) (w / v) is gradually added to 100 ml, that is, to a final concentration of 12.5%. The operation is all performed below at 4 ° C. After standing for 12 hours, the precipitate obtained by centrifugation (10,000 xg, 10 minutes) was dissolved in 120 ml of 10 mM Hepes-potassium hydroxide buffer (pH 7.0), and again standing for 2 hours, and then the resultant insoluble substance was again centrifuged. Separation (10,000 × g, 10 min) removes to give a supernatant (127 ml) containing enzyme-I.

(2) The active fraction obtained in (1) above was subjected to a column (1.6 × 15 cm) packed with Heparin Sepharose CL-6B (from Pharmacia) equilibrated with 10 mM Hepes-potassium hydroxide buffer (pH 7.0). The nonadsorbed material was washed out with 96 ml of the same buffer and then eluted with 10 mM Hepes-Potassium hydroxide buffer (pH 7.0) containing 0.5 M sodium chloride (flow rate 30 ml / hour). The elution pattern is shown in FIG. Enzyme-I elutes with 0.5 M sodium chloride containing buffer [fraction no. Collect 14-16 (100 ml)].

(3) The fractions were gel filtered using Sephadex G-25 Fine (Pharmacia) column chromatography (5 cmφ × 23 cm). The solvent is eluted with 10 mM hepes-potassium hydroxide) (pH 7.0) at a flow rate of 2 ml / min. The protein is detected at absorbance of 280 nm to collect 100 ml of fractions containing the protein.

(4) 5 ml of Apigel 10-Phe-Gly-Phe-Gly gel prepared according to Example 1 was charged to a column (1.0 × 6.3 cm), and 10 mM Hepes-Potassium hydroxide buffer containing 0.1 M sodium chloride (pH Equilibrate to 7.0). The sample (18.1 ml) obtained in the above (3) is hanged on this column. The liquid passed through the column is circulated through the column several times (flow rate 20 ml / hour) to sufficiently adsorb enzyme-I to the gel. After 12 hours, the circulation was stopped and the nonadsorbed material was washed with 35 ml of the buffer used for equilibration and then eluted with 8 mM Hepes-Potassium hydroxide buffer (pH 7.0) containing 0.4 M sodium chloride, 20% acetonitrile (pH 7.0). Flow rate 20 ml / hour). The activity of enzyme-I is confirmed only in the elution fraction (10 ml).

(5) Mono Q column (0.5x5cm from Pharmacia Co., Ltd.), wherein the purified product obtained in (4) was again subjected to the treatment of (3) and then equilibrated with 10 mM Hepes-potassium hydroxide buffer (pH 7.0). In the same buffer, the linear concentration gradient of NaCl is walked as shown in FIG. 3 to elute the protein. At this time, the flow rate is 0.5 ml / min.

Table 1 shows the shear back mass, preenzyme activity, inactivity, yield, and purification ratio in each step of the purification performed in the above (1) to (5).

Preparation of Enzyme-I from Equine Serum step Shear protein (mg) Fully active (U) Inactive (U / mg) yield(%) Tablet magnification serum 7,500 10,500 ^* 1.4 100 (1) polyethylene glycol 6000 precipitation 4,100 9,020 2.2 86 1.6 (2) Heparin Sepharose CL-6B 1,400 5,740 4.1 55 2.9 (3) Sephadex G-25 1,100 3,960 3.6 38 2.6 (4) Apigel 10-Phe-Gly-Phe-Gly 2.0 800 400 8 290 (5) Mono Q column 0.5 350 700 3 500 It is not known whether it is due to the effects of proteolytic enzymes present in serum, but relatively low activity due to partial degradation of substrate and product.

The activity assay is performed by performing a reaction according to the method for preparing FGFhyG from FGFG described in Example 2 for each purified product and quantifying FGFhyG by HPLC described herein. Enzymatic activity 1U is the amount produced by 1 nmol of FGFhyG at 37 ℃ for 1 hour.

In addition, the measurement of the amount of protein was carried out by Lowry's improvement method [Bensadoun et al., Anal. Biochem., 70, 265, 1976, the standard curve is constructed with bovine serum albumin (fraction V, Sigma).

As shown in Table 1, the enzyme can be purified about 500-fold in yield 2%. Moreover, when refinement | purification is needed, said process (3)-(5) is repeated, but what is necessary is just to repeat any of these processes.

Example 4

Preparation of Enzyme-II from Equine Serum

Equivalent serum was treated in the same manner as in Example 3 except that each purification step was performed while tracking the activity of enzyme-II.

