KR100496748B1 - Novel amino-terminal deblocking enzyme - Google Patents

Abstract

An enzyme having an activity of releasing this protecting group by acting on a peptide blocked by the amino terminal by a protecting group, the enzyme comprising: an amino terminal protecting group free enzyme exhibiting the above activity for two or more protecting groups, or a functional equivalent thereof; DNA encoding this enzyme; A method for producing this enzyme; A method for removing an amino terminal protecting group comprising the step of activating the enzyme to release the amino terminal protecting group; And methods for interpreting amino acid sequences. Such enzymes are useful for amino acid sequencing of peptides, particularly proteins and peptides whose amino ends are blocked by unidentified protecting groups.

Description

New amino terminal protecting group free enzyme {NOVEL AMINO-TERMINAL DEBLOCKING ENZYME}

The present invention relates to an amino terminal protecting group freease having the activity of releasing a protecting group present at the amino terminal of a protein or peptide. Moreover, this invention relates to DNA which codes such an amino terminal protecting group free enzyme. Moreover, this invention relates to the manufacturing method by the genetic recombination technique of such amino terminal protecting group free enzyme. Moreover, this invention relates to the removal method of an amino terminal protecting group using such an amino terminal protecting group free enzyme. The present invention also relates to a method for analyzing amino acid sequences using such a removal method. Moreover, this invention relates to the kit used for the analysis of an amino acid sequence containing the said amino terminal protective group freease. The present invention also relates to an antibody or fragment thereof that specifically binds to the amino terminal protecting group freease or a functional equivalent thereof. The present invention also relates to a synthetic oligonucleotide probe or a synthetic oligonucleotide primer capable of hybridizing with the DNA.

Determining amino terminal amino acid sequences of proteins and peptides is an essential assay for their identification or identification.

However, more than 60% of eukaryotic cell-derived soluble proteins are known to block the α-amino group of the amino terminal with an acetyl group or other protecting group, and have already been established and widely used by the amino acid sequence determination method from the amino terminal. In the Edman decomposition method, it is not possible to analyze proteins and peptides in which the amino terminal is blocked by a protecting group.

As protecting groups blocking the amino ends of proteins and peptides, formyl groups, acetyl groups, myristoyl groups, pyroglutamyl groups, dimethyl groups, glucuronyl groups, glycosyl groups, trimethyl groups and the like have been reported. As the amino terminal amino acid sequence analysis method of these blocked proteins, the method of applying an Edman decomposition method after removing a protecting group is used.

There are two methods for removing the protecting group, a chemical method and an enzyme method. The enzyme method is acylamino acid free enzyme for acetyl group and pyroglutamyl peptidase for pyroglutamyl group. Different enzymes have the drawback of lack of versatility, and no chemical method has been developed. In addition, there is no known method for identifying the protecting group when the amino terminal is blocked. Therefore, when actually removing the protecting group, there is no method other than testing the removing method according to each protecting group one by one. In addition, there is no effective protecting group removal method for proteins and peptides blocked with myristoyl groups, and it is impossible to obtain sequences from the amino terminus.

Moreover, as a peptidase which acts on the amino terminal part of a peptide isolated so far from Pyrococcus furiosu s, an aminopeptidase (Japanese Patent Laid-Open No. Hei 6-319566), a pyroglutamyl peptidase (Japanese Patent Laid-Open Publication No. 6-13,064). JP-A-7-298881) and methionine aminopeptidase (JP-A-8-9979) are known. Among these, pyroglutamyl peptidase has an activity of releasing an amino terminal pyroglutamyl group, but does not act on other protective groups such as acetyl groups. In addition, the other two enzymes cannot act on the amino terminal blocked by the protecting group.

As described above, the current amino terminal amino acid sequence analysis for proteins and peptides in which the amino terminal is blocked by a protecting group has a problem that there is no universality.

1 is a diagram showing a restriction map of plasmid pDAP3.

Fig. 2 shows the restriction map of the plasmid pDAP.

Figure 3 is a graph showing the optimum temperature of the amino terminal protecting group free enzyme in the present invention.

Figure 4 is a graph showing the optimal pH of the amino terminal protecting group free enzyme in the present invention. In the figure, Δ represents the data using sodium acetate buffer, PIPES-Na buffer, ○ is sodium borate buffer, ▲ is sodium dihydrogen phosphate-sodium hydroxide buffer.

Fig. 5 is a graph showing the temperature stability at 75 ° C. of the amino terminal protecting group free enzyme of the present invention. In the figure,? Represents data in a buffer containing 0.1 mM CoCl ₂ , and ○ represents data in a buffer containing no CoCl ₂ .

Figure 6 is a view showing the effect of the amino terminal protecting group free enzyme of the present invention over time with respect to neurotensin.

7 is a view showing the action of the amino-terminal protecting group free enzyme of the present invention over α-MSH over time.

8 is a view showing the action of the amino terminal protecting group free enzyme of the present invention over Ac-Gly-Asp-Val-Glu-Lys over time.

9 is a view showing the action of the amino terminal protecting group free enzyme of the present invention over time for For-Met-Leu-Phe-Lys.

10 is a view showing the effect of the amino terminal protecting group free enzyme of the present invention over time against Myr-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln.

11 is a view showing the effect of the amino terminal protecting group freease of the present invention over time against Myr-Gly-Ala-Gly-Ala-Ser-Ala-Glu-Glu-Lys.

12 is a view showing the action over time of the reduced lysozyme of the amino-terminal protecting group free enzyme of the present invention.

Best Mode for Carrying Out the Invention

1.Amino-terminal protecting group free enzyme of the present invention

The amino terminal protecting group free enzyme of the present invention is an enzyme having an activity (amino terminal protecting group free activity) that acts on a peptide blocked by the amino terminal by a protecting group and releases the protecting group. It features.

As used herein, "peptide" indicates that two or more amino acids are linked by peptide bonds and includes those described as "proteins".

Examples of the protecting group present at the amino terminal of the peptide include an acetyl group, a pyroglutamyl group, a formyl group, a myristoyl group, and the like. The amino terminal protecting group-releasing enzyme of the present invention includes these protecting groups from the amino terminal of the substrate peptide. It has an activity which liberates 2 or more types of protecting groups. Such amino-terminal protecting group free activity can be measured by the method of mentioning later by using synthetic peptide etc. which an amino terminal was block | blocked by the said arbitrary protecting groups as a board | substrate.

Examples of the amino terminal protecting group-releasing enzyme of the present invention include an enzyme having an amino terminal protecting group-releasing activity and an enzyme having an amino terminal protecting group-releasing activity and having an aminopeptidase activity which liberates amino acids sequentially at the amino terminal of the peptide. As an example, the enzyme which consists of an amino acid sequence shown to SEQ ID NO: 1 of a sequence list is mentioned. In the case of further having an aminopeptidase activity, it is also possible to analyze the amino acid sequence of the peptide without the need to use another peptide amino terminal decomposition method. Such aminopeptidase activity can be measured by a known method commonly used (Arch. Biochem. Biophys., Vol. 274, pp. 241 to 250 (1989)).

As an example of the enzyme which has an amino terminal protective group free activity, and has aminopeptidase activity, the enzyme which consists of an amino acid sequence shown to SEQ ID NO: 1 of a sequence list is mentioned specifically ,. In the enzyme of the present invention, the amino terminus may be in a free state, or the amino terminus of the enzyme may be blocked by a protecting group. Thus, unless otherwise specified herein, the amino terminus of this enzyme includes any form. Therefore, although the amino terminal was shown to be free in SEQ ID NO: 1, it is also within the scope of the present invention to have the sequence of SEQ ID NO: 1 and that the amino terminal is blocked by a protecting group such as an acetyl group.

The amino terminal protecting group freease of the present invention is, for example, 1) from a culture of a microorganism producing the enzyme of the invention, 2) from a culture of a transformant containing DNA encoding the enzyme of the invention. It can manufacture by the method of purification | purification, etc.

1) Purification from Culture of Microorganisms Producing the Enzyme of the Invention

As for the microorganism producing the enzyme of the present invention, such microorganisms can be found by screening with enzyme activity as an index. As a method for detecting enzyme activity, there is a method of confirming the release of a protecting group from the substrate by using high-performance liquid chromatography, amino acid analysis, mass spectrometry, etc., by using an amino acid or peptide whose amino terminal is blocked with a protecting group as a substrate. . Moreover, the synthetic substrate etc. which added the suitable coloring group and fluorescent group to the amino acid blocked with the protecting group can also be used.

In this manner, the enzyme can be produced by culturing a microorganism in which the production of the amino terminal protecting group free enzyme is confirmed. The microorganisms may be cultured under conditions that are optimal for the growth of the microorganisms. Preferably, culture conditions in which the expression level of the target enzyme is increased are used. In this way, the target enzyme produced in a cell or culture medium can be purified by the method used for normal enzyme purification.

As a microorganism which produces the enzyme of this invention selected by said screening, Pyrococcus furiosus DSM 3638 is mentioned.

Hereinafter, although the pyrococcus puriosus DSM 3638 is described as an example, the manufacturing method of the enzyme of this invention is described, but it is not limited to this. In addition, Pyrococcus puriosus DSM 3638 is a strain available from Deutsch Sammlung von Mikroorganismen und Zellkulturen und GmbH.

In culturing this strain, the method generally used for culturing super heat-resistant bacteria can be used, and the nutrient source to be added to the medium may be one that can be used by this strain. As the carbon source, for example, starch or the like can be used. As the nitrogen source, for example, tryptone, peptone, yeast extract or the like can be used. In the medium, metal salts such as magnesium salt, sodium salt and iron salt may be added as trace elements. For example, it is advantageous to use artificial seawater for the production of the medium. The medium is preferably a transparent medium that does not contain solid sulfur. When the medium is used, the growth of the cells can be easily monitored by measuring the turbidity of the culture solution. In culture, it can be carried out by standing culture or by stirring culture. For example, as described in [Applied and Environmental Microbiology, Vol. 55, pp. 2086 to 2088 (1992)], a dialysis culture method can be used. do. Generally, the incubation temperature is preferably around 95 ° C., and amino terminal protecting group free enzyme is usually accumulated in a significant amount in the culture for about 16 hours. The culture conditions are preferably set so that the production amount of the amino terminal protecting group free enzyme is maximized depending on the cells and the medium composition to be used.

