WO1997049819A1 - Nouvelle enzyme de deblocage des terminaisons amino - Google Patents
Nouvelle enzyme de deblocage des terminaisons amino Download PDFInfo
- Publication number
- WO1997049819A1 WO1997049819A1 PCT/JP1997/002121 JP9702121W WO9749819A1 WO 1997049819 A1 WO1997049819 A1 WO 1997049819A1 JP 9702121 W JP9702121 W JP 9702121W WO 9749819 A1 WO9749819 A1 WO 9749819A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- amino
- protecting group
- enzyme
- activity
- amino acid
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
Definitions
- the present invention relates to an amino terminal protecting group releasing enzyme having an activity of releasing a protecting group present at the amino terminal of proteins and peptides.
- the present invention also relates to DNA encoding such an amino terminal protecting group releasing enzyme.
- the present invention also relates to a method for producing such an amino terminal protecting group releasing enzyme by a genetic recombination technique.
- the present invention also relates to a method for removing an amino terminal protecting group using such an amino terminal protecting group releasing enzyme.
- the present invention relates to a method for analyzing an amino acid sequence using such a removing method.
- the present invention relates to a kit containing the amino terminal protecting group releasing enzyme and used for analyzing an amino acid sequence.
- the present invention relates to an antibody or a fragment thereof which specifically binds to the amino terminal protecting group releasing enzyme or a functional equivalent thereof. Furthermore, the present invention relates to a synthetic oligonucleotide probe or a synthetic oligonucleotide primer which hybridizes with the above-mentioned DNA. Background art
- Determining the amino-terminal amino acid sequence of proteins and peptides is an indispensable step for their identification or confirmation.
- Protecting groups that block the amino terminal of proteins and peptides include: Formyl, acetyl, myristoyl, pyroglutamyl, dimethyl, glucuronyl, glycosyl, and trimethyl groups have been reported.
- a method of analyzing the amino terminal amino acid sequence of these blocked proteins a method of applying the Edman degradation method after removing the protecting group is used.
- an object of the present invention is to provide an amino-terminal protecting group-enzyme having functional activity for protecting two or more protecting groups with an amino-terminal protecting group, a functional equivalent thereof, a DNA encoding the enzyme, and the enzyme. It is an object of the present invention to provide a method for producing an amino acid, a method for removing an amino terminal protecting group which acts on the enzyme, a method for analyzing an amino acid sequence using the method, and a kit for amino acid sequence analysis containing the enzyme.
- an object of the present invention is to provide an antibody or a fragment thereof that specifically binds to the amino-terminal protecting group releasing enzyme or a functional equivalent thereof, and a synthetic oligonucleotide probe or a synthetic oligonucleotide primer that hybridizes with the DNA. To provide.
- the present inventors searched for a cosmid protein library derived from Pyrococcus furiosus, and obtained one cosmid clone expressing the activity of releasing an amino-terminal protecting group.
- the present inventors isolated the amino-terminal protecting group releasing enzyme gene contained in this clone and determined its nucleotide sequence.
- a recombinant plasmid that expresses the enzyme in a large amount in a microorganism was constructed, the enzyme was successfully produced, and various enzymatic properties of the enzyme were clarified.
- the enzyme has an activity to release a plurality of amino terminal protecting groups including an acetyl group.
- the functional equivalent of the enzyme was successfully produced, and the present invention was completed. That is, the gist of the present invention is:
- amino terminal protecting group releasing activity An enzyme having an activity of acting on a peptide whose amino terminal is blocked by a protecting group to release the protecting group (abbreviated as “amino terminal protecting group releasing activity”), and two or more enzymes.
- amino-terminal protecting group-releasing enzyme which exhibits the above activity for a protecting group of
- the enzyme according to (1) which exhibits an amino-terminal protecting group-releasing activity for at least two or more protecting groups selected from the group consisting of an acetyl group, a virogultamyl group, a formyl group, and a myristoyl group. ,
- amino acid residues are deleted, added, inserted or substituted in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and at least one amino acid residue is deleted, and the amino terminal protecting group releasing activity A functional equivalent of the enzyme according to the above (5),
- one or more amino acid residues are deleted, added, inserted, or substituted in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and at least one amino acid residue is released, and the amino-terminal protecting group is released DNA encoding a protein that exhibits activity
- the amino-terminal protecting group is blocked by the amino-terminal protecting group-releasing enzyme or the functional equivalent thereof according to any of (1) to (9) above, on the peptide whose amino terminal is blocked by the protecting group.
- FIG. 1 is a diagram showing a restriction map of plasmid pDAP3.
- FIG. 2 is a diagram showing a restriction map of brassmid pDAP.
- FIG. 3 is a graph showing the optimum temperature of the amino terminal protecting group releasing enzyme of the present invention.
- FIG. 4 is a graph showing the optimum pH of the amino-terminal protecting group-enzyme in the present invention.
- ⁇ is a sodium acetate buffer solution. I PES-Na buffer, ⁇ indicates data using sodium borate buffer, and ⁇ indicates data using sodium hydrogen phosphate sodium hydroxide.
- FIG. 5 is a graph showing the temperature stability of the amino terminal protecting group releasing enzyme of the present invention at 75 ° C.
- Qin Isseki data of a buffer containing 0.101! ⁇ Of Flip 0 Ji 1 2, ⁇ indicates the data in buffer containing no CoC l 2.
- FIG. 6 is a graph showing the time-dependent action of an amino-terminal protecting group releasing enzyme on neurotensin in the present invention.
- FIG. 7 is a graph showing the aging effect of the amino terminal protecting group releasing enzyme on MSH in the present invention.
- FIG. 8 is a view showing the time-dependent action of the amino-terminal protecting group releasing enzyme of the present invention on Ac-G1y-Asp-Va1-G1u-Lys.
- FIG. 9 is a graph showing the time-dependent action of the amino-terminal protecting group-transferring enzyme of the present invention on Fo r -Me t -L e ⁇ -Phe-Ls.
- FIG. 10 is a graph showing the time-dependent action of the amino-terminal protecting group releasing enzyme of the present invention on Myr-Phe-A1a-Arg-Lys-GlyA1a-Leu-ArgGln. .
- FIG. 11 shows that the amino-terminal protecting group releasing enzyme of the present invention is Myr-G1y-A1a-G1y-A1a-Ser-A1a-Gl ⁇ -Glu-L.
- FIG. 4 is a view showing the time-dependent action on ys.
- FIG. 2 is a graph showing the time-dependent action of the amino terminal protecting group releasing enzyme of the present invention on reduced ribzyme.
- the amino-terminal protecting group releasing enzyme of the present invention is an enzyme having an activity of acting on a peptide having an amino terminal blocked by a protecting group to release the protecting group (amino terminal protecting group releasing activity), It is characterized by exhibiting activity on more than one kind of protecting group.