Table 2 shows the shear protein, preenzyme activity, inactivity, yield and purification ratio of each purification step (1) to (5).

Preparation of Enzyme-II from Equine Serum step Shear protein (mg) Fully active (U) Inactive (U / mg) yield(%) Tablet magnification serum 7,500 2,100 ^* 0.28 (1) polyethylene glycol 6000 precipitation 4,000 5,300 1.3 250 4.6 (2) Heparin Sepharose CL-6B 1,500 3,800 2.5 180 9.0 (3) Sephadex G-25 1,200 3,000 2.5 140 9.0 (4) Apigel 10-Phe-Gly-Phe-Gly 1.2 360 300 17 1070 (5) Mono Q column 0.1 50 500 2 1790 It is not known whether it is due to the effects of proteolytic enzymes present in serum, but relatively low activity due to partial degradation of substrate and product.

Activity measurement was performed by dissolving phenylalanylglyciylphenylalanylhydroxyglycine (FGFhyG) obtained in Example 1 in 10 mM Hepes-potassium hydroxide (pH 6.5) at a concentration of 5 mM and adding a sample of each step to a total amount of 100 µl. Reaction at 30 ° C. After reacting for 1 hour, the reaction is stopped by adding 10% formic acid, and the reaction product is quantified by HPLC using the conditions of Example 2. At this time, the reaction of the sample-free control was also performed, and it was confirmed that all of the non-enzymatic conversion did not proceed. The HPLC pattern of the reaction solution is shown in FIG. Activity is defined as the amount of enzyme that 1 U expressed in units (U) produces 1 nmol of FGF-NH ₂ at 30 ° C. for 1 hour.

In addition, the measurement of protein amount is performed similarly to Example 3. As shown in Table 2, the enzyme can be purified about 1800-fold in yield 2%.

In the following examples, the production of sugar enzymes using peptide C terminal amidation enzyme cDNA derived from rat pituitary gland is described.

Example 5

Preparation of Expression Plasmids

CDNA cloning is performed using poly A ⁺ RNA derived from the rat pituitary to give five cDNAs of different molecular weights (FIG. 6, FIG. 7, Biochemistry, 61, 842 (1989)). 2.58kbp DNA fragment digested with EcoR I-Xma I of cDNA clone 205 was obtained from animal culture cell line expression vector pSV2 vector [S. Subramani, R. Mulligan, P. Berg, Mol. Cell. biol. 1, 854 (1981), inserted via a synthetic linker via the Hind III-Bgl II site, and this plasmid is named SV-205. Subsequently, the Nsi I (700) -Xma I fragment of SV-205 is substituted with the Nsi I (700) -Xma I fragments of cDNA clones 201, 202, 203, and 204, respectively. These expression plasmids are referred to as SV-201, SV-202, SV-203, and SV-204. Of these, it was confirmed that the portion of SV-203 corresponding to the transmembrane region of cDNA was removed. This constructs an expression plasmid SV-A expressing an enzyme that acts on the peptide C terminal glycine adduct from the obtained SV-203 plasmid DNA and converts it to the peptide C terminal α-hydroxyglycine adduct. DNA portions other than the BamH I site [FIG. 7, B (1386)], which exist near the cDNA region encoding the KK sequence portion near the center, are digested with BamH I, Xma I [FIG. 7, X (2948)]. Removed by a synthetic DNA linker at the cleavage site Insert and link, then complete the SV-A plasmid. Synthetic DNA is purified by synthesizing according to a conventional method using a DNA synthesizer manufactured by ABI. This synthetic DNA consists of the BamH I cleavage site-purification codon-Xma I cleavage site.

Next, an expression plasmid SV-B according to the present invention expressing an enzyme converting the C-terminal α-hydroxyglycine adduct into a C-terminal amidate and glyoxylic acid is constructed. The SV-203 DNA is cleaved into a Kpn I site (Fig. 7, N (175)) located below the region encoding the signal peptide and a BamH I site located at a position corresponding to the central KK site. Between synthetic DNA It connects by and it is set as expression plasmid SV-B. As a result, the signal peptide region and the frame of the second half of the cDNA are joined together.