In extracting the amino terminal protecting group free enzyme, for example, cells are collected from the culture medium by centrifugation, filtration or the like, and then the cells are crushed to prepare a crude enzyme solution. As a method for pulverizing the cells, a method having high extraction effect of the target enzyme may be selected from ultrasonic crushing, bead crushing, and lytic enzyme treatment. In addition, when this enzyme is secreted in a culture liquid, an enzyme is concentrated by the sulfur eye salt method, an ultrafiltration method, etc., and this is used as a crude enzyme liquid. In isolating the amino terminal protecting group free enzyme from the crude enzyme solution thus obtained, a method usually used for purification of the enzyme can be used. For example, it can use combining methods, such as a sulfur salt salt treatment, ion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography.

2) Purification from Cultures of Transformants Containing DNA Encoding Enzymes of the Invention

DNA encoding the enzyme according to the present invention can be obtained by screening gene libraries of suitable microorganisms. Microorganisms can be found by screening using the enzyme activity as an index as in the above.

More specifically, the target DNA can be obtained by screening for example a cosmid library of a bacterium belonging to the genus Pyrococcus, for example, the Pyrococcus furiosus genome. Pyrococcus puriosus DSM 3638 can be used as the pyrococcus puriosus.

In addition, methods such as cloning desired DNA from such microorganisms, producing and culturing transformants containing DNA encoding the enzyme of the present invention, and purifying target proteins from the culture are known methods. Can be used. Hereinafter, although the pyrococcus puriosus DSM 3638 is demonstrated about the manufacturing method of this invention as an example, it is not limited to this.

The cosmid library of the pyrococcus furiosus genome is obtained by introducing a DNA fragment obtained by partial cleavage of the pyrococcus furiosus genome DNA with a restriction enzyme Sau3AI (manufactured by Takarashu Corporation) into a triple helix cosmid vector (produced by Stratagene). After that, it can be produced by packaging in lambda phage particles by in vitro packaging. Next, a clone containing the DNA of this enzyme was obtained by transforming a suitable E. coli, for example, E. coli DH5αMCR (manufactured by BRL) using the library thus obtained and examining the enzymatic activity according to the present invention in the transformant. Can be obtained.

Furthermore, the inventors pay attention to the fact that the enzyme producing pyrococcus furiosus as a material has high heat resistance. At the time of screening for this enzymatic activity, the present inventors individually cultured transformants obtained using cosmid libraries. And the process of manufacturing the degradation product containing only heat-resistant protein from the obtained cells was combined. This group of degradation products is termed cosmid protein library, and the cosmid protein library is used for the detection of enzymatic activity, so that the detection sensitivity is higher than that of the method using colonies of transformants, The adverse effects of background levels and enzyme activity inhibition can be eliminated by thermal denaturation.

That is, first, the cells obtained by culturing the transformant obtained using the cosmid library are subjected to heat treatment (100 ° C. for 10 minutes), sonication, and reheat treatment (100 ° C. for 10 minutes), followed by centrifugation to supernatant. (Degradation product) is collect | recovered and a cosmid protein library is produced.

Next, each degradation product contained in this library is examined for the presence of peptidase activity and screened for clones expressing peptidase. For detection of peptidase activity, amino acid-4-methylcoumariyl-7-amide (hereinafter referred to as amino acid-MCA), which is a synthetic peptide, can be used as a substrate, for example, Met-MCA, Leu-MCA, Ala. -MCA, His-MCA (manufactured by Peptide Research Institute), and the like are useful. The digested product whose peptidase activity was recognized was also an activity of releasing the protecting group of the amino terminal by using α-MSH (α-melanin cell stimulating hormone: manufactured by Peptide Research Institute), which is a peptide whose amino terminal was blocked by an acetyl group, as a substrate. It can be investigated whether or not Thereby, the cosmid clone containing DNA which expresses the activity which liberates a protective group can be obtained.

In addition, the cosmid prepared in the cosmid clone thus obtained can be cleaved with a suitable restriction enzyme to prepare a recombinant plasmid in which each fragment is inserted. Next, the recombinant plasmid containing the DNA encoding the target enzyme can be obtained by investigating the Met-MCA degradation activity produced by the transformant obtained by introducing the plasmid into a suitable microorganism.

The recombinant plasmid which cut | disconnected the cosmid manufactured by said cosmid clone with BamHI (made by the Takarashu irradiation), and inserted the DNA fragment obtained in the BamHI site | part of the plasmid vector pUC18 (made by Takarashu Research) can be produced. Next, after culturing Escherichia coli JM109 (manufactured by Takarashu Research Co., Ltd.) having introduced this plasmid, the plasmid containing the target DNA can be obtained by examining the peptidase activity in the digested product prepared from the obtained cells. This plasmid is named plasmid pDAP1.

Next, the recombinant plasmid can be produced by digesting the above plasmid pDAP1 with EcoRI (manufactured by Takarashu Irradiation) and self-ligating. The plasmid containing the DNA of interest can be obtained by confirming the peptidase activity of the degradation product of the cells obtained by culturing E. coli JM109 into which this plasmid has been introduced. This plasmid is named pDAP2.

In addition, the DNA fragment containing no amino terminal protecting group freease gene in the plasmid pDAP2 can be removed as follows. That is, a DNA fragment of about 1.7 kb obtained by cleaving the plasmid pDAP2 by SacI (manufactured by Takarashu Irradiation) is inserted into the SacI site of plasmid vector pUC18 (manufactured by Takarashu Irradiation), and introduced into E. coli JM109. Peptidase activity of the degradation products prepared from the obtained transformants is measured to prepare plasmids from the transformants exhibiting activity. This plasmid is named pDAP3. 1 shows the restriction map. In the figure, the thick solid line shows the inserted DNA fragment in the plasmid vector pUC18.

In addition, the DNA fragment containing no amino terminal protecting group freease gene in the plasmid pDAP3 can be removed as follows. That is, about 1.2 kb of the SnaBI-SacI DNA fragment obtained by cleaving the plasmid pDAP3 by SnaBI (manufactured by Takarashu Irradiation) and SacI (manufactured by Takarashu Irradiation) was inserted into the SmaI-SacI site of the plasmid vector pUC19 and introduced into E. coli JM109. do. Peptidase activity of digests prepared from the resulting transformants is measured. In addition, the activity of lysate liberating the acetyl group at the amino terminus was confirmed using α-MSH as a substrate, and a plasmid was prepared from the transformant having the activity. This plasmid is named plasmid pDAP. The restriction map is shown in FIG. In the figure, the thick solid line shows the inserted DNA fragment in the plasmid vector pUC19. E. coli JM109 introduced with plasmid pDAP was named E. coli JM109 / pDAP, and it was designated as FERM BP-5804 in the Biotechnology Research Institute of the Ministry of Commerce, Industry and Technology of Japan, Ibaragi-chan Tigubashi Higashi 1-chome 3 (Postal Code 305). Deposited (original date: March 29, 1996, transfer to international deposit: January 30, 1997).

The base sequence of the DNA fragment derived from pyrococcus puriosus contained in the plasmid pDAP can be determined using a known method, for example, the dideoxy method.

SEQ ID NO: 3 in Sequence Listing shows the base sequence of a DNA fragment of about 1.2 kb inserted into the plasmid pDAP. The base sequence of the open reading frame present in this sequence is shown in SEQ ID NO: 2 of the sequence listing. That is, SEQ ID NO: 2 in the Sequence Listing is an example of the nucleotide sequence of the amino terminal protecting group free enzyme gene obtained by the present invention. In addition, the amino acid sequence of the amino terminal protecting group free enzyme of this invention estimated by the base sequence shown to SEQ ID NO: 1 and SEQ ID NO: 2 is shown. Namely, SEQ ID NO: 1 in the Sequence Listing is an example of an amino acid sequence of an amino terminal protecting group free enzyme obtained by the present invention.

The amino terminal protecting group-releasing enzyme of the present invention can be obtained using Escherichia coli JM109 / pDAP having plasmid pDAP introduced as described above.

That is, E. coli JM109 / pDAP was subjected to normal culture conditions, for example, LB medium containing 100 μg / ml of ampicillin (tryptone 10 g / liter, yeast extract 5 g / liter, NaCl 5 g / liter, pH7.2 ), The amino terminal protecting group free enzyme can be expressed in the cultured cells by culturing at 37 ° C.

After completion of the culture, the cells obtained by collecting the cultured cells were heat-treated at 100 ° C. for 10 minutes. The crude enzyme solution was obtained as a supernatant obtained by centrifugation after heat treatment of the heat-treated cells, and the enzyme solution was usually subjected to denatured protein, gel filtration chromatography, ion exchange chromatography, etc. The amino terminal protecting group free enzyme can be purified by using a combination of methods used for purification.

The enzymatic and physicochemical properties of the amino terminal protecting group-releasing enzyme of the present invention obtained in E. coli JM109 / pDAP by the above method are shown below.

(1) action

The enzyme of the present invention releases at least an acetyl group, a pyroglutamyl group, a formyl group, and a myristoyl group among various protecting groups present at the amino terminal of the peptide. The enzymes of the invention also liberate amino acids sequentially at the amino terminus of the peptide.

(2) enzyme activity assay

The activity of the enzyme of the present invention is measured by the operation shown below using Met-MCA as a substrate.

a. reaction

Appropriately dilute the enzyme standard for which the enzyme activity is to be measured, add 100 µl of 20 mM PIPES-Na buffer solution (pH7.6) to 5 µl of the enzyme solution, and again dissolve in 1 mM Met-MCA (dimethylsulfoxide). 5 µl of) is added and reacted at 75 ° C for 20 minutes, and then the reaction is stopped by adding 10 µl of 30% acetic acid.

b. Measure

The 7-amino-4-methylcoumarin produced | generated by reaction is quantitatively determined by using titatech fluoroscan II (made by Dainippon Seiyaku Co., Ltd.) at an excitation wavelength of 355 nm and a measurement wavelength of 460 nm. One unit of enzyme is Met-MCA as a substrate, and it is set as the amount of enzyme which can produce 1 micromol of 7-amino-4-methylcoumarin in 1 minute at pH7.6 and 75 degreeC. The enzyme of the present invention had Met-MCA degrading activity at the measured pH7.6 and 75 ° C. Equally, the enzyme of the present invention acted not only on Met-MCA but also on Leu-MCA, Ala-MCA, His-MCA and the like to liberate amino acids.