- peptide refers to one in which two or more amino acids are connected by a peptide bond, and includes those described as “protein J”.
- Protecting groups present at the amino terminus of this peptide include acetyl, pyrrogylyl, formyl, myristoyl, and the like.
- the amino-terminal protecting group-releasing enzyme of the present invention is an amino acid of the peptide of the substrate. It has an activity to release at least two or more of these protecting groups from the terminal. Such an amino terminal protecting group releasing activity can be measured by a method described later by using a synthetic peptide or the like whose amino terminal is blocked by the above protecting group as a substrate.
- the amino-terminal protecting group releasing enzyme of the present invention includes an enzyme having an amino-terminal protecting group releasing activity, an enzyme having an amino-terminal protecting group releasing activity and an aminopeptidase activity of sequentially releasing amino acid from the amino terminal of the peptide.
- Specific examples include an enzyme having an amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. If the peptide has additional aminopeptidase activity, the amino acid sequence of the peptide should be analyzed without the need to use other peptide amino terminal digestion methods. Both are possible.
- Such an aminopeptidase activity can be determined by a generally known method (Arch. Biochera. Biophys., Vol. 274, Vol. 241 to 250 (pp. 1989)). Can be measured.
- Examples of an enzyme having an amino terminal protecting group releasing activity and an aminopeptidase activity include, specifically, an enzyme having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
- the enzyme of the present invention may have its amino terminal in a free state, or the amino terminal of the enzyme itself may be blocked by a protecting group. Therefore, unless otherwise specified in the present specification, the amino terminus of the enzyme includes any state. Therefore, although SEQ ID NO: 1 shows a state in which the amino terminal is free, a compound having the sequence of SEQ ID NO: 1 and whose amino terminal is blocked by a protecting group such as a cetyl group is also described in the present invention. Is within the range.
- the amino-terminal protecting group releasing enzyme of the present invention can be obtained, for example, from 1) purification from a culture of a microorganism producing the enzyme of the present invention, and 2) from a culture of a transformant containing DNA encoding the enzyme of the present invention. Can be produced by a method such as purification.
- microorganism producing the enzyme of the present invention such a microorganism can be found by screening using the enzyme activity as an index.
- a method for detecting the enzymatic activity an amino acid or a peptide whose amino terminal is blocked with a protecting group is used as a substrate, and the release of the protecting group from the substrate is determined by high performance liquid chromatography / amino acid analysis. There is a method of confirming by mass spectrometry.
- a synthetic substrate obtained by adding an appropriate coloring group or fluorescent group to an amino acid blocked with a protecting group can also be used.
- the enzyme can be produced by culturing a microorganism in which production of the amino-terminal protecting group-releasing enzyme has been confirmed.
- the cultivation of the microorganism may be performed under conditions suitable for the growth of the microorganism, and is preferably such that the expression level of the target enzyme is high. Culture conditions are used.
- the target enzyme produced in the cells or the culture solution in this manner can be purified by a method generally used for enzyme purification.
- Microorganisms that produce the enzyme of the present invention include Pyrococcus furiosus DSM 368.
- Viroko Cass Friosus DSM 3638 can be obtained from Deutsch Sung Lung von Mikroorgani smen und Zellkulturen und GmbH, Germany. It is a strain.
- a method usually used for culturing hyperthermostable bacteria can be used, and the nutrient added to the medium may be any one that can be used by the strain.
- the carbon source for example, starch and the like can be used, and as the nitrogen source, for example, tryptone, leptone, yeast extract, and the like can be used.
- Metal salts such as magnesium salts, sodium salts, and iron salts may be added to the medium as trace elements.
- the medium is desirably a transparent medium containing no solid sulfur. If the medium is used, the growth of the cells can be easily monitored by measuring the turbidity of the culture solution.
- the culture can be performed by static culture or stirring culture. For example, Applied and Environmental Microbiology, Vol. 55, pp. 286-208, pp. (1992) As described in (2), a through-culture method may be used.
- the culture temperature is preferably around 95 ° C, and usually about 16 hours, a significant amount of the amino-terminal protecting group-releasing enzyme accumulates in the culture.
- the culture conditions are preferably set so that the production amount of the amino-terminal protecting group-releasing enzyme is maximized according to the cells used and the medium composition.
- a method for crushing the cells a method having a high effect of extracting the target enzyme may be selected from ultrasonic crushing, bead crushing, and lytic enzyme treatment.
- the enzyme is secreted into the culture solution, the enzyme is concentrated by ammonium sulfate precipitation / ultrafiltration or the like to obtain a crude enzyme solution.
- a method used for ordinary enzyme purification can be used. For example, a combination of ammonium sulfate salting-out treatment, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography and the like can be used in combination.
- the DNA encoding the enzyme according to the present invention can be obtained by screening a gene library of an appropriate microorganism. As described above, microorganisms can be found by screening using enzyme activity as an index.
- the desired DNA can be screened and obtained from, for example, a genomic cosmid library of bacteria belonging to the genus Pyrococcus, for example, Pyrococcus flyosus.
- Pyrococcus furiosus DSM3638 can be used as Pyrococcus furiosus.
- the Pyrococcus furiosus genome cosmid library is obtained by introducing DNA fragments obtained by partially digesting Pyrococcus furiosus genomic DNA with the restriction enzyme Sau3AI (Takara Shuzo) into Triblehelix Cosmid Vector-1 (Stratagene). After that, lambda phage particles It can be manufactured by packaging inside. Next, a suitable E. coli, for example, Escherichia coli DH5 MCR (manufactured by BRL) is transformed using the thus obtained library, and the enzymatic activity according to the present invention in this transformant is examined. Clones containing the DNA of the enzyme can be obtained.
- the present inventors have focused on the fact that the enzyme produced by Pyrococcus furiosus used as a material in screening the enzyme activity has high heat resistance, and individually transformed the transformants obtained using the cosmid library.
- the steps of culturing and preparing a lysate containing only heat-resistant protein from the obtained cells were combined.
- This group of lysates is called a cosmid protein library, and by using the cosmid protein library for detecting enzyme activity, the detection sensitivity is improved as compared with the method using a colony of a transformant.
- adverse effects such as inhibition of background II enzyme activity due to host-derived proteins and the like can be eliminated by heat denaturation.
- the cells obtained by culturing the transformant obtained using the cosmid library are subjected to heat treatment (100, 10 minutes), ultrasonic treatment, and re-heat treatment (100 ° C, After 10 minutes), centrifuge and collect the supernatant (lysate) to prepare a cosmid protein library.
- amino acid-MCA synthetic peptide amino acid-4-methylcoumalil 7-amide
- Met-MCA Leu-MCA. A1a-MCA. His-MCA (manufactured by Peptide Institute) can be used.