Example 6

Expression in Animal Culture Cells

Cultured cell COS-7 is grown in synthetic medium (DMEM) containing 10% bovine placental serum and transformed using the expression plasmid of Example 1 by a known method [C. Chen and H. Okayama, Mol. Cell, Biol. 7, 2745 (1987). 20 μg of expression plasmid is used for 5 × 10 ⁵ cells. After 24 hours of incubation at 35 ° C. under 3% carbon dioxide, the cells were washed twice with 10 ml of DMED medium containing 0.2% bovine serum albumin (BSA), and then, in 10 ml of DMEM medium containing 0.2% BSA, 5 Incubate again for 48 hours under conditions of% carbon dioxide, 37 ° C.

Example 7

C-terminal amidase activity produced by recombinant cells

The cell culture expressed in Example 6 is divided into cells and supernatant (medium) by centrifugation. Enzyme activity is measured against the supernatant. Activity measurements are basically described in J. Biol. Chem., 265, 9602-9605, 1990, according to the method using HPLC. In other words, the conversion activity of the C-terminal glycine adduct to the α-hydroxyglycine adduct is proceeded to the reaction solution composition (A) as follows. After a certain time reaction, the substrate (PheGlyPheGly) and the product (PheGlyPhehydroxyGly) Quantify

Reaction liquid composition (A)

15 μM PheGlyPheGly

5 mM CuSO ₄

5 μl / reaction solution 1 ml catalase (Sigma)

100 mM MES buffer (pH 5.6)

1 mM ascorbic acid

+ Culture supernatant (medium)

Incidentally, the conversion activity of the α-hydroxyglycine adduct to amidide and glyoxylic acid is measured in the same manner using the following reaction solution composition (B).

Reaction liquid composition (B)

15 μM PheGlyPhehydroxyGly ^*

100 mM MES buffer (pH 5.6)

+ Culture supernatant (medium)

* Produced by proceeding with the reaction solution composition (A) and fractionating α-hydroxyl glycine adduct by HPLC. HydroxyGly also represents α-hydroxyglycine.

The measurement results are shown in Table 3.

Enzyme activity nmol / hour / ml medium Plasmid Substrate PheGlyPheGly PheGlyPhehydroxyGly Product PheGlyPhe-hydroxyGly PheGlyPhe-NH ₂ + glyoxylic acid SV-203 (signal sequence + N terminal domain + C terminal domain; invention) 2.5 4.2 SVa (signal sequence + N terminal domain; the present invention) 2.8 2 SVb (signal sequence + C-terminal domain; the present invention) 0.4 10.8 PSV2 (Control) 0.3 2 No plasmid (control) 0.5 2

Significantly improved α-hydroxyglycine adduct production activity was observed in the transformed strain by the SV-a plasmid, and was not involved in the reaction using the α-hydroxyglycine adduct as a substrate. In contrast, the strain transformed with the SV-b plasmid did not react completely with the C-terminal glycine adduct and only the activity of converting the α-hydroxyglycine adduct to an amidate was confirmed. In the strain transformed with plasmid SV-203, which has almost the entire region of cDNA, bienzyme activity was confirmed, but the enzyme activity was lower than that of SV-a and SV-b.

Next, in these transformants, it is confirmed by gel filtration chromatography whether the expressed enzyme is a single strain. Separcryl S-200 (available from Pharmacia) column (1 × 95 cm) is used and equilibrated with elution buffer 10 mM HEPES-KOH (pH 7.0), 50 mM NaCl. Elution rate is 6 ml / hour and 1 ml fraction is collected. The results of measuring both enzyme activity and protein amount are shown in FIGS. 8 to 10. The enzymatic activity of SV-a-derived (FIG. 8) and SV-b-derived (FIG. 9) becomes a single peak, respectively, and its measured molecular weight was 36 KDa and 54 KDa, respectively, and the molecular weight of the cDNA encoded by the cDNA of each plasmid. Corresponds to However, proteins derived from the SV-203 plasmid have activity (□-□) and α-hydroxyglycine adducts that act on C-terminal glycine to produce α-hydroxyglycine adducts as shown in FIG. It is separated into two peaks of the enzymatic activity (○-○) that produces a cargo and glyoxylic acid. In addition, these molecular weights are the same as what expressed each enzyme shown to FIG. 8, FIG. 9 independently. The results show that the KK sequence located in the center of the protein encoding the cDNA in the cultured cells was cleaved by processing. Therefore, the expression of cDNA having such an entire cDNA region also indicates that two enzymes according to the present invention can be produced.