(3) substrate specificity

Even if the protecting group is present at the amino terminus of the peptide, the enzyme of the present invention releases the protecting group at the amino terminus, and then releases the amino acids sequentially.

The activity can be confirmed by using a synthetic peptide having an amino terminal blocked as a substrate. That is, the change over time of the amount of amino acid liberated from the substrate peptide by the enzymatic reaction can be confirmed by measuring amino acid analysis and analyzing the degradation profile of the peptide. Among the peptides shown below, those which are not commercially available can be synthesized chemically by a known method, for example, by using a peptide synthesizer.

The activity of releasing pyroglutamyl groups can be investigated using neurotensin as a substrate. SEQ ID NO: 4 in Sequence Listing shows an amino acid sequence of neurotensin. Namely, 10 μl of 1 mM neurotensin (manufactured by Peptide Research Institute) was added with 0.12 milliliters of enzyme standard (5 μl), 40 μl of 40 mM PIPES-Na buffer solution (pH7.6), and 35 μl of distilled water. After reacting for 5 hours, the free amino acid in the reaction solution was measured by amino acid analysis. As a result, it was confirmed that leucine and pyroglutamyl-free leucine are liberated sequentially at the amino terminus of neurotensin as the reaction time elapses, and the enzyme of the present invention releases the pyroglutamyl-free activity. Appeared to have.

The activity of releasing an acetyl group can be investigated by using α-MSH and Ac-Gly-Asp-Val-Glu-Lys as a substrate. The amino acid sequences of α-MSH and Ac-Gly-Asp-Val-Glu-Lys are shown in SEQ ID NO: 5 and SEQ ID NO: 6 in the Sequence Listing, respectively. In other words, 0.1 μl of enzyme standard product (5 μl), 40 μm 40 mM PIPES-Na (pH7.6) buffer, and 35 μl of distilled water were added to 10 μl of the 1 mM peptide substrate solution. After reacting for some time, free amino acid in the reaction solution was measured by amino acid analysis. As a result, even when using any substrate, the amino terminal of the amino terminal in which the acetyl group was liberated first was released, and it turned out that the enzyme of this invention has the activity which liberates an acetyl group. In addition, it was confirmed that the following amino acids are sequentially released as the reaction time elapses.

The activity which liberates a formyl group can be investigated using For-Met-Leu-Phe-Lys (made by BACHEM) as a substrate. SEQ ID NO: 7 in Sequence Listing shows an amino acid sequence of For-Met-Leu-Phe-Lys. In other words, 0.5 μl of 10 mM For-Met-Leu-Phe-Lys (30% acetic acid solution) was added with 4 milligrams of enzyme standard (3.5 μl), 25 μl of 0.1M N-ethylmorpholine and 21 μl of distilled water. After reacting at 37 degreeC for 0 to 2 hours, the free amino acid in the reaction liquid was measured by amino acid analysis. As a result, it was confirmed that methionine free of formyl group was released in the reaction solution, and it was shown that the enzyme of the present invention has an activity of releasing formyl group. It was also confirmed that the amino acid subsequent to this was sequentially released as the reaction time elapsed.

Activities that release the myristoyl group are Myr-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (manufactured by BACHEM) and Myr-Gly-Ala-Gly-Ala-Ser-Ala-Glu-Glu Can be investigated using -Lys as a substrate. SEQ ID NO: 8 and SEQ ID NO: 9 in the Sequence Listing are Myr-Phe-Ala- Arg-Lys-Gly-Ala-Leu-Arg-Gln, Myr-Gly-Ala-Gly-Ala-Ser-Ala-Glu- The amino acid sequence of Glu-Lys is shown. That is, to 5 µl of the 1 mM peptide substrate solution, 4 µm of enzyme standard (3.5 µl), 25 µl of 0.1 M N-ethylmorpholine acetate buffer (pH9.0) and 16.5 µl of distilled water were added. After reacting for 0 to 9 hours at, the free amino acid in the reaction solution was measured by amino acid analysis. As a result, even when any substrate was used, first, the amino terminal amino acid free of myristoyl group was released, and it was shown that the enzyme of the present invention has the activity of releasing myristoyl group. In addition, it was confirmed that the following amino acids are sequentially released as the reaction time elapses.

The enzyme of the present invention also acts on the amino terminus of peptides in which the amino terminus is not blocked by a protecting group, and has an aminopeptidase activity that liberates amino terminal amino acids. This activity is described by Arch. Biochem. Biophys., Vol. 274, pp. 241 to 250 (1989)].

In addition, the enzyme of the present invention can act not only on the single-stranded polypeptide but also on the amino terminal of the high molecular weight protein. For example, chicken egg white reduction lysozyme can be identified as a substrate. That is, 50 mM sodium hydrogen phosphate containing 8 mM enzyme standard (1.6 µl) and 0.1 mM CoCl ₂ in 5 µl of 1 mM chicken egg white reducing lysozyme (water soluble, molecular weight about 14000, manufactured by Wako Pure Chemical Industries, Ltd.). 50 µl of sodium hydroxide buffer (pH11.0) and 43.4 µl of distilled water were added, followed by reaction at 50 ° C for 0 to 240 minutes, followed by measurement of the free amino acid in the reaction solution by amino acid analysis. From the change over time of the amino acid produced in the reaction liquid, it was shown that the amino acid was liberated sequentially at the amino terminal of the reducing lysozyme, and it was confirmed that the enzyme of the present invention also acts on the amino terminal of the protein.

(4) optimum temperature

From Figure 3, the optimum temperature of the enzyme standard of the present invention is 75 to 95 ℃ at pH7.6. In the figure, the vertical axis represents the relative activity (%) with respect to the maximum activity (95 ° C), and the horizontal axis represents the reaction temperature (° C).

(5) optimum pH

As shown in FIG. 4, the optimum pH of the enzyme of the present invention was around 6.5 to 9.5. In the figure, the vertical axis shows relative activity (%) with the activity at pH 7.2 as 100, and the horizontal axis shows the pH at 75 ° C.

(6) Influence of various reagents

The activity of the enzyme of the present invention is inhibited by asmatitin (manufactured by Peptide Research Institute). For example, when the enzyme of the present invention in units of 0.3 milliliters is treated with 5 nmol of amastatin, and its activity is measured by a substrate, Met-MCA is almost completely lost.

In addition, the activity of the enzyme of the present invention is promoted by CoCl ₂ .

(7) molecular weight

The enzyme of the present invention exhibits a molecular weight of about 40,000 by SDS-polyacrylamide electrophoresis and about 400,000 by ultracentrifugal method.

(8) Temperature stability

The residual enzyme activity after heat treatment of the enzyme standard at 75 ° C. was investigated to investigate the temperature stability of the enzyme of the present invention. As shown in Fig. 5, in the enzyme of the present invention, 0.1 mM Tris-HCl buffer (pH8.0) containing 0.1 mM CoCl ₂ showed no degradation of enzyme activity even after 5 hours of treatment (in the drawing). In addition, 80% or more of activity was maintained even when treated for 5 hours in a buffer containing no CoCl ₂ (o). In the figure, the vertical axis shows the remaining enzyme activity (%), and the horizontal axis shows the time (hour) of heat treatment.

The amino-terminal protecting group free enzyme in the present invention, in addition to the above-mentioned enzyme, contains an enzyme or a functional equivalent thereof containing all or a part of the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and showing amino-terminal protecting group free activity. Can be mentioned.

As used herein, "functional equivalent" refers to the following. In addition to polymorphisms or mutations in DNA encoding the proteins present in nature, modifications such as deletion, insertion, addition and substitution of amino acids may occur in the amino acid sequence by modification reactions in vivo and purification of the protein. However, it is known that such mutations exhibit substantially the same physiological and biological activity as proteins that do not have mutations when they exist in parts that are not important for the activity or maintenance of the structure of the protein. In this specification, even if there is a slight difference in structure, the difference in function and activity is not seen as a "functional equivalent" in the present specification.

The above-mentioned mutations are artificially introduced into the amino acid sequence of the protein, and in this case, various variants can be produced. For example, it is known that a polypeptide in which a specific cysteine residue in the amino acid sequence of human interleukin 2 (IL-2) is substituted with serine maintains interleukin 2 activity (Science, Vol. 224, p. 1431 (1984)). Therefore, even if the amino acid sequence disclosed by the present invention (SEQ ID NO: 1 of the sequence listing) is represented by the amino acid sequence in which deletion, insertion, addition, or substitution of one or more amino acid residues occurs, functional differences cannot be seen. If it is within the scope of the present invention.

It is also known that certain types of proteins have peptide regions that are not essential for activity. For example, a signal peptide present in a protein secreted out of the cell, a pro sequence shown in a precursor of a protease, or the like corresponds to this, and most of these regions are removed after translation or upon conversion to an active protein. Such proteins exist in different forms in their primary structure, but are proteins that finally express equivalent functions.

When genetically producing a protein, a peptide chain irrelevant to the activity of the protein may be added to the amino or carboxyl terminus of the protein of interest. For example, in order to increase the expression level of the protein of interest, a fusion protein may be produced in which a part of the amino terminal region of the protein that is highly expressed in the host used is added to the amino terminal of the protein of interest. Alternatively, in order to facilitate purification of the expressed protein, a peptide having affinity for a specific substance is added to the amino or carboxyl terminus of the target protein. When these added peptides do not adversely affect the activity of the protein of interest, the added peptide may be added, and if necessary, may be removed from the protein of interest by appropriate treatment, for example, limited degradation by a protease.

As described above, even if the peptide is not essential to the function of the protein or is added to the peptide, it is within the scope of the "functional equivalent" as long as it can express the equivalent function.

As the functional equivalent of the amino terminal protecting group freease, for example, at least one amino acid residue is deleted, added, inserted or substituted in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing, and amino And enzymes exhibiting terminal protecting group free activity. Although the aspect of a functional equivalent in this invention is not specifically limited, The modified enzyme which changed the amino acid sequence in order to block the N terminal of the amino terminal protecting group freease of this invention by a protecting group is illustrated. For example, the enzyme in which valine of amino acid number 2 of the amino acid sequence of SEQ ID NO: 1 in the sequence listing is substituted with aspartic acid is mentioned.