- the lysate in which peptidase activity was observed was further purified using Hiichi MSH (Nippon Melanocyte Stimulating Hormone: Peptide Laboratories), a peptide whose amino terminus was blocked by an acetyl group. Whether it has the activity of releasing a protecting group at the amino terminal. You can find out. As a result, a cosmid clone containing DNA expressing the activity of releasing a protecting group can be obtained.
- Hiichi MSH Natural Melanocyte Stimulating Hormone: Peptide Laboratories
- cosmids prepared from the cosmid clones thus obtained can be fragmented with an appropriate restriction enzyme to produce a recombinant plasmid into which each fragment has been inserted.
- a transformant obtained by introducing the plasmid into a suitable microorganism is produced.
- a recombinant plasmid containing DNA encoding the enzyme of interest is obtained.
- the cosmid prepared from the above cosmid clone was digested with BamHI (Takara Shuzo), and the obtained DNA fragment was inserted into the BamHI site of the plasmid vector pUC18 (Takara Shuzo). Can be made.
- the peptidase activity in the lysate prepared from the obtained cells is examined to contain the DNA of interest. You can get Brasmid.
- the plasmid is named plasmid pDAP1.
- the above plasmid pDAP1 is digested with EcoRI (manufactured by Takara Shuzo Co., Ltd.), and self-ligation can be performed to prepare a recombinant plasmid.
- EcoRI manufactured by Takara Shuzo Co., Ltd.
- self-ligation can be performed to prepare a recombinant plasmid.
- Escherichia coli JM109 into which the plasmid has been introduced, the bactidase activity of the lysate obtained from the cells can be confirmed, and a plasmid containing the target DNA can be obtained.
- the plasmid has been named pDAP2.
- a DNA fragment containing no amino-terminal protecting group releasing enzyme gene from the above plasmid pDAP2 can be removed as follows. Specifically, an approximately 1.7 kb DNA fragment obtained by digesting the plasmid pDAP2 with Sacl (Takara Shuzo) was inserted into the SacI site of the plasmid vector pUC18 (Takara Shuzo), and Introduce into fungus JM109. The peptidase activity of the lysate prepared from the obtained transformant is measured, and a plasmid is prepared from the transformant showing the activity.
- the plasmid has been named Brasmid p DAP3.
- Fig. 1 shows the restriction enzyme map. Show. In the figure, the thick solid line is the DNA fragment inserted into the plasmid vector pUC18.
- a DNA fragment containing no amino-terminal protecting group releasing enzyme gene from the plasmid pDAP3 can be removed as follows. That is, the plasmid pD eight? 3 was digested with 311 & 8 1 (Takara Shuzo) and Sacl (Takara Shuzo) digested. About 1.2 kb of SnaBI—SacI DNA fragment was digested with SmaI of plasmid vector pUC19. — Insert into SacI site and introduce into E. coli JM109. The peptidase activity of the lysate prepared from the obtained transformant is measured.
- plasmid pDAP The plasmid is named plasmid pDAP.
- Fig. 2 shows the restriction enzyme map.
- the thick solid line is the DNA fragment inserted into plasmid vector pUC19.
- Escherichia coli JM109 into which plasmid pDAP has been introduced is named Escherichia coli J109 / pDAP. Deposited with the National Institute of Technology, FERM BP-5804 (Original deposit date: March 29, 1996, transfer to international deposit: January 30, 1997).
- the nucleotide sequence of the DNA fragment derived from Pyrococcus furiosus contained in the plasmid pDAP can be determined by a known method, for example, the dideoxy method.
- SEQ ID NO: 3 in the sequence listing shows the nucleotide sequence of a DNA fragment of about 1.2 kb inserted into plasmid pDAP.
- the nucleotide sequence of the open reading frame present in the sequence is shown in SEQ ID NO: 2 in 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 releasing enzyme gene obtained by the present invention.
- SEQ ID NO: 1 shows the amino acid sequence of the amino terminal protecting group releasing enzyme of the present invention deduced from the nucleotide sequence shown in SEQ ID NO: 2. Ie the sequence listing SEQ ID NO: 1 is an example of the amino acid sequence of the amino terminal protecting group releasing enzyme obtained by the present invention.
- the Escherichia coli JM109 / pDAP into which the plasmid pDAP has been introduced, obtained as described above, can be used to obtain the amino-terminal protecting group releasing enzyme of the present invention. That is, Escherichia coli JM109 / pDAP was cultured under normal culture conditions, for example, LB medium containing 100 g / mL of ampicillin (10 liters of tryptone, 5 g of yeast extract, 5 g of yeast extract, 5 g of ZCI By culturing at 37 in pH 7.2), the amino-terminal protecting group-transferring enzyme can be expressed in the cultured cells.After completion of the culture, the cultured cells were collected and obtained. Heat the cells at I 00 for 10 minutes.
- the crude enzyme solution is obtained as a supernatant obtained by centrifuging the heat-treated cells after sonication, denaturing contaminating proteins by heat treatment at 100 ° C for 10 minutes, gel filtration chromatography, ion exchange.
- An amino-terminal protecting group-releasing enzyme can be purified by using a combination of methods commonly used for purification of enzymes such as chromatography.
- 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. Further, the enzyme of the present invention releases amino acids sequentially from the amino terminus of the peptide.
- the enzyme activity of the present invention is measured by the following operation using Met-MCA as a substrate.
- 7-Amino-4-methylcoumarin produced by the reaction is quantified using a titer tech fluoroscan [ ⁇ (Dainippon Pharmaceutical Co., Ltd.) at an excitation wavelength of 355 nm and a measurement wavelength of 450 nm.
- One unit of enzyme uses Met-MC ⁇ as a substrate, ⁇ 7.6, 75. Determine the amount of enzyme capable of producing 1 jmo 1 of 7-amino-4-methyltamarin per minute in C.
- the enzyme of the present invention had met-MCA decomposition activity at the measured pH of 7.6 and 75 ° C.
- the enzyme of the present invention acted on Leu-MCA, A1a-MCA, His-MCA, etc. in addition to Met-MCA to release amino acid.
- the enzyme of the present invention releases a protecting group from the amino terminal even if a protecting group is present at the amino terminal of the peptide, and further sequentially transmits amino acids.
- the above activity can be confirmed by using a synthetic peptide having a blocked amino terminal as a substrate. That is, it can be confirmed by measuring the time-dependent change in the amount of amino acid released from the substrate peptide by the enzymatic reaction by amino acid analysis, and analyzing the decomposition mode of the peptide.
- a synthetic peptide having a blocked amino terminal as a substrate. That is, it can be confirmed by measuring the time-dependent change in the amount of amino acid released from the substrate peptide by the enzymatic reaction by amino acid analysis, and analyzing the decomposition mode of the peptide.