Next, in the peptide C terminal amidation reaction, the synergistic effect by using two kinds of enzymes in the present invention is shown using Figs. 11 and 12. 11 and 8 show changes with time of conversion to amidide when PheGlyPheGly is used as a substrate. An enzyme sample is prepared by purifying the SV-a, SV-b plasmid and the media supernatant obtained by expression by gel filtration described above, and concentrating each active fraction. Fig. 11 shows that derived from SV-a, but only α-hydroxyl adduct is produced and no amidide is produced. In FIG. 12, the case where only Sv-b origin is used (☆) and the case where SV-a origin and SV-b origin are used together are shown. It is understood that neither α-hydroxyl adduct nor amidide is produced from SV-b alone, but both enzymes (amount of enzyme added) are used to simultaneously produce both α-hydroxyl adduct and amidate. Further, it should be noted further that the reaction efficiency is increased when used in combination within 4 hours, and the reaction time is 9 times higher than the case of SV-a-derived alone shown in FIG. Thus, the combination of both enzymes is a very effective means because the C-terminal amidation reaction is efficiently performed.

In the following Examples, the production of DNA sequences encoding polypeptides having the activity of enzyme-I and enzyme-II (horse C-terminal amidase) derived from horses will be described.

Example 8

Poly A from the Horse Atrial ⁺ Preparation of RNA

(1) Preparation of Whole RNA

After extracting the horse atrium, it is quickly dissected and placed in a 50 ml plastic tube (Falcon Product No. 2070) and frozen in liquid nitrogen. 20 ml guanidine solution (4M thiocyanate-guanidine, 25 mM sodium quenoate, pH 7.0) 0.5% sodium laurylzarcosine sodium, 0.1% antiform A, 0.1M 2-mercapto ethanol) was added, After cell crushing using (Central Science Trade), the crushed liquid is drawn out several times with a 10 ml syringe (product of Termosa) equipped with an 18 G needle. The precipitate was removed by low speed centrifugation (300xg, 5 minutes), and 7.3 ml of the supernatant was poured into a 3.7 ml Cs TFA vessel (Pharmacia), an aqueous cesium trifluoroacetic acid solution containing 0.5 M EDTA, and the density was 1.64 g / layer) and treated for 16 hours at 33,000 rpm with an ultracentrifuge (product SCP85H) using Swing Rota RPS-40T. The precipitate is washed with 3 ml of 4M guanidine solution followed by 3 ml of 95% ethanol and then dissolved in 1.5 ml of Cs TFA solution. 60 μl of 5M NaCl solution and 3.9 ml of ethanol were added to carry out ethanol precipitation at −80 ° C. for 30 minutes, centrifuged at 16,000 × g for 15 minutes to obtain a precipitate, washed with 70% ethanol, and then concentrated (Sakuma Corporation). , EC -57C). After dissolving in sterile distilled water, the absorbance at 260 nm is measured to quantify the amount of RNA. This method yields 350 μg of RNA from about 2 g of horse atrial tissue.

(2) poly A ⁺ Preparation of RNA

Preparation of poly A ⁺ RNA from whole RNA is performed according to its attachment protocol using an mRNA purification kit (from Pharmacia). Affinity chromatography with an oligo (dT) column was performed twice to yield 13 μg of polyA ⁺ RNA than 350 μg of horse atrial pre-RNA.

Example 9

Construction of cDNA Library

(1) Construction of cDNA

cDNA synthesis is carried out using the cDNA synthesis system Phras (Amersham, RPN 1256Y) and 5 μg of atrial polyA ⁺ RNA. The order of synthesis adheres faithfully to the attached protocol. When polyoligo dT nucleotides were used as a primer and the efficiency of cDNA synthesis by radioactivity was calculated using a synthetic system containing [α- ³² P] -dCTP, the reverse transcription efficiency was about 20% and the second chain synthesis efficiency was 90% or more. to be.

(2) cDNA library construction

As for phage DNA, cDNA cloning system λgt 10, version 2.0 (manufactured by Amersham, RPN1257), and for packaging into phage, gigapak; CDNA libraries are constructed by synthetic cDNA using Gold (produced by Stratagene) and according to their attachment protocol.

(3) Escherichia coli infection

As a host bacterium, Escherichia coli Y1089 (ATCC37196) is used, and competent cells are prepared as follows. Single colony cells were inoculated in 5 ml of NZY medium (0.5% NaCl, 1% NZ amine, type A 0.5% yeast extract (DIFCO), 0.2% magnesium sulfate, pH 7.5) with 0.2% maltose overnight. Shake culture at 37 ℃.

100 μl are transplanted into 5 ml of the same fresh medium, followed by incubation at 37 ° C. until OD ₆₆₀ = 0.5, followed by centrifugation. It is suspended in 1 ml of 10 mM magnesium sulfate solution to prepare competent cells.