This modifier can be obtained, for example, by using genetic engineering techniques. That is, the modification can be carried out by modifying the nucleotide sequence in the DNA region encoding the enzyme on the pDAP plasmid DNA using a known nucleic acid mutation introduction technique, and then expressing the protein using an appropriate host. The N-terminal amino acid sequence Met-Asp of this modifier is known to act as an acetyl group addition signal sequence in baker's yeast [Journal of Biochemistry, Vol. 265, pp. 19638 to 19643 (1990)]. Therefore, the N-terminal acetylated protein can be obtained by inserting the DNA region encoding this modifier into an appropriate yeast expression vector and expressing in an appropriate host yeast. For example, pVT103-L [Gene, Vol. 52, pp. 225 to 233 (1987)] as an expression vector for yeast, BJ2168 [FEMS Microbiology Review, Vol. 54, pp. 17 to 46 ( 1988). In addition, by measuring the amino-terminal protecting group free activity of such a protein using the method described in the above-described substrate specificity, it can be confirmed that such a protein is the amino-terminal protecting group free enzyme of the present invention. PVT103-L into which DNA encoding the enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 10 of the Sequence Listing is inserted is named pAcDAP, and the yeast BJ2168 transformed with this plasmid is Saccharomyces cerevisiae. It is named BJ2168 / pAcDAP and has been deposited as FERM BP-5952 to the Institute of Biotechnology, Institute of Industrial Technology, Japan Industrial Technology Research Institute, 1-room, 3-cho, Higashi 1-chome, Tsukuba-shi, Ibaragi, Japan. 23 May 1997). Even if it is such a modifier, if it is an enzyme which has the activity which releases 2 or more types of amino terminal protecting groups, it will be included in the functional equivalent in this specification.

The method of blocking the N terminus of a protein by a protecting group by modification of the amino acid sequence is not limited to this method, for example, other acetylation methods [Journal of Biological Chemistry, Vol. 260, pp. 5382 to 5391 (1985). ) Or the myristoylation method [Proceedings of the National Academy of Sciences of the United States of America, Vol. 84, pp. 2708-2712 (1987)]. In addition, the method of blocking the N-terminus of a protein by a protecting group is not limited to the method by substitution of such amino acids, for example, chemical methods [Methods in Enzymology, Vol. 11, pp. 565 to 570 (1967) )] May be used.

2. DNA of the present invention

The DNA of the present invention is a DNA encoding the amino terminal protecting group free enzyme of the present invention as described above, specifically 1) the amino terminal protecting group containing all or part of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. DNA encoding free activity, 2) DNA containing all or part of the DNA shown in SEQ ID NO: 2 of Sequence Listing, 3) DNA encoding the functional equivalent of the present invention, and 4) the above 1) to And DNA encoding a protein exhibiting amino terminal protecting group free activity capable of hybridizing to DNA of 3).

Such DNA of this invention can be obtained as follows, for example.

First, the DNAs of 1) and 2) can be obtained from pyrococcus puriosus DSM3638 as described in the description of the amino terminal protecting group freease of the present invention.

It is also possible to obtain DNA of a protein having the same activity as the enzyme of the present invention based on the nucleotide sequence of the DNA encoding the amino terminal protecting group free enzyme provided by the present invention. That is, by using a DNA encoding the enzyme of the present invention or a part of its nucleotide sequence as a hybridization probe or a primer for gene amplification such as PCR, a protein having a functionally equivalent activity to that of the enzyme is encoded. DNA can be screened. By this method, DNAs of 3) and 4) can be obtained.

In the above method, a DNA fragment containing only a part of the target DNA may be obtained. At this time, after examining the nucleotide sequence of the obtained DNA fragment and confirming that it is a part of the target DNA, the DNA fragment or part thereof is used as a probe. The entire target DNA can be obtained by performing hybridization or PCR using a primer synthesized based on the nucleotide sequence of the DNA fragment.

Said hybridization can be performed, for example on the following conditions. In other words, the DNA-immobilized membrane was treated with 6 × SSC (1 × SSC) containing 0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400, and 0.01% denatured salmon sperm DNA. Incubated with probe at 50 ° C. for 12-20 hours in 0.15 M NaCl, 0.015 M sodium citrate, pH7.0). After completion of incubation, fixed DNA, starting with washing at 37 ° C. in 2 × SSC containing 0.5% SDS, changing the SSC concentration in the range up to 0.1 times, and the temperature in the range up to 50 ° C. The membrane is washed until the signal of origin can be distinguished from the background, and then the probe is detected. Further, the new DNA obtained in this way can be checked whether or not the DNA obtained is intended by examining the activity of the protein encoded therein in the same manner as described above.

In addition, it is also possible to produce a transformant containing such DNA and to produce the above amino terminal protecting group free enzyme on an industrial scale.

In addition, even if it is not the same base sequence as the base sequence disclosed in this specification, as long as it codes for the protein which shows the amino terminal protective group free activity disclosed in this specification, such a base sequence is as above-mentioned in the scope of the present invention. It should be noted. The reason for this is as follows.

It is known that codons (combinations of three bases) that designate amino acids on genes are present in one to six kinds of amino acids. Accordingly, a large number of DNAs encoding amino acid sequences may be present even in the amino acid sequence. DNA is by no means stable in nature, and mutations in its sequence are not uncommon. In some cases, the amino acid sequence encoded by the mutation on the DNA is not changed (called a silent mutation), and in this case, other DNA encoding the same amino acid sequence is generated. Therefore, even if the DNA encoding a specific amino acid sequence is isolated, the possibility of the generation of multiple types of DNA encoding the same amino acid sequence cannot be denied while the organism containing the same continues. In addition, it is not difficult to artificially produce a plurality of DNAs encoding the same amino acid sequence using various genetic engineering techniques.

For example, in the production of genetically engineered proteins, when the codons used on the original DNA encoding the protein of interest are less frequently used in the host, the expression level of the protein may be low. In such a case, the high expression of the target protein is aimed at by artificially converting the codon into one frequently used in the host without changing the encoded amino acid sequence (for example, Japanese Patent Application Laid-Open No. 7-). 102146). Thus, it goes without saying that the various types of DNA encoding a specific amino acid sequence can be produced in nature, not to mention artificially.

Moreover, even if DNA which introduce | transduced the mutation artificially in order to have a specific characteristic to a protein, if it codes the enzyme which has the amino terminal protective group free activity of this invention, it will be included in DNA of this invention. Examples of such DNA include DNA having a nucleotide sequence shown in SEQ ID NO: 11 in Sequence Listing. This DNA has a nucleotide sequence in which the base sequence TG present in base numbers 5 to 6 of the nucleotide sequence set forth in SEQ ID NO: 2 of the sequence listing is substituted with the nucleotide sequence AT, and when the protein encoded in the DNA is expressed in yeast, The amino-terminal protecting group free enzyme in which the valine of amino acid number (2) of the amino acid sequence of SEQ ID NO: 1 of the sequence list is substituted with aspartic acid, and whose N-terminal is acetylated is obtained.

3. Method for removing amino terminal protecting group of the present invention

The method for removing the amino terminal protecting group of the present invention is characterized in that the amino terminal protecting group-releasing enzyme of the present invention, or a functional equivalent thereof, is applied to a peptide in which the amino terminal is blocked by a protecting group to release the protecting group of the amino terminal.

As a specific removal method, for example, in the case of using an enzyme consisting of the amino acid sequence of SEQ ID NO: 1 in the Sequence Listing, the protecting group is released by reacting the substrate with the substrate at 50 ° C. in 50 mM PIPES-Na buffer (pH7.6). You can. However, it is natural that the reaction conditions differ depending on the type of protecting group and the peptide. Moreover, CoCl2 which raises the activity of this enzyme can also be added as needed.

4. Analysis method of amino acid sequence of the present invention

The method for analyzing the amino acid sequence of the present invention is a method for analyzing the amino acid sequence of a peptide in which the amino terminal is blocked by a protecting group, wherein the protecting group is liberated by the removal method, and then used for the analysis of the amino acid sequence. .

As a method for analyzing the amino acid sequence of a peptide whose amino terminal is blocked by a protecting group using the method for removing a protecting group of the present invention, for example, the amino terminal protecting group of the peptide whose sequence is to be determined is the amino terminal protecting group free enzyme of the present invention. After removal using, the sequence is determined from the newly generated amino terminal by Edman analysis.

In addition, when the sample subjected to the enzyme treatment using the amino terminal protecting group free enzyme of the present invention was used for the amino acid sequence analysis by Edman's analysis, an excess amount of enzyme was used to treat the amino acid sequence information of the sample for enzyme treatment. It may be considered that amino acid sequence information of the enzyme is incorporated into noise. In this case, for example, by using an amino terminal protecting group-releasing enzyme having an amino acid sequence shown in SEQ ID NO: 10 in the sequence listing, the amino terminal protecting group-releasing enzyme itself can be prevented from being incorporated without undergoing Edman degradation. In addition, the N-terminal protecting group in this enzyme is not specifically limited to an acetyl group.

In addition, when the amino terminal protecting group free enzyme has an aminopeptidase activity, the amino acid is liberated sequentially by the action of the enzyme following the liberation of the protecting group, but only if the amino terminal protecting group is controlled to control the action of the enzyme. The amino acid sequence may be determined by Edman decomposition after removing the protecting group and the subsequent amino acid residues.

In addition, by utilizing this aminopeptidase activity, amino acid sequence analysis can be performed by a method as described below. That is, after the amino-terminal protecting group free enzyme of the present invention is applied to the peptide to determine the sequence, a method of determining the sequence by analyzing the change over time of the amino acid liberated in the reaction solution, and a variety of occurrences in the reaction solution There is a method of determining the amino acid composition of the partially cleaved peptide and comparing the same, and in particular, the latter method is advantageous in identifying the amino acid composition of the peptide by molecular weight measurement using mass spectrometry. In the method using mass spectrometry, the kind of free amino acid can be easily determined from the difference in molecular weight of the partially cleaved peptide, and the kind of protecting group present at the amino terminus can be determined simultaneously.