- those not commercially available can be chemically synthesized and used by a known method, for example, using a peptide synthesizer.
- SEQ ID NO: 4 in the sequence listing shows the amino acid sequence of neurotensin . That is, 0.1 mM enzyme sample (5 L) in 10 L of ImM new mouth tensin (manufactured by Peptide Research Institute), 4 OmM PIPES-Na buffer solution (pH 7.6) 5 After adding 0 ⁇ L and 35 ml of distilled water and reacting at 37'C for 0 to 5 hours, the amount of free amino acids in the reaction solution was measured by amino acid analysis.
- the activity of releasing the formyl group can be examined using For-Me-Leu-Phe-Lys (manufactured by BA Chem) as a substrate.
- SEQ ID NO: 7 in the sequence listing shows the amino acid sequence of For-Me-Leu-Phe-Lys. That is, 1 OmM
- the activity to release the myristoyl group is as follows: Myr—Phe—Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (manufactured by BACHEM) and Myr-G1y- A1a-G1y-A1a-Ser-A1a-G1u-Glu-Lys can be used as a substrate to examine.
- 1 shows the amino acid sequence of 1y-A1a-Gly-A1a-Ser-A1a—Glu1G1u—Lys. That is, 4 L units of enzyme preparation (3.5 L), 0.1 M N-ethyl morpholine acetate buffer (pH 9.0) 25 L, distilled water 1 After adding 6.5 L and reacting at 0 to 9 hours at 37 ° C, the amount of free amino acid in the reaction solution was measured by an amino acid analysis method. As a result, when either substrate was used, the amino acid at the amino terminus from which the myristoyl group had been lost was first released, indicating that the enzyme of the present invention had the activity to release the myristoyl group. Indicated. It was also confirmed that the amino acids following this were sequentially released over the reaction time.
- the enzyme of the present invention also acts on the amino terminal of the peptide whose amino terminal is not blocked by the protecting group, and has an aminopeptidase activity to release the amino terminal amino acid. This activity can be confirmed by the method described in Arch. Biochem. Bioptys .. Vol. 274, Vol. 24, p. 250 (1989).
- the enzyme of the present invention can act on the amino terminus of high-molecular-weight proteins in addition to the short-chain polypeptides described above. For example, this can be confirmed using reduced chicken egg white lysozyme as a substrate. That is, ImM reduced chicken egg white rib team (water-soluble, molecular weight about 14000, manufactured by Wako Pure Chemical Industries, Ltd.) 5 milliliters of enzyme product (1.6 // L), 0.1 mM CoC l 2 containing 5 OmM disodium hydrogen phosphate-sodium hydroxide buffer (pH 11.0) 50 L and 43.4 L of distilled water were added, and the mixture was reacted at 50 for 0 to 240 minutes.
- ImM reduced chicken egg white rib team water-soluble, molecular weight about 14000, manufactured by Wako Pure Chemical Industries, Ltd.
- 5 milliliters of enzyme product 1.6 // L
- the optimum temperature of the enzyme preparation of the present invention is 75 to 95 ° C. at pH 7.6.
- the vertical axis shows the relative activity (%) with respect to the maximum activity (95 “C), and the horizontal axis shows the reaction temperature (in).
- the optimum pH of the enzyme of the present invention was around pH 6.5 to 9.5.
- the vertical axis shows the relative activity (%) with the activity at pH 7.2 as 100, and the horizontal axis shows the pH at 75'C.
- the activity of the enzyme of the present invention is inhibited by amycin (manufactured by Peptide Research Institute). For example, when 0.3 milliunit of the enzyme of the present invention is treated with 5 nmol of amastatin and its activity is measured using Met-MCA as a substrate, the enzyme activity is almost completely lost.
- the enzyme of the present invention has a molecular weight of about 40,000 by SDS-polyacrylamide electrophoresis and about 400,000 by ultracentrifugation. (8) Temperature stability
- the residual enzyme activity after heat treatment of the enzyme product at 75'C was examined, and the temperature stability of the enzyme of the present invention was examined. 5 As shown, after the enzyme is 0. 1 mM C o C 1 2 containing 0. 1 M Bok squirrel one HC 1 buffer (PH 8. 0) 5 hours treatment in the present invention also not seen at all decrease in enzyme activity (in FIG Qin), also C oC l If 2 for 5 hours at buffer without also had retained 80% or more activities (in the figure ⁇ ). In the figure, the vertical axis shows the remaining enzyme activity (%), and the horizontal axis shows the heat treatment time (hour).
- the amino-terminal protecting group-releasing enzyme in the present invention includes, in addition to the above enzymes, all or a part of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and exhibits an amino-terminal protecting group releasing activity. Or its functional equivalents.
- “functional equivalent” refers to: Naturally occurring proteins have polymorphisms and mutations in the DNA encoding them, as well as deletions and insertions of amino acids in their amino acid sequences due to modification reactions in vivo and during purification of the produced proteins. Mutations such as addition, substitution and the like may occur. However, when such a mutation is present in a portion that is not important with respect to the activity and structure retention of the protein, some of them show physiological and biological activities substantially equivalent to those of the protein having no mutation. It is known. In the present specification, those having little difference in function even if there is a slight difference in structure are called “functional equivalents”. The same applies to the case where the above-mentioned mutation is artificially introduced into the amino acid sequence of a protein.
- proteins are known to have peptide regions that are not essential for activity. For example, signal peptides present in extracellularly secreted proteins, pro-sequences found in protease precursors, and the like. Most of these regions are post-translationally or converted to active proteins. Removed. Such proteins are forces that exist in different forms in their primary structure, and ultimately express the same function.
- a peptide chain irrelevant to the activity of the protein may be added to the amino or carboxyl terminus of the target protein.
- a fusion protein in which a part of the amino terminal region of the protein highly expressed in the host used is added to the amino terminal of the target protein is produced.
- a peptide having an affinity for a specific substance has been added to an amino terminal or a carboxyl terminal of a target protein.
- Functional equivalents of the above-mentioned amino terminal protecting group mobilizing enzyme include, for example, a deletion, addition, insertion or substitution of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. At least one enzyme may be used, and the enzyme may exhibit amino terminal protecting group releasing activity. In the present invention, aspects of functional equivalents are not particularly limited. Although there is no modified amino acid sequence, an amino acid sequence modified by blocking the N-terminus of the amino-terminal protective group releasing enzyme of the present invention with a protecting group is exemplified. An example is an enzyme in which valine at amino acid number 2 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing has been replaced with aspartic acid.