0.1 ml of the phage solution prepared in (2) was added to 0.2 ml of competent cell suspension, and NZY containing top agarose (0.7% type I-Low EEO-agarose (manufactured by Sigma)) was insulated at 56 ° C. After mixing with 3 ml of medium, 30 ml of NZY medium containing 1.5% Bacta agar (produced by DIFCO) is added to the Falcon 1005 plate. After solidification of top agarose, the cells are left to incubate overnight at 37 ° C. Check for plaque to identify phage infected cells.

In the same manner as described above, a 2.0 × 10 ⁷ atrial cDNA library can be constructed.

Example 10

Isolation of the C-terminal amidation enzyme cDNA

(1) Construction of DNA Probe

Rat-derived peptide C terminal amidation enzyme cDNA has already been isolated and its nucleotide sequence has been reported (DA Soffer et al., Proc. Natl. Acad. Sci. USA, 86, 735-739 (1989), Gaodo et al. , Biochemistry, 61, 842 (1989)). The rat cDNA and the horseradish-derived peptide C terminal amidation enzyme cDNA are considered to have some degree of homology, and a part of the rat cDNA is obtained and used as a probe to proceed with the isolation of the horse cDNA. Rat cDNA was obtained from the Department of Medicine, Doo Recovery University (Kadoo et al., Biochemistry, 61, 842 (1989)) and digested with restriction enzymes EcoR I and Hin II and NsiI and Sph I, respectively, shown in FIGS. 14 and 15. The DNA fragments are isolated and labeled with ³² P using a multiprime DNA labeling kit (manufactured by Amersham) to make probes.

(2) plaque hybridization

Example 9 (3) Approximately 500,000 plaques were formed per sheet of 15 cm diameter (FALCON, No. 1058) according to the method indicated for Escherichia coli infection. At this time, the culture for plaque formation is carried out for 37 ℃, 4 hours. The plaques were allowed to stand at 4 ° C. for 2 hours, and then nitrocellulose filters (Schleicher Schuell, BA85) were adhered and phage DNA was transferred to the filters, followed by DNA in alkaline solution (0.5 M caustic soda, 1.5 M sodium chloride). Denature After neutralization with neutralization solution (1.5M saline, 0.5M tris hydrochloric acid buffer, pH 7.0), washed with 2xssc solution (0.3M sodium chloride, 30 mM sodium citrate buffer pH 7.0), and then heated to 80 ° C. under reduced pressure for 2 hours after ventilation drying. Then, the DNA is fixed to the filter.

Plaque hybridization is performed on the nitrocellulose filter to which phage DNA is immobilized using the probe prepared in (1). Place the filter in a wrap-bag (manufactured by Iwadani Co.) and 30 ml of preliminary hybridization solution (0.75 M sodium chloride, 50 mM sodium phosphate buffer, pH 7.4, 5 mM EDTA, 0.05% filter, 0.05% polyvinylpyrrolidone, 0.05% bovine) Serum albumin (Sigma Plaque V), 0.1% SDS, 0.2 mg / ml salmon sperm DNA) is added and sealed with a sealant and warmed at 65 ° C. for 4 hours. Discard the preliminary hybridization solution and contain 30 ml of hybridization solution (0.75 M sodium chloride, 50 mM sodium phosphate buffer, pH 7.4, 5 mM EDTA, 0.02% picol, 0.02% polyvinylpyrrolidone) containing a probe having a radioactivity of about 10 million cpm , 0.02% bovine serum albumin, 0.1% SDS, 0.1 mg / ml salmon sperm DNA) were added and hybridization was performed at 65 ° C. for 15 hours after sealing. The filter is washed twice with 250 ml of washing solution (0.3 M sodium chloride, 20 mM sodium phosphate buffer, pH 7.4, 2 mM EDTA, 0.1% SDS) and again 250 ml of washing liquid (30 mM sodium chloride, 2 mM sodium phosphate buffer 7.4, 0.2 mM EDTA, 0.1% SDS). Wash twice with) and dry with ventilation. Detection of positive clones is carried out by autoradiography using X-ray film (Fuji, HR-H) under conditions that are photoexposed for -80 ° C. day.

As for the two probes used, about 2 million plaques were screened, and about 1,000 positive clones were obtained. Phage DNA is recovered by positive plaque, infected again with E. coli according to the above method, plaque hybrid is performed again and the operation is repeated until plaque becomes single. Usually, a single plaque is obtained by repeating twice.