5. Amino Acid Sequence Analysis Kit of the Present Invention

The kit for amino acid sequence analysis of the present invention is a kit used for the analysis of the amino acid sequence of a peptide whose amino terminal is blocked by a protecting group, and is characterized by containing the enzyme of the present invention described above. Such kits can be used for amino acid sequencing of peptides with amino terminal blocked, regardless of the type of protecting group. Moreover, in this kit, the standard product of a protecting group compound for identifying the protecting group which blocks the amino terminal of a peptide can also be added.

6. Antibodies, Probes or Primers of the Invention

By a conventional method, an antibody or fragment thereof that specifically binds to the amino terminal protecting group free enzyme or a functional equivalent thereof can be obtained. Such antibodies are useful for the purification and detection of the enzyme of the present invention. Moreover, the synthetic oligonucleotide probe or synthetic oligonucleotide primer which can hybridize with said DNA can be obtained by a conventional method. Such probes and primers are useful for detecting or amplifying the DNA of the present invention.

Although the Example of this invention is shown below, this invention is not limited only to a following example. In addition,% in an Example means weight%.

Accordingly, an object of the present invention is to provide an amino-terminal protecting group-releasing enzyme or a functional equivalent thereof that exhibits an amino-terminal protecting group free activity with respect to two or more protecting groups, a DNA encoding the enzyme, a method for producing the enzyme, and a method for causing this enzyme to act. There is provided a method for removing an amino terminal protecting group, a method for analyzing an amino acid sequence using the method, and a kit for amino acid sequence analysis containing the enzyme. It is also an object of the present invention to provide an antibody or fragment thereof that specifically binds to the amino terminal protecting group free enzyme or a functional equivalent thereof, and a synthetic oligonucleotide probe or a synthetic oligonucleotide primer capable of hybridizing with the DNA. have.

The present inventors screened a cosmid protein library derived from Pyrococcus puriosus and obtained one cosmid clone expressing the activity of releasing the protecting group at the amino terminal. The present inventors isolated the gene of the amino terminal protecting group free enzyme contained in this clone, and determined the base sequence. Furthermore, by constructing a recombinant plasmid expressing a large amount of this enzyme in microorganisms, the enzyme was produced successfully, and various enzymatic properties of the enzyme were revealed. It has also been found that this enzyme has the activity of releasing a plurality of amino terminal protecting groups including an acetyl group. In addition, the present invention has been completed by successfully producing functional equivalents of this enzyme.

That is, the gist of the present invention,

[1] An enzyme having an activity of releasing a protecting group by acting on a peptide blocked by an amino terminal by a protecting group (hereinafter referred to as "amino-terminal protecting group free activity"), wherein the enzyme exhibits the above-described activity against two or more protecting groups. Amino terminal protecting group free enzyme,

[2] the enzyme according to the above (1), which exhibits an amino terminal protecting group free activity to at least two protecting groups selected from the group consisting of an acetyl group, a pyroglutamyl group, a formyl group and a myristoyl group;

[3] the enzyme according to the above (1) or (2), which also has amino peptidase activity;

[4] the enzyme according to any one of (1) to (3), having the following physicochemical properties:

(1) Optimum temperature: 75 to 95 ℃ at pH7.6

(2) Optimum pH: pH6.5 to 9.5

(3) Influence of various reagents: The activity is inhibited by amastatin and promoted by CoCl 2,

[5] The enzyme according to any one of (1) to (4), containing all or part of an amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and exhibiting amino terminal protecting group free activity;

[6] In the above (5), the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is one or more amino acid residues deleted, added, inserted or substituted, and exhibits an amino terminal protecting group free activity. Functional equivalents of the enzymes described,

[7] the functional equivalent of (6) above, wherein the amino terminus is blocked by a protecting group,

[8] the functional equivalent of (7) above, wherein the protecting group is an acetyl group;

[9] a functional equivalent of (8) above having an amino acid sequence as set forth in SEQ ID NO: 10 in Sequence Listing,

[10] DNA encoding the amino terminal protecting group freease according to any one of (1) to (4);

[11] DNA encoding a polypeptide which contains all or part of the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing and exhibits amino terminal protecting group free activity;

[12] DNA encoding all or part of the DNA shown in SEQ ID NO: 2 of the Sequence Listing and encoding a polypeptide having amino terminal protecting group free activity;

[13] a DNA encoding at least one amino acid residue at least one of deletion, addition, insertion or substitution in the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, and also having an amino terminal protecting group free activity;

[14] DNA according to the above (13), having the nucleotide sequence shown in SEQ ID NO: 11 in Sequence Listing;

[15] DNA encoding a protein having amino terminal protecting group free activity capable of hybridizing to DNA according to any one of (10) to (14);

[16] a recombinant DNA containing the DNA according to any one of (10) to (14);

[17] an expression vector having a recombinant DNA according to the above (16) inserted therein, the microorganism, an animal cell or a plant cell as a host cell;

[18] a transformant transformed with the expression vector described in (17) above,

[19] An amino terminal characterized by culturing the transformant according to the above (18), and extracting a protein having an amino terminal protecting group free activity or a polypeptide having a functionally equivalent activity with the activity of the protein in the culture. Process for preparing protecting group free enzyme or functional equivalent thereof.

[20] The amino terminal protecting group is freed by reacting the amino terminal protecting group free enzyme according to any one of (1) to (9) or a functional equivalent thereof with a peptide blocked by the protecting terminal. Method of removing amino terminal protecting group.

[21] A method for analyzing an amino acid sequence of a peptide whose amino terminal is blocked by a protecting group, wherein the peptide produced by releasing the protecting group by the removing method described in (20) above is used for analyzing the amino acid sequence. How to interpret amino acid sequences,

[22] A kit used for the analysis of the amino acid sequence of a peptide whose amino terminal is blocked by a protecting group, the amino terminal protecting group-releasing enzyme or the functional equivalent of any one of (1) to (9). Kit,

[23] an antibody or fragment thereof that specifically binds to an amino-terminal protecting group-releasing enzyme according to any one of (1) to (9), or a functional equivalent thereof;

[24] The present invention relates to a synthetic oligonucleotide probe or a synthetic oligonucleotide primer capable of hybridizing with the DNA according to any one of (10) to (15).

Example 1

(Production of amino terminal protecting group free enzyme)

(1) Preparation of pyrococcus puriosus genomic DNA

The culture of Pyrococcus furiosus DSM 3638 was performed as follows.

The composition of the medium used is shown below: 1% tryptone, 0.5% yeast extract, 1% soluble starch, 3.5% jamarin S solids (jamarin laboratory), 0.5% jamarin S liquid (jamarin laboratory) , 0.003% MgSO ₄ , 0.001% NaCl, 0.0001% FeSO ₄ · 7H ₂ O, 0.001% CoSO ₄ , 0.0001% CaCl ₂ · 7H ₂ O, 0.0001% ZnSO ₄ , 0.1 ppm CuSO ₄ · 5H ₂ O, 0.1 ppm KAl (SO ₄ ) ₂ , 0.1 ppm H ₃ BO ₃ , 0.1 ppm Na ₂ MoO ₄ · 2H ₂ O, 0.25 ppm NiCl ₂ · 6H ₂ O.

2 liters of the medium of the above composition was placed in an intermediate bottle for 2 liters, sterilized at 120 ° C. for 20 minutes, and nitrogen gas was blown to remove dissolved oxygen. Subsequently, the cells were inoculated and incubated at 95 ° C. for 16 hours. After incubation, the cells were collected by centrifugation.

The aggregates were then suspended in 4 ml of 0.05 M Tris-HCl (pH 8.0) containing 25% sucrose, and 0.8 ml of lysozyme [5 mg / ml, 0.25 M Tris-HCl (pH 8.0) in this suspension. )], 2 ml of 0.2 M EDTA (pH8.0) was added, followed by warming at 20 ° C for 1 hour, followed by 24 ml of SET solution [150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH8.0)]. 4 ml of 5% SDS and 400 µl of proteinase K (10 mg / ml) were further added, and the mixture was reacted at 37 ° C for 1 hour. After completion of the reaction, phenol chloroform extraction followed by ethanol precipitation yielded about 3.2 mg of genomic DNA.

(2) Construction of Cosmid Protein Library

400 µg of pyrococcus furiosus genomic DNA was partially cut into Sau3AI, and fractionated by concentration gradient ultracentrifugation to obtain a DNA fraction corresponding to 35 to 50 kb. Next, 1 μg of the triple helix cosmid vector was BamHI cleaved, mixed with 140 μg of the 35-50 kb DNA fraction described above, and ligated thereto, followed by an in vitro packaging method using "Gigapack Gold" (manufactured by Staratazine). A cosmid library was prepared by packaging fragments of pyrococcus furiosus genomic DNA into lambda phage particles.

E. coli DH5αMCR was transformed using a part of the obtained phage solution to obtain a cosmid clone. After selecting several of the obtained transformants to prepare a cosmid, and confirming that there was an insertion fragment of an appropriate size, a new 500 transformants were added to 150 ml of LB medium containing 100 µg / ml of ampicillin (trip) 10 g / liter, 5 g / liter yeast extract, 5 g / liter NaCl, pH7.2). The culture was centrifuged, the recovered cells were suspended in 1 ml of 20 mM Tris-HCl (pH 8.0), and heat-treated at 100 ° C for 10 minutes. Subsequently, the cell lysate generated by the ultrasonic heat treatment was further heat treated at 100 ° C. for 10 minutes. The digested product obtained from the supernatant after centrifugation was used as a cosmid protein library.

(3) Selection of cosmids containing amino terminal protecting group freease genes

First, a cosmid clone having peptidase activity in the cosmid protein library was selected by quantifying the amount of 7-imino-4-methylkhamline produced using amino acid-MCA as a substrate. That is, 10-30 μl of digests were extracted from the cosmid protein library, and 100 μl of 0.1 M PIPES-Na buffer (pH7.6) and 5 μl of 18 5 mM amino acid-MCA (dissolved in dimethyl sulfoxide) were added. It added and made it react at 90 degreeC for 1-3 hours. Thereafter, 7-amino-4-methylcoumarin produced using TiterTech Fluorocan II (manufactured by Dainippon Seiyaku Chemical Co., Ltd.) was measured at an excitation wavelength of 355 nm and a measurement wavelength of 460 nm. The degradation products having degradation activity against MCA, Leu-MCA, Ala-MCA, His-MCA were selected. Furthermore, using α-MSH as a substrate, one having an activity of sequentially releasing an acetyl group and an amino acid at the amino terminus in these degradation products was selected to obtain a cosmid clone corresponding to this degradation product.