- the modified enzyme can be obtained, for example, by using a genetic engineering technique. That is, the modified enzyme is prepared by modifying the nucleotide sequence of the DNA region encoding the above enzyme on pDAP plasmid DNA using a known nucleic acid mutagenesis technique, and then expressing the protein using an appropriate host. Can be implemented. It is known that the N-terminal amino acid sequence Met-Asp of the modified enzyme serves as an acetyl-added signal sequence in baker's yeast [Journal of Biochemistry], No. 265. Vol., Pp. 19638-196643 (1990)].
- a protein having an N-terminal acetylated protein can be obtained by incorporating the DNA region encoding the modified enzyme into an appropriate yeast expression vector and expressing it in an appropriate host yeast.
- pVT103-L Gene, Vol. 52, pp. 225-233 (1987)
- BJ2168 BJ2168
- the activity of releasing the amino-terminal protecting group of the protein can be confirmed by using the method described in the above section on substrate specificity to confirm that the protein is the amino-terminal protecting group-releasing enzyme of the present invention. I can do it.
- PVT103-L into which DNA encoding the enzyme consisting of the amino acid sequence described in SEQ ID NO: 10 was inserted which was named pAc DAP
- pAc DAP DNA encoding the enzyme consisting of the amino acid sequence described in SEQ ID NO: 10
- pAc DAP plasmid BJ2168
- Saccharomyces cerevisiae BJ2168 / pAcDAP FERM BP- Deposited as 595 (Original deposit date: May 23, 1997).
- the method of blocking the N-terminus of the protein with a protecting group by modifying the amino acid sequence is not limited to this method, and examples thereof include an alternative method of acetylation (Journal of Biological Chemistry, Vol. 260, pp. 5382-5391 (19985)] and the method of myristoylation (Procedings of the National Academy of Sciences of the USA (Proceedings of the USA) Vol. 84, pp. 2708-2712 (19987)] may be used, and a method for blocking the N-terminus of a protein with a protecting group may be used.
- the method is not limited to the method by amino acid substitution as described above. For example, a chemical method (Methods in Enzymology, Vol. 11, pp. 565-570 (1967)) can be used. Good.
- the DNA of the present invention is a DNA encoding the above-mentioned amino-terminal protecting group releasing enzyme of the present invention, and specifically, the whole or a part of the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
- DNA encoding an amino acid having an amino terminal protecting group releasing activity (2) DNA containing all or a part of the DNA shown in SEQ ID NO: 2 in the sequence listing, (3) Functionality of the present invention DNA encoding an equivalent, and (2) DNA encoding a protein capable of hybridizing to DNA described in (1) to (3) above and having an amino terminal protecting group releasing activity.
- Such a DNA of the present invention can be obtained, for example, as follows.
- the DNAs 1) and 2) can be obtained from Pyrococcus furiosus DSM3638, as described in the description of the amino-terminal protecting group releasing enzyme of the present invention. It is also possible to obtain a DNA of a protein having the same activity as the enzyme of the present invention based on the base sequence of DNA 6 encoding the amino-terminal protecting group releasing 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 probe for hybridization or a primer for a gene amplification method such as PCR, a functionally equivalent to the present enzyme is obtained. The DNA encoding the active protein can be screened. The DNA of (3) and (4) can be obtained by power, power, and method.
- a DNA fragment containing only a part of the target DNA may be obtained.
- the nucleotide sequence of the obtained DNA fragment is examined, and the DNA sequence of the target DNA is obtained.
- the PCR is performed using a primer that has been synthesized based on the ability to perform hybridization or a primer synthesized based on the nucleotide sequence of the DNA fragment. By doing so, the entire target DNA can be obtained.
- the above hybridization can be performed, for example, under the following conditions. That is, the membrane on which the DNA was immobilized was treated with 0.5% SDS, 0.1% serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% polyester 400, 0.01 6 x SSC containing% denatured salmon sperm DNA (1 XSSC is 0.15M
- nucleotide sequence is not the same as the nucleotide sequence disclosed in the present specification, as long as it encodes a protein having an amino-terminal protecting group releasing activity disclosed in the present specification, such a nucleotide sequence may be used in the present invention. Is included in the range as described above. The reason is as follows.
- DNA is not stable in nature, and mutations in its nucleotide sequence are not uncommon. In some cases, mutations that occur in DNA do not change the amino acid sequence encoded by them (called silent mutations), in which case different DNAs encoding the same amino acid sequence have been generated. . Therefore, even if a DNA encoding a specific amino acid sequence is isolated, there is a possibility that many types of DNA that encode the same amino acid sequence will be produced as the organism containing it is passaged. Cannot be denied. Furthermore, it is not difficult to artificially produce various kinds of DNAs encoding the same amino acid sequence by using various genetic engineering techniques.
- the expression level of the protein may decrease. May be low.
- high expression of the target protein can be achieved by artificially converting codons into those commonly used in the host without changing the encoded amino acid sequence.
- DNA that is artificially mutated to impart specific properties to the protein is included in the DNA of the present invention as long as it encodes the enzyme having the amino-terminal protecting group releasing activity of the present invention.
- Examples of such DNA include a DNA having the base sequence shown in SEQ ID NO: 11 in the sequence listing.
- the DNA has a nucleotide sequence in which the nucleotide sequence TG present at nucleotide numbers 5 to 6 in the nucleotide sequence described in SEQ ID NO: 2 in the sequence listing has been substituted with the nucleotide sequence AT, and was encoded by the DNA.
- the valine at amino acid number 2 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is replaced with aspartic acid and the N-terminal is acetylated. Enzymes can be obtained.
- the method for removing the amino-terminal protecting group of the present invention comprises protecting the amino-terminal by reacting the amino-terminal-protected peptide with the protecting group with the amino-terminal protecting group-releasing enzyme of the present invention or a functional equivalent thereof. It is characterized by liberating a group.
- the substrate is mixed with the substrate at 50 ° C. in 5 OmM PI PES-Na buffer (pH 7.6) By reacting, the protecting group can be released.
- the reaction conditions differ depending on the type of the protecting group and the peptide.
- Ru can also be added CoC 1 2 to increase the activity of the enzyme needed.
- the method for analyzing an amino acid sequence according to the present invention is a method for analyzing an amino acid sequence of a peptide having an amino terminal blocked by a protecting group, comprising the steps of: It is characterized by being used for analysis of
- an amino acid sequence analysis method of the obtained peptide includes, for example, removing the amino terminal protecting group of the peptide whose sequence is to be determined using the amino terminal protecting group releasing enzyme of the present invention, and then starting from the newly generated amino terminal. The sequence is determined by the Edman decomposition method.
- the use of an excess amount of the enzyme caused the enzyme to degrade the amino acid sequence information of the sample. It is conceivable that the amino acid sequence information of the enzyme of the present invention used in the treatment is mixed as noise. In this case, for example, when an amino-terminal protecting group releasing enzyme having the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing is used, the amino-terminal protecting group releasing enzyme itself is not subjected to Edman degradation, so that noise contamination can be prevented.