Example 11

cDNA sequencing

DNA is isolated and purified by cloned phage according to the method described in the Molecular Cloning A Laboratory Manual (T. Maniatis, EF Fritsch, J. Sambrook, Cold Spring Harbor Laboratory, 1982), 371-372. . DNA is digested by restriction enzyme EcoR I (Housozo) and cDNA insert DNA fragment is isolated from phage DNA by 1.5% agarose gel electrophoresis. The cDNA fragment is extracted from the gel and recombined by the ligation reaction to the EcoR I site of E. coli plasmid pUC119 (Housezo Co.). If there is an EcoR I cleavage site in the cDNA fragment, cDNA fragments are obtained by partial digestion of phage DNA with EcoR I. After amplifying the plasmids, the cDNA fragments were cloned into M13 phage mp18, mp19 (House Products) and single chain DNA was obtained according to a conventional method. Sequenase (trade name, manufactured by Toyoobo Seki Co., Ltd.) is used, and the DNA sequence is determined according to the instruction manual. The base sequence of the Japanese DNA chain is determined to be about 400 bases, and DNA fragments longer than that are sequenced by acloning using an appropriate restriction enzyme site. The cDNA fragment also determines the nucleotide sequence with both strands of the double strand.

The horse C terminal amidation enzyme cDNA nucleotide sequence determined in FIG. 13 (this nucleotide sequence is shown for the longest cDNA clone as a result of the analysis of many DNAs) and the amino acid sequence predicted from this nucleotide sequence (in one letter). CDNAs with one or both of the portions indicated by [] in the drawings may be identified. It is believed that these cDNAs are derived from different mRNAs in the manner of mRNA splicing.

The present invention can be used for the preparation of corresponding peptide C terminal amidates from peptide C terminal glycine adducts, and these peptide C terminal amidates include bioactive substances of great interest.

Claims (13)

It acts on the peptide C terminal α-hydroxyl glycine adduct of formula (II) to produce peptide C terminal amidide and glyoxylic acid of formula (III), each of which is the 443th residue of the human, horse, bovine and rat of FIG. An enzyme involved in the C terminal amidation of a peptide C terminal glycine adduct having an amino acid sequence selected from those corresponding to the amino acid sequence from D to K, the 830th residue. Formula II Formula III In the above formula, A represents a residue other than an α-amino group or an imino group and an α-carboxyl group derived from a natural α-amino acid, X represents the residue of the amino acid derivative couple | bonded with N atom through a hydrogen atom or a carbonyl group. The enzyme of claim 1 wherein (a) the appropriate pH is about 5-6 and the stable pH is 4-9, and (b) the operating temperature is in the range of 15-35 ° C. The enzyme of claim 1, wherein the enzyme has a molecular weight of about 40 KDa or about 43 KDa. The method of claim 1, comprising culturing the host cell transformed with a plasmid containing the cDNA encoding the enzyme according to claim 1, and collecting the enzyme from the culture in which the enzyme is accumulated. Manufacturing method. The method of claim 4, wherein the cDNA is derived from a mammal. The method of claim 5, wherein the mammal is a rat or a horse. The peptide C terminal amidate of formula (III) according to claim 1, characterized by treating the peptide C terminal α-hydroxyglycine adduct of formula II according to claim 1 with the enzyme according to claim 1. Manufacturing method. (a) buffering a subject predicted to have enzymatic activity according to claim 1 at pH 4 to 8, (b) incubating the buffer with the addition of the peptide C terminal α-hydroxyglycine adduct of formula (II) as defined in claim 1, and (c) detecting the resultant peptide C terminal amidate of formula (III) according to claim 1, characterized in that the activity of the enzyme according to claim 1. A method for searching for an enzyme according to claim 1, wherein the measuring method according to claim 8 is applied to a subject. The peptide C according to claim 1, characterized in that the enzymatic activity content according to claim 1 is subjected to substrate affinity chromatography with a ligand as the peptide C terminal glycine adduct of formula (I), and anion exchange chromatography. A method for producing an enzyme involved in terminal amidation. Formula I In the above formula, A and X are as defined in claim 1. The method of claim 10, wherein the enzymatic active content is a homogenate of an organ selected from the group consisting of the brain, the pituitary gland and the heart of a mammal or blood of a mammal. The method of claim 10, wherein the enzymatic active content is horse serum. The method of claim 4, wherein the membrane spanning region of the cDNA is removed.