(4) Preparation of plasmid pDAP1 containing amino terminal protecting group freease gene

Cosmids were prepared from cosmid clones having the activity of sequentially releasing acetyl groups and amino acids at the amino terminus obtained as described above, and DNA fragments obtained by BamHI cleavage were inserted into the BamH site of the plasmid vector pUC18. This recombinant plasmid was introduced into Escherichia coli JM109, followed by LB plate containing 100 μg / ml of ampicillin (10 g / liter of tryptone, 5 g / liter of yeast extract, 5 g / liter of NaCl, 15 g / liter of agar, pH 7.2). Scattered above, the resulting transformants were individually cultured in 5 ml LB medium containing 100 μg / ml ampicillin. The cultured cells were centrifuged and suspended in 50 µl of 50 mM Tris-HCl (pH 8.0), heat-treated at 100 ° C for 10 minutes, and the cells were crushed by sonication. The cell lysate was once again heat treated at 100 ° C. for 10 minutes, and centrifuged to obtain a digested product. Peptidase activity was measured for this digest.

That is, 50 µl of 0.1 M PIPES-Na buffer (pH7.6) and 5 µl of 5 mM Met-MCA (dissolved in dimethyl sulfoxide) were added to 15 µl of the digested product, and the mixture was reacted at 90 ° C for 1.5 hours. Then, the amount of 7-amino-4-methyl coumarin produced | generated was measured with excitation wavelength 355 nm and measurement wavelength 460 nm using the Titatech Fluoro sCan II (made by Dainippon Seiyaku Co., Ltd.). A plasmid was prepared from a transformant having Met-MCA degrading activity and named plasmid pDAP1.

(5) Preparation of plasmid pDAP2 containing amino terminal protecting group freease gene

EcoRI digestion was performed on the plasmid pDAP1 and self-ligated. This recombinant plasmid was introduced into Escherichia coli JM109, and the peptidase activity was examined for the digested product prepared with the obtained transformant by the above-described method. A plasmid was prepared from a transformant whose enzyme activity was recognized and named plasmid pDAP2.

(6) Preparation of plasmid pDAP3 containing amino terminal protecting group freease gene

A DNA fragment of about 1.7 kb obtained by SacI cleavage of the plasmid pDAP2 was inserted into the SacI site of the plasmid vector pUC18. This recombinant plasmid was introduced into Escherichia coli JM109, and the peptidase activity was examined for the digested product prepared from the obtained transformant by the above-described method. A plasmid was prepared from a transformant whose enzyme activity was recognized and named plasmid pDAP3. 1 shows a restriction map of the plasmid pDAP3.

(7) Preparation of plasmid pDAP containing amino terminal protecting group freease gene

The plasmid pDAP3 was digested with SnaBI and SacI, and then subjected to agarose electrophoresis to recover a DNA fragment of about 1.2 kb from agarose. This DNA fragment was inserted into the plasmid vector pUC19 using the SmaI-SacI site. This recombinant plasmid was introduced into Escherichia coli JM109, and the peptidase activity was examined for the digested product prepared from the obtained transformant by the above-described method. It was confirmed that this degradation product further had an activity of sequentially releasing the acetyl group and the amino acid at the amino terminal with respect to α-MSH having an acetyl group at the amino terminal.

Plasmids were prepared from colonies showing this enzymatic activity and named plasmid pDAP. 2 shows a restriction map of the plasmid pDAP. Escherichia coli JM109 transformed with plasmid pDAP was designated as E. coli JM109 / pDAP, and was applied to the Biotechnology Industrial Technology Research Institute of the Ministry of Trade, Industry and Energy Deposited as FERM BP-5804 (Original Deposit Date: March 29, 1996, Transfer to International Deposit: January 30, 1997).

(8) Determination of base sequence of amino-terminal protecting group free enzyme gene

A DNA fragment of about 1.2 kb containing the amino terminal protecting group freease gene inserted into the plasmid pDAP was fragmented into a suitable size with various restriction enzymes, and the fragment was inserted into a plasmid vector. Decided. The base sequence was determined by the dideoxy method using the BcaBEST dideoxy sequencing kit (manufactured by Takarashu Research Co., Ltd.). The base sequences of the respective fragments were compared and synthesized to determine the total base sequence of the 1.2 kb DNA fragment.

SEQ ID NO: 3 in the Sequence Listing shows the base sequence of a DNA fragment of about 1.2 kb that contains DNA encoding the amino terminal protecting group freease inserted into the plasmid pDAP. Among the sequence numbers, base numbers 86 to 88 were start codons, base numbers 1130 to 1132 were stop codons, and an open reading frame therebetween was assumed as the structural gene region of the enzyme.

SEQ ID NO: 2 in Sequence Listing shows the base sequence of the open reading frame. In addition, the amino acid sequence of the amino terminal protecting group free enzyme of this invention estimated from the base sequence shown by SEQ ID NO: 1 in SEQ ID NO: 2 in Sequence Listing is shown.

(9) Preparation of enzyme standard

E. coli JM109 / pDAP was inoculated into 12 test tubes each containing 5 ml of LB medium containing 100 μg / ml of ampicillin, and cultured at 37 ° C. under normal culture conditions. Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 1 mM and incubated at 37 ° C. for 16 to 18 hours.

A total of 60 ml of the culture solution obtained above was centrifuged to recover the cells. The obtained cells were suspended in 0.6 ml of Buffer A (20 mM Tris-HCl pH8.0), heat-treated at 100 ° C. for 10 minutes, and then crushed cells by sonication, and then again at 100 ° C. for 10 minutes. Heat treatment was carried out and centrifuged to obtain a decomposed product. The obtained digested product was subjected to gel filtration on a 50 ml Sephacryl S-300 HR (Pharmacia Co., Ltd.) column equilibrated with buffer B (50 mM Tris-HCl: pH8.0) in advance to obtain a fraction having Met-MCA degradation activity. Collected. Further, this active fraction was adsorbed onto a 5 ml Econopack highQ cartridge (manufactured by Biorad), which had previously been equilibrated with Buffer B, and sufficiently washed with Buffer B, followed by Buffer B having a NaCl linear concentration gradient of 0 to 0.5 M. Eluted. Met-MCA degradation activity was measured for the eluate, and the active fractions were collected to obtain a purified enzyme standard.

One unit of the obtained purified enzyme standard product was Met-MCA as a substrate, and the amount of enzyme capable of producing 1 μmol of 7-amino-4-methylcoumarin in 1 minute at pH7.6 and 75 ° C.

Example 2

(Physicochemical Properties of Enzyme Standard Prepared in Example 1)

1) Optimum temperature

The measurement of the optimum temperature was performed by the operation shown below. That is, the activity was measured at various temperatures by the enzyme activity measuring method using Met-MCA as a substrate using an enzyme standard of 18 micro units. As shown in Fig. 3, the enzyme of the present invention was active in the range of 25 to 95 DEG C at the measured pH7.6, and exhibited the highest activity at 95 DEG C, which was the highest temperature measured. In FIG. 3, the optimum temperature of the enzyme standard of the present invention was 75 to 95 ° C at pH7.6. In the figure, the vertical axis represents relative activity (%) with respect to the maximum activity (95 ° C), and the horizontal axis represents reaction temperature (° C).

2) optimum pH

The measurement of optimum pH was performed by the operation shown below. That is, in the enzyme activity measuring method using Met-MCA shown above as a substrate using an enzyme standard product of 18 micro units, the buffer solution added to the reaction solution was changed to a complete solution of various pHs to measure the activity. As shown in FIG. 4, the optimum pH of the enzyme of the present invention was about 6.5 to 9.5. In the figure, the vertical axis represents relative activity (%) with the activity at pH 7.2 as 100, and the horizontal axis represents the pH at 75 ° C. The buffer used for the activity measurement includes 20 mM sodium acetate complete solution at pH4.1 to pH5.1, 20 mM PIPES-Na buffer at pH5.8 to pH7.2, 20 mM sodium borate buffer at pH8.0 to 9.5, pH9 In .9 to 10.6, 20 mM disodium hydrogen phosphate-sodium hydroxide buffer was used. In addition, said pH is a value of 75 degreeC.

3) Influence of various reagents

The activity of the enzymes of the present invention was inhibited by amastatin. For example, 3 milliseconds of the enzyme of the present invention was treated in 50 µl of 20 mM boric acid buffer solution (pH10.0) containing 1 mM amastatin at 37 ° C. for 30 minutes, and 5 µl of the enzyme according to the above 2). When the activity was measured by the method shown in the activity assay, a value of 1.5% was shown for the treatment with the buffer containing no amastatin.

In addition, the activity of the enzyme of the present invention was promoted by CoCl ₂ . For example, when CoCl ₂ was added to the measurement system using Met-MCA so as to have a final concentration of 91 μM, the activity was about 6 times higher than that of the non-addition.

4) molecular weight

The enzyme of the present invention exhibited a molecular weight of about 40,000 and ultracentrifugation by about 400,000 by SDS-polyacrylamide electrophoresis.

5) Temperature stability

The residual enzyme activity after heat treatment of the enzyme standard was investigated to investigate the temperature stability of the enzyme of the present invention. That is, 0.1 M Tris-HCl buffer containing pH of 26 milliliters (pH8.0), or a final concentration of 0.1 mM CoCl ₂ was added thereto at 75 ° C. for 0 to 5 hours, and then partially The residual enzyme activity was measured using. In addition, activity measurement was performed by the enzyme activity measurement method by the system which used said Met-MCA as a substrate.

As shown in Fig. 5, the enzyme of the present invention showed no degradation of the enzyme activity even after treatment for 5 hours in 0.1M Tris-HCl buffer (pH8.0) containing 0.1 mM CoCl ₂ . _The activity was maintained at 80% or more even when treated for 5 hours in a buffer containing no ₂ . In the figure, the vertical axis shows the remaining enzyme activity (%), and the horizontal axis shows the time (hour) of heat treatment.