- the N-terminal protecting group is not particularly limited to an acetyl group.o
- the amino acid is also sequentially released by the action of the enzyme following the release of the protecting group, but under conditions where the action of the enzyme can be controlled. Then, the amino group can be determined by Edman degradation method after removing only the protecting group at the amino terminal or the protecting group followed by a few residues of amino acid.
- amino acid sequence analysis can be performed by the following method. That is, a method of determining the sequence by reacting the amino acid protecting group-releasing enzyme of the present invention on the peptide whose sequence is to be determined, and then analyzing the change over time of the amino acid released in the reaction solution, Alternatively, there is a method in which the amino acid composition of various partially digested peptides generated in the reaction solution is identified and the sequence is determined by comparing the amino acid compositions.In the latter case, the molecular weight is determined by mass spectrometry. A method for identifying the amino acid composition of a peptide is advantageous. In the method using mass spectrometry, the type of amino acid released can be easily determined from the difference in molecular weight of the partially digested peptide, and the protecting group existing at the amino terminus can be easily determined. Can be determined at the same time.
- the kit for analyzing an amino acid sequence of the present invention is a kit used for analyzing the amino acid sequence of a peptide whose amino terminal is blocked by a protecting group, and contains the enzyme of the present invention described above. It is characterized by doing. By using such a kit, it can be used for amino acid sequence analysis of a peptide whose amino terminus is blocked, regardless of the type of protecting group. In addition, a standard product of a protecting group compound for identifying a protecting group blocking an amino terminal of a peptide can be added to the kit.
- An antibody or a fragment thereof which specifically binds to the above amino terminal protecting group releasing enzyme or a functional equivalent thereof can be obtained by a conventional method. Such antibodies and the like are useful for purification and detection of the enzyme of the present invention.
- a synthetic oligonucleotide probe or a synthetic oligonucleotide primer that hybridizes with the above-mentioned DNA can be obtained by a conventional method. Such probes and primers are useful for the detection and amplification of DNA of the present invention. Examples of the present invention will be described below, but the present invention is not limited to the following examples. Incidentally,% in the examples means% by weight.
- the culture of Pyrococcus furiosus DSM36638 was performed as follows.
- the composition of the medium used is as follows: 1% tryptone, 0.5% yeast extract, 1 % Soluble starch, 3.5% Jamarin S ⁇ Sori' de (Jamarin Laboratory, I), 0.5% Jamarin S ⁇ Riki' de (Jamarin Laboratory), 0. 0 0 3% MgS0 4, 0. 00 1% Na C 0. OOO l ⁇ F e SO * ⁇ 7 ⁇ 2 0, 0. 000 1% C o S O4, 0. 000 1% C a C 12 ⁇ 7 ⁇ 2 ⁇ , 0. 0 0 0 1% Zn S0 4 . 0. 1 p pmCu SO * ⁇ 5 ⁇ 2 0, 0. 1 p pmKA 1 (S Rei_4) 2, 0. lp pmH 3 B0 3, 0. 1 pmN a 2 Mo 0 4 ⁇ 2 ⁇ 2 ⁇ , 0. 25 p pmN i C 12 ⁇ 6 H 20 .
- the collected cells were suspended in 4 mL of 0.05 M Tris-HC1 (pH 8.0) containing 25% sucrose, and 0.8 mL of lysozyme (5 mg ZmL, 0.25 M Tris-HC) was added to this suspension.
- 1 (pH 8.0)] add 2 mL of 0.2 M EDTA (pH 8.0), incubate at 20'C for 1 hour, then add 24 mL of the SET solution (150 mM NaCl, 1 mM EDTA, HC1 (pH 8.0)), add 4 mL of 5% SDS and 400 L of proteinase K (10 mg / mL), and react at 37 ° C for 1 hour.
- phenol-chloroform was extracted, followed by ethanol precipitation to prepare about 3.2 mg of genomic DNA.
- Cosmids were prepared by selecting several of the obtained transformants, and after confirming that an insert fragment of an appropriate size was present, 500 transformants were re-added with 100 ⁇ g ZmL of ampicillin.
- LB medium 10 liters of tryptone, 5 liters of yeast extract, 5 g of NaC5 gZ liter, pH 7.2
- the culture was centrifuged, and the collected cells were suspended in 1 mL of 20 mM Tris-HC1 (pH 8.0) and heat-treated at 100 for 10 minutes. Subsequently, ultrasonic treatment was performed, and the cell lysate was heat-treated again at 100 for 10 minutes.
- the lysate obtained as the supernatant after centrifugation was used as the cosmid protein library.
- a cosmid clone having peptidase activity in the cosmid protein library was selected by quantifying the amount of 7-amino-4-methylcoumarin produced using amino acid-MCA as a substrate. Specifically, 10 to 30 L of the lysate was taken from the above cosmid protein library, and 0.1 MPI PES—100 L of Na buffer (pH 7.6) and 18 5 mM amino acids—MC A ( (Dissolved in dimethyl sulfoxide) was added, and reacted at 90 eC for 1-3 hours.
- the generated 7-amino-4-methylcoumarin was measured using Titertec Fluoroscan II (manufactured by Dainippon Pharmaceutical Co., Ltd.) at an excitation wavelength of 355 nm and a measurement wavelength of 460 nm, and met-MCA, A lysate having degrading activity against Leu-MCA, A1a-MCA, and His-MCA was selected. Furthermore, acetyl group and amino acid were converted from these lysates to amino terminal using MSH as a substrate.
- Cosmid was obtained from the cosmid clone obtained as described above and having an activity of sequentially releasing an acetyl group and an amino acid from the amino terminal. It was prepared and the DNA fragment obtained by BamHI digestion was inserted into the BamH site of plasmid vector pUC18.
- an LB plate containing 100 xz gZmL of ambicilin (10 g of tryptone / liter, 5 g of yeast extract / liter, NaC 15 g / litre, agar 15 g / litre, pH 7.2) Spread on top, and transform the resulting transformants individually into 5 mL containing 100 gZmL of ampicillin.
- LB medium The cells collected by centrifugation of the culture were suspended in 50 mM Tris-HC1 ( ⁇ 8.0) 50 ⁇ L, heat-treated at 100 ° C for 10 minutes, and then sonicated. The cells were disrupted. The lysate was again heat-treated at 100 ° C. for 10 minutes and centrifuged to obtain a lysate. Peptidase activity was measured for this lysate.
- plasmid pDAP1 50 L of 0.1 MPI PES-Na buffer (pH 7.6) and 5 L of 5 mM Met-MCA (dissolved in dimethyl sulfoxide) were added to 15 L of lysate, and the mixture was reacted at 90 for 1.5 hours. I let it. Thereafter, the amount of 7-amino-4-methyl coumarin produced was measured using Titertec Fluoroscan II (manufactured by Dainippon Pharmaceutical Co., Ltd.) at an excitation wavelength of 355 nm and a measurement wavelength of 460 nm.
- a plasmid was prepared from a transformant having Met-MCA degrading activity and named plasmid pDAP1.
- plasmid p DAP2 containing amino-terminal protecting group releasing enzyme gene
- the above plasmid pDAP1 was digested with Ec0RI and self-ligated. Was done.
- the recombinant plasmid was introduced into Escherichia coli JM109, and the lysate prepared from the obtained transformant was used to examine the peptidase activity by the method described above.
- a plasmid was prepared from the transformant in which the enzyme activity was observed, and this was named plasmid pDAP2.
- FIG. 1 shows a restriction map of plasmid pDAP3.
- FIG. 1 A plasmid was prepared from a colony in which the enzyme activity was observed, and this was named plasmid pDAP.
- Figure 2 shows a restriction map of plasmid PDAP.
- Escherichia coli JM109 transformed with the plasmid pDAP is named and displayed as Escherichia coli JM109 / pDAP, and 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan (zip code 30 5) Deposited with FERM BP-5804 at the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry of the Ministry of International Trade and Industry of Japan (Original deposit date: 1992, March 29, 2006 Transfer to international deposit: 19 January 30, 1997).
- nucleotide sequence of each fragment was determined.
- the nucleotide sequence was determined by the dideoxy method using Bca BEST dideoxy sequencing kit (Takara Shuzo Co., Ltd.).
- the nucleotide sequences of the obtained fragments were compared and integrated, and the above 1.2 kb DNA fragment was analyzed. The entire nucleotide sequence was determined.
- SEQ ID NO: 3 in the sequence listing shows the nucleotide sequence of a DNA fragment of about 1.2 kb containing the DNA encoding the amino-terminal protecting group-releasing enzyme inserted into plasmid pDAP.
- base numbers 86 to 88 are the start codon
- base numbers 113 to I132 are the stop codon
- the open reading frame between them is assumed to be the structural gene region of the enzyme.
- SEQ ID NO: 2 in the sequence listing shows the nucleotide sequence of the above open reading frame. Further, SEQ ID NO: 1 in the sequence listing shows the amino acid sequence of the amino terminal protecting group releasing enzyme of the present invention deduced from the base sequence shown in SEQ ID NO: 2.
- Escherichia coli JM109 / pDAP was inoculated into each of two test tubes containing 5 mL of LB medium supplemented with 100 ⁇ g ZmL of ampicillin under normal culture conditions and cultured at 37 ° C. A 68 .
- When 1, add isopropyl yS-D-thiogalactopyranoside to a final concentration of ImM, and then add 37 to 16 to 18 hours
- the active fraction is adsorbed to a 5 mL Econopack hig hQ cartridge (manufactured by BioRad) column previously equilibrated with buffer B, washed thoroughly with buffer B, and then washed with 0.5 to 0.5 M NaCl. 1Eluted with buffer B having a linear concentration gradient.
- the met-MCA degrading activity of the eluate was measured, and the active fraction was collected to obtain a purified enzyme preparation.
- One unit of the obtained purified enzyme preparation was defined as the amount of enzyme capable of producing 7-amino-4-methylcoumarin with 1 mol per minute at pH 7.6 and 75 ° C using Me-MCA as a substrate. .
- Example 2 Example 2
- the measurement of the optimum temperature was performed by the following operation. That is, the activity was measured at various temperatures by an enzyme activity measurement method using Met-MCA as a substrate, using an enzyme sample of 18 micro units. As shown in FIG. 3, the enzyme of the present invention has an activity in the range of 25 to 95 at the measured pH of 7.6, and has the highest activity at 95 ° C., which is the highest temperature at which the measurement was performed. Indicated. As shown in FIG. 3, the optimal temperature of the enzyme preparation of the present invention was 75 to 95 ° C. at pH 7.6. In the figure, the vertical axis shows the relative activity (%) relative to the maximum activity (95 ° C), and the horizontal axis shows the reaction temperature (° C). 2Optimal pH
- the measurement of the optimum pH was performed by the following operation. That is, using the enzyme sample of 18 micro units and using the above-mentioned method of measuring enzyme activity using Met-MCA as a substrate, the buffer added to the reaction solution was changed to various pH buffers. Activity measurements were performed. As shown in FIG. 4, the optimum pH of the enzyme of the present invention was around pH 6.5 to 9.5. In the figure, the vertical axis shows the relative activity (%) with the activity at pH 7.2 as 100, and the horizontal axis shows the pH at 75 ° C.
- the buffer used for activity measurement is 2 OmM sodium acetate buffer at pH 4.1 to pH 5.1, 20 mM PI PES-Na buffer at ⁇ 5.8 to pH 7.2, pH 8.0 to 9.5. In the above, 20 mM sodium borate buffer was used, and in the case of pH 9.9 to 10.6, 20 mM disodium hydrogen phosphate-sodium hydroxide buffer was used. The above pH is a value at 75.
- the activity of the enzyme of the present invention was inhibited by amastin.
- amastin For example, after treating the enzyme of the present invention in 3 milliliters in 50 L of 2 OmM borate buffer (pH 10.0) containing 1 mM amastatin for 37 minutes (:, 30 minutes, When the activity of 5 L was measured by the method described in (2) above, the value was 1.5% of that of the buffer treated with amastatin-free buffer.
- the activity of the enzyme of the present invention is facilitated by C oC 1 2.
- C oC 1 2 the addition of C 0 C 1 2 to a final concentration of 9 1 M in the measurement system using the above-mentioned M et-MCA, was about 6 times the activity as compared to those not added.
- the enzyme of the present invention showed a molecular weight of about 40,000 by SDS-polyacrylamide electrophoresis and about 400,000 by ultracentrifugation. 5 Temperature stability
- the residual enzyme activity after heat treatment of the enzyme preparation was examined, and the temperature stability of the enzyme of the present invention was examined. That, 26 0. 1 M Tris containing millimeter units of enzyme preparation - HC 1 buffer (. PH8 0), or which was also added to C 0 C 1 2 at a final concentration of 0. 1 mM to 75 After treating for 0 to 5 hours, the remaining enzyme activity was measured using a part thereof. The activity was measured by the enzyme activity measurement method using the above-mentioned system using Met-MCA as a substrate.
- 0 enzyme of the present invention including C oC 1 2 of 0. 1 mM.