Example 3

(Confirmation of the amino terminal protecting group free activity of the enzyme standard prepared in Example 1)

Using the enzyme standard prepared in Example 1, the action of this enzyme on the synthetic peptide with the amino terminal blocked was examined.

(1) Action on pyroglutamyl group

To 10 µl of 1 mM neurotensin, 0.1 µm of enzyme standard (5 µl), 50 µl of 40 mM PIPES-Na buffer solution (pH7.6), 35 µl of distilled water were added, and 0, 1, 3, The reaction was carried out for 5 hours. A part of the reaction solution was subjected to amino acid analysis using an L-8500 type high speed amino acid analyzer (manufactured by Hitachi Seisakusho Co., Ltd.) to quantify the free amino acid generated in the reaction solution. The result is shown in FIG. As shown in Fig. 6, leucine, tyrosine, and glutamic acid present at the amino terminal side of neurotensin are free from the reaction solution, and the enzyme acts on the amino terminal side of neurotensin from changes over time. It was shown that the amino acids were released sequentially. In addition, since leucine was detected as a free amino acid, it became clear that this enzyme cleaved the bond between pyroglutamyl and leucine.

(2) action on acetyl groups

To 1 mM α-MSH (10 µl), 0.12 milligrams of enzyme standard (5 µl), 40 µm of 40 mM PIPES-Na (pH7.6) buffer and 35 µl of distilled water were added. After reacting for 1, 3 and 5 hours, the free amino acid produced by the reaction was analyzed in the same manner as described above. The result is shown in FIG. As shown in Fig. 7, the amount of amino acid liberated in α-MSH is higher as it exists on the amino terminal side (serine is present at the amino terminal and in the second two places at the amino terminal, and thus, compared with other amino acids). It is confirmed that this enzyme acts on the amino terminal side of α-MSH and cleaves the bond between the acetyl group and serine.

Moreover, the result of having carried out the same experiment as the case of (alpha) -MSH by replacing a substrate with Ac-Gly-Asp-Val-Glu-Lys is shown in FIG. As shown in FIG. 8, it was suggested that glycine was liberated in the reaction solution, and the enzyme cleaved the bond between the acetyl group and the glycine, followed by the glycine-aspartic acid. In addition, in the reaction solution, the presence of trace amounts of aspartic acid and valine in addition to glycine was also confirmed.

(3) action on formyl groups

To 0.5 µl of 10 mM For-Met-Leu-Phe-Lys (30% acetic acid solution) was added 4 ml of enzyme standard (3.5 µl), 25 µl of 0.1 M N-ethylmorpholine and 21 µl of distilled water. After reacting for 0, 20, 40, 60, 120 minutes at 37 degreeC, the free amino acid produced | generated by reaction was analyzed by the same operation as the above. The result is shown in FIG. As shown in Fig. 9, first, methionine free of formyl group was liberated in the reaction solution, and the enzyme of the present invention was found to have an activity of releasing formyl group. It has also been shown that the enzyme releases amino acids sequentially on the amino terminal side of the substrate peptide from the change over time of the production of other amino acids (leucine, phenylalanine, lysine).

(4) action on myristoyl group

To 5 μl of 1 mM Myr-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln solution, 4 milligrams of enzyme standard (3.5 μl), 0.1 M N-ethylmorpholine acetic acid buffer (pH9 .0) 25 µl and 16.5 µl of distilled water were added and reacted at 37 ° C for 0, 1.5, 3, 6, and 9 hours, and the free amino acid produced by the reaction was analyzed in the same manner as described above. The result is shown in FIG. As shown in FIG. 10, first, phenylalanine in which the myristoyl group was liberated was released in the reaction solution, and it was shown that the enzyme of the present invention had the activity of releasing the myritriyl group. In addition, other changes in the amino acid generation over time showed that the enzyme liberated amino acids sequentially on the amino terminal side of the substrate peptide.

In addition, the result of performing the same experiment as above by replacing the substrate with Myr-Gly-Ala-Gly-Ala-Ser-Ala-Glu-Glu-Lys is shown in FIG. Since the substrate peptide has two residues of glycine and three residues of alanine in the molecule, it is difficult to determine the free order of amino acids from the result of FIG. 11, but the enzyme of the present invention acts from the amino acid terminal side of the substrate. And it was suggested that the enzyme of this invention has the activity which liberates a myristoyl group.

Example 4

(Confirmation of aminopeptidase activity on the protein of the enzyme standard prepared in Example 1)

The action of the enzyme standard on the protein was investigated using a chicken egg white reducing lysozyme as a substrate.

50 mM disodium hydrogen phosphate containing 8 mM enzyme standard (1.6 µl) and 0.1 mM CoCl ₂ in 5 µl of 1 mM chicken egg white reducing lysozyme (water soluble, molecular weight about 14000, manufactured by Okun Koyaku Co., Ltd.) 50 µl of sodium hydroxide buffer (pH11.0) and 43.4 µl of distilled water were added and reacted at 50 ° C for 0, 40, 80, 120, 180, 240 minutes. A part of the reaction solution was subjected to amino acid analysis using an L-8500 type high-speed amino acid analyzer (manufactured by Hitachi Seisakusho Co., Ltd.) to quantify the free amino acid generated in the reaction solution. The result is shown in FIG. As shown in Fig. 12, lysine, valine, phenylalanine, glycine, and arginine present in the amino terminus of the reduced lysozyme are free from the reaction solution, and the amino terminus of the reduced lysozyme is shown by the change in the amount of its production over time. It was shown that the amino acid was released sequentially by acting on the side.

In addition, the amino acid sequence from the amino terminal of the reduced lysozyme used here is Lys-Val-Phe-Gly-Arg. Was in order.

Example 5

(Preparation of amino terminal protecting group free enzyme whose N terminal is blocked by acetyl group)

(1) Modification of amino terminal protecting group free enzyme gene

A mutation was introduced into pDAP so that the N-terminal amino acid sequence of the enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing became the acetyl group addition signal Met-Asp. Specifically, in the sequence within the multicloning site on the plasmid pDAP and the synthetic DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 12 of the sequence listing for substitution of the second amino acid valine codon with the aspartic acid codon from the N-terminus of this enzyme. PCR was carried out using another synthetic DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 13 for the destruction of each HindIII and SphI restriction enzyme site as a primer pair, and pDAP DNA as a template. After the PCR reaction, the reaction solution was enzymatically treated with SphI (manufactured by Takarashu Irradiation) to cleave the plasmid without introducing the mutation, and then introduced into Escherichia coli JM109 (manufactured by Takarashu Irradiation) to obtain the desired transformant. Mutated plasmids were prepared. Next, the plasmid DNA is used as a template, and the synthetic DNA comprising the nucleotide sequence set forth in SEQ ID NO: 14 of the sequence listing for the introduction of the BamHI site immediately upstream of the protein start codon in the protein code region of the DNA; PCR was performed using another synthetic DNA consisting of a nucleotide sequence complementary to the region downstream of the protein code region of this DNA according to SEQ ID NO: 15 as a primer pair. The reaction solution was used for agarose electrophoresis to recover the amplified DNA fragment of about 1.2 kb in agarose gel. This DNA fragment was digested with BamHI (manufactured by Takarashu Irradiation) and HindIII (manufactured by Takarashu Irradiation) and inserted using the BamHI, HindIII site of the plasmid vector pVT103-L, which is a shuttle vector of Escherichia coli and yeast. This recombinant plasmid was introduced into Escherichia coli JM109, and plasmid DNA was prepared from the resulting transformant.

(2) Expression of the modifying enzyme

After introducing the obtained plasmid DNA into yeast BJ2168, the nutrient-containing plate (yeast nitrogen base 6.7g / liter, glucose 20g / liter, tryptophan 20mg / liter, histidine 20mg / liter, uracil 20mg / liter, agar 15g / The transformants obtained on the liter) were individually inoculated into test tubes containing 5 ml of YPD medium (10 g / liter yeast extract, 20 g / liter peptone, 20 g / liter glucose) and incubated at 30 ° C for 16 hours. Each culture was individually inoculated into an Erlenmeyer flask containing 500 ml of the above nutrient-containing media and incubated at 30 ° C for 16 hours. The culture was centrifuged to recover the cells. The obtained cells were suspended in 50 ml of buffer C (1M sorbitol, 50 mM Tris-HCl, pH7.5, 30 mM DTT), and kept at 30 ° C. for 10 minutes, and then the cells obtained by centrifugation were further washed with 10 ml of buffer. Suspended in D (1 M sorbitol, 50 mM Tris-HCl, pH7.5, 2 mM DTT), Zymoriace 100T (manufactured by Namakaga Co., Ltd.) was added to a final concentration of 0.5 mg / ml, and 30 at 30 ° C. After warming for a minute, centrifugation was carried out to obtain a protoplast. The obtained protoplasts are suspended in 20 ml of Buffer E (50 mM Tris-HCl, pH7.5, 2 mM DTT) and EDTA, KCl, Triton X-100 are added to a final concentration of 1 mM, 0.2 M and 0.2%, respectively. And it kept warm at 37 degreeC for 5 minutes. The suspension was heat-treated at 100 ° C. for 15 minutes, and then centrifuged to obtain a decomposed product. From the obtained digested product, a modified enzyme standard was prepared according to the purification method equivalent to the amino-terminal protecting group free enzyme described in (9) of Example 1, using Met-MCA degradation activity as an index. In addition, 1 unit of this modifier standard was made into the quantity of enzyme which can produce 1 micromol of 7-amino-4- methyl coumarin in 1 minute at pH7.6 and 75 degreeC using Met-MCA as a substrate. Yeast expressing the protein is named and labeled BJ2168 / pAcDAP in Saccharomyces cerevisiae, and is the life of the Ministry of Trade, Industry and Energy It is deposited with the Institute of Engineering and Technology as FERM BP-5952 (original date: May 23, 1997).

(3) Determination of amino terminal protecting group free activity of modifier

The amino terminal protecting group free activity of the modified enzyme standard prepared in (2) was measured according to the methods described in (2) and (4) of Example 3. As a result, this modifier showed free activity against the acetyl group and myristoyl group at the N-terminal of the peptide, and also showed activity to liberate amino acids sequentially at the N-terminal of the peptide.