- Tris - HC 1 buffer (p H 8. 0) was not observed at all low under the enzymatic activity after 5 hours treatment in, also 5 hours at C o C l 2 does not include a buffer In this case, the activity was maintained at 80% or more.
- the vertical axis indicates the remaining enzyme activity (), and the horizontal axis indicates the heat treatment time (hour).
- FIG. 8 shows the results of an experiment similar to Hiichi MSH, in which the substrate was changed to Ac—Gly-Asp-Va1—Glu-Lys.
- glycine was released in the reaction solution, indicating that the enzyme cleaved the bond between the acetyl group and glycine, and subsequently the bond between daricin and aspartic acid. It was suggested.
- trace amounts of aspartic acid and parin were also confirmed in the reaction solution.
- the reaction solution contained lysine, valin, phenylalanine, and glycine at the amino terminal of reduced lysozyme.
- Arginine is released, and from the change in the amount of arginine produced over time, this enzyme acts from the amino terminal side of reduced lysozyme to produce amino acids. It was shown that sequential release.
- the pDAP DNA is a type II, and consists of the nucleotide sequence shown in SEQ ID NO: 12 in the sequence listing for replacing the codon of valine, which is the second amino acid from the N-terminal of the enzyme, with the codon of aspartic acid.
- Synthetic DNA and plasmid p PCR was performed using a synthetic DNA consisting of the nucleotide sequence shown in SEQ ID NO: 13 for crushing each of the Hind III and Sph I restriction enzyme sites with the sequence in the multi-cloning site as a primer pair. After the PCR reaction, the reaction mixture is digested with Sph I (Takara Shuzo) to digest the non-mutated plasmid, and then introduced into Escherichia coli J Ml09 (Takara Shuzo) to obtain the resulting transformant. The desired mutagenized plasmid was prepared from the body.
- this plasmid DNA was designated as type III, and the base described in SEQ ID NO: 14 of the sequence listing for introducing the BamHI site immediately upstream of the protein initiation codon in the protein coding region of the DNA PCR was performed using a synthetic DNA having a nucleotide sequence complementary to the downstream region of the protein coding region of the DNA described in SEQ ID NO: 15 in SEQ ID NO: 15 as a primer pair. .
- the reaction solution was subjected to agarose electrophoresis, and the amplified DNA fragment of about 1.2 kb was recovered from the agarose gel.
- This DNA fragment was digested with BamHI (Takara Shuzo) and HindIII (Takara Shuzo), and the plasmid vector PVT1033-L BamHI and Hind of the E. coli and yeast shuttle vectors were digested. Inserted using the i II site. This recombinant plasmid was introduced into Escherichia coli JM109, and plasmid DNA was prepared from the obtained transformant.
- an auxotrophic plate (6.7 g / litre of yeast nitrogen base, 20 g of glucose, 3 g of glucose, tritophan 2 OmgZ little, histidine) Transformants obtained on 2 OmgZ liter, Peracil 2 OmgZ liter, agar 15 gZ liter) were individually transformed into YPD medium (10 liter yeast extract, 20 gZ liter yeast peptone, 20 gZ liter glucose, 20 gZ glucose). (Little) A test tube containing 5 ml was inoculated and cultured at 30 ° C for 16 hours.
- Each culture was individually inoculated into a triangular flask containing 50 Om1 of the auxotrophic medium described above, and cultured at 30 ° C for 16 hours. The culture is centrifuged and the cells are recovered. Received. The obtained cells are suspended in 5 Om 1 of buffer C (1 M sorbitol, 5 OmM Tris-HC1, pH 7.5, 3 OmMDTT), incubated at 30 ° C for 10 minutes, and centrifuged. The cells were further suspended in 1 Oml of buffer D (1 M sorbitol, 5 OmM Tris-HC1, pH 7.5, 2 mM DTT), and Zymoryase 100 T (manufactured by Seikagaku Corporation) was finally added.
- buffer C 1 M sorbitol, 5 OmM Tris-HC1, pH 7.5, 3 OmMDTT
- Zymoryase 100 T manufactured by Seikagaku Corporation
- the solution was added to a concentration of 0.5 mgZm 1, incubated at 30 ° C for 30 minutes, and centrifuged to obtain protoplasts.
- the obtained protoplasts were suspended in 20 ml of buffer E (50 mM Tris-HC1, pH 7.5, 2 mM DTT), and EDTA, KC1, and TritonX-100 were added to final concentrations of 1 mM and 0.2 M, respectively. , 0.2%, and kept at 37 ° C for 5 minutes.
- the suspension was heat-treated at 100 ° C. for 15 minutes, and then centrifuged to obtain a lysate.
- a modified enzyme preparation was prepared using Met-MCA degrading activity as an index according to the same purification method as the amino terminal protecting group releasing enzyme described in (9) of Example 1.
- One unit of the modified enzyme preparation is the amount of enzyme capable of producing 1 / mol of 7-amino-4-methylcoumarin per minute at pH 7.6 and 75'C using met-MCA as a substrate.
- the yeast expressing the above protein was named and displayed as Saccharomyces cerevisiae BJ2168 / pA cDAP, and it was designated as 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan (zip code: 2005).
- FERM BP-5952 (Original date of deposit: May 23, 1997).
- the amino terminal protecting group releasing activity of the modified enzyme preparation prepared in (2) was measured according to the method described in (3) and (4) of Example 3. As a result, the modified enzyme showed a releasing activity for the acetyl group and a myristyl group at the N-terminal of the peptide, and further showed an activity of allowing the amino acid to migrate sequentially from the N-terminal of the peptide.
- the molecular weight difference 57 between the two enzymes was equal to the sum of the molecular weight difference 15 when palin was replaced by asparagine and the molecular weight increase 42 due to the addition of the acetyl group. Further, the modified enzyme was subjected to HP G1000A Protein Sequencer (manufactured by Hewlett-Packard) employing an N-terminal amino acid sequence analysis method by Edman degradation, and a signal was obtained. Thus, it was found that the modified enzyme did not undergo Edman degradation.
- a carboxymethylated product of N-terminally acetylated erythrocyte superoxydoximesidase (molecular weight: 1,551, manufactured by Washington Biochemical Corp.—Shion) was used.
- the solution 51 prepared so that the substrate had a concentration of 0.4 mM was added to the modified enzyme preparation (52 1) of 600 milliunits prepared in (2), and 10 OmM of trimethylamine-HC1.
- buffer 1 0 0 ⁇ 1, 1 Omm C 0 C 1 2 of 2 // and distilled water 4 1 1 was added, after 48 hours reaction at 5 0 ° C, the reaction solution was directly subjected to HP G1000A protein sequencer to perform amino acid sequence analysis.
- the obtained amino acid sequence data is shown in SEQ ID NO: 16 in the sequence listing.
- the sequence was consistent with the known amino acid sequence