(4) Confirmation of N-terminal acetylation of the modifier

It was confirmed by mass spectrometry whether the N terminus of this modifier was acetylated. That is, the mass of the enzyme prepared in Example 1 and this modifier was measured by API / 300 (manufactured by PE-Sciex) to determine the mass difference. As a result, the molecular weight of the enzyme expressed in Escherichia coli and prepared in Example 1 was 38586, and the molecular weight of this modifier was 38643. The molecular weight difference 57 between the two enzymes coincided with the sum of the molecular weight increase 42 in which valine was substituted with aspartic acid and the addition of an acetyl group 42.

In addition, this modifier was used in the HP G1000A protein sequencer (manufactured by Hewlett Packard) employing the N-terminal amino acid sequence analysis method by Edman's decomposition, but it was clear that no signal was obtained and the modifier was not subjected to Edman analysis. Done

(5) Analysis of amino terminal sequence of peptide using modifier

As a substrate of the modifying enzyme, a carboxymethylated product of red blood cell superoxide dismutase (molecular weight 15551, manufactured by Washington Biochemical Corporation) of N-terminal acetylated cow was used. To 5 µl of the solution prepared so that the substrate became 0.4 mM, 600 µm of the reformate standard (52 µl) prepared in (2), 100 µl of 100 mM trimethylamine-HCl (pH11.0) buffer, 10 mM 2 µl of CoCl ₂ and 41 µl of distilled water were added, and after 48 hours of reaction at 50 ° C, the reaction solution was subjected to amino acid sequencing using the HP G1000A protein sequencer as it was. The obtained amino acid sequence data is shown in SEQ ID NO: 16 in Sequence Listing. This sequence was consistent with the known amino acid sequence of the superoxide dismutase used as the substrate. In the amino acid sequence analysis, no signal other than the amino acid sequence of the superoxide dismutase was mixed.

Example 6

(Interpretation of amino terminal sequence of peptide using mass spectrometry)

For-Met-Leu-Phe-Lys was used as the enzyme standard and substrate prepared in Example 1, and the amino terminal sequence of the peptide was analyzed by mass spectrometry.

1.5 μl of 10 mM For-Met-Leu-Phe-Lys (30% acetic acid solution), 2.4 milligrams of enzyme standard (2.1 μl), 0.1 μl of ammonium bicarbonate, 75 μm of 1 mM CoCl ₂ (15 μl), After adding 56.4 µl of distilled water and reacting at 30 ° C for 4 hours, 15 µl of formic acid was added to stop the reaction. Next, the reaction solution was subjected to reverse phase HPLC (high-performance liquid chromatography) using a DEVELOSIL ODS-HG-5 column (manufactured by NOMURA CHEMICAL Co., Ltd.), and four peaks containing peptides in the reaction solution were separated and extracted. In addition, the molecular weight of the peptide contained in each peak was measured using the JMS-HX100 double convergent FAB mass spectrometer (made by JEOL). The elution time of each peak, the molecular weight of the peptide contained in the peak, and the sequence of each peptide estimated from this molecular weight are shown in Table 1.

Peak number Dissolution time (minutes) Molecular weight Estimated array One 12.125 Not measurable - 2 21.008 407.3           Leu-Phe-Lys 3 27.358 538.3       Met-Leu-Phe-Lys 4 32.583 566.4   For-Met-Leu-Phe-Lys

As shown in Table 1, it was the unreacted substrate peptide separated by peak 4 among the peptides produced in the reaction solution. In addition, the molecular weight of the peptide contained in the peak 3 was different from the formyl group in the substrate peptide, and the molecular weight of the one contained in the peak 2 was consistent with the release of the formyl group and methionine in the substrate peptide. From this result, it became clear that the enzyme of the present invention acts on the amino terminal side of the substrate peptide, thereby releasing formyl group and methionine sequentially. As described above, by accurately measuring the molecular weights of various partially digested peptides obtained by acting the enzyme of the present invention on peptides and comparing them, it was found that the amino terminal sequence of the peptides can be determined including the type of the protecting group of the amino terminal. .

The amino terminal protecting group free enzyme of the present invention exhibits an amino terminal protecting group free activity with respect to two or more kinds of protecting groups. In addition, the present invention provides a method for removing an amino terminal protecting group of a peptide using such an enzyme. This enzyme is useful for amino acid sequencing of peptides, particularly proteins and peptides whose amino ends are blocked by unidentified protecting groups. In addition, the present invention provides a DNA encoding such an enzyme and a method for producing the enzyme. The N-terminal acetylated amino-terminal protecting group free enzyme produced by modifying this DNA is not subjected to Edman degradation, and is particularly useful because the amino acid sequence information derived from this enzyme does not become noise in an amino acid sequence analysis method using Edman degradation.

[Sequence list]

SEQ ID NO: 1

Sequence length: 348

Type of sequence: amino acid

Number of chains: 1 chain

Topology: linear

Type of molecule: peptide

Description of Sequences: 1:

[SEQ ID NO: 1a]

[SEQ ID NO: 1b]

SEQ ID NO: 2

Sequence length: 1044

Type of sequence: nucleic acid

Number of chains: 2 prints

Topology: linear

Type of molecule: genomic DNA

Description of sequence: 2:

[SEQ ID NO 2]

SEQ ID NO: 3

Length of sequence: 1247

Type of sequence: nucleic acid

Number of chains: 2 prints

Topology: linear

Type of molecule: genomic DNA

Description of sequence: 3:

[SEQ ID NO 3]

SEQ ID NO: 4

Sequence length: 12

Type of sequence: amino acid

Number of chains: 1 chain

Topology: linear

Type of molecule: peptide

Sequence Features: 1 (N-pyroglutamyl-leucine)

Description of sequence: 4:

[SEQ ID NO 4]

SEQ ID NO: 5

Sequence length: 13

Type of sequence: amino acid

Number of chains: 1 chain

Topology: linear

Type of molecule: peptide

Sequence Features: 1 (N-acetyl-serine)

Description of sequences: 5:

[SEQ ID NO: 5]

SEQ ID NO: 6

Sequence length: 5

Type of sequence: amino acid

Number of chains: 1 chain

Topology: linear

Type of molecule: peptide

Sequence Features: 1 (N-acetyl-glycine)

Description of sequence: 6:

[SEQ ID NO: 6]

SEQ ID NO: 7

Sequence length: 4

Type of sequence: amino acid

Number of chains: 1 chain

Topology: linear

Type of molecule: peptide

Sequence Features: 1 (N-formyl-methionine)

Description of sequence: 7:

[SEQ ID NO: 7]

SEQ ID NO: 8

Sequence length: 9

Type of sequence: amino acid

Number of chains: 1 chain

Topology: linear

Type of molecule: peptide

Sequence Features: 1 (N-myristoyl-phenylalanine)

Description of sequences: 8:

[SEQ ID NO: 8]

SEQ ID NO: 9

Sequence length: 9

Type of sequence: amino acid

Number of chains: 1 chain

Topology: linear

Type of molecule: peptide

Sequence Features: 1 (N-myristoyl-glycine)

Description of sequence: 9:

[SEQ ID NO: 9]

SEQ ID NO: 10

Sequence length: 348

Type of sequence: amino acid

Number of chains: 1 chain

Topology: linear

Type of molecule: peptide

Sequence Features: 1 (N-acetyl-methionine)

Description of sequence: 10:

[SEQ ID NO: 10a]

[SEQ ID NO: 10b]

SEQ ID NO: 11

Sequence length: 1044

Type of sequence: nucleic acid

Number of chains: 2 prints

Topology: linear

Type of molecule: genomic DNA

Description of sequence: 11:

[SEQ ID NO: 11]

SEQ ID NO: 12

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of molecule: other nucleic acids (synthetic DNA)

Description of sequence: 12:

[SEQ ID NO: 12]

SEQ ID NO: 13

Sequence length: 20

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of molecule: other nucleic acids (synthetic DNA)

Description of sequence: 13:

[SEQ ID NO: 13]

SEQ ID NO: 14

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of molecule: other nucleic acids (synthetic DNA)

Description of sequence: 14:

[SEQ ID NO: 14]

SEQ ID NO: 15

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: 1 chain

Topology: linear

Type of molecule: other nucleic acids (synthetic DNA)

Description of sequence: 15:

[SEQ ID NO: 15]

SEQ ID NO: 16

Sequence length: 151

Type of sequence: amino acid

Number of chains: 1 chain

Topology: linear

Type of molecule: peptide

Description of sequence: 16:

[SEQ ID NO: 16]

Claims (13)

It has the activity of releasing this protecting group by acting on the peptide blocked by the amino terminal by the protecting group (hereinafter referred to as "amino-terminal protecting group free activity"). Or the amino terminal protecting group freease of (2): (1) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1; or (2) A polypeptide having the amino acid sequence shown in SEQ ID NO: 10. The enzyme of claim 1, wherein the amino terminus of the enzyme is blocked by a protecting group. The enzyme according to claim 2, wherein the protecting group is an acetyl group. DNA encoding the amino terminal protecting group freease of claim 1. DNA encoding a polypeptide showing amino terminal protecting group free activity having the entire sequence of DNA shown by SEQ ID NO: 2 in Sequence Listing. The DNA according to claim 4, having a nucleotide sequence shown as SEQ ID NO: 11 in Sequence Listing. An expression vector in which the DNA according to any one of claims 4 to 6 is inserted, wherein the microorganism, animal cell or plant cell is used as a host cell. A transformant transformed with the expression vector of claim 7. A method for producing an amino terminal protecting group free enzyme, comprising culturing the transformant of claim 8 and collecting a protein having amino terminal protecting group free activity from the culture. A method for removing an amino terminal protecting group, wherein the amino terminal protecting group-releasing enzyme is reacted with a peptide having an amino terminal blocked by a protecting group to release the protecting group of the amino terminal. A method for analyzing the amino acid sequence of a peptide whose amino terminal is blocked by a protecting group, wherein the peptide generated by releasing the protecting group by the elimination method according to claim 10 is used for analysis of the amino acid sequence. Way. A kit used for analysis of an amino acid sequence of a peptide whose amino terminal is blocked by a protecting group, wherein the kit contains the amino terminal protecting group free enzyme of claim 1. An antibody or fragment thereof that specifically binds to the amino terminal protecting group freease of claim 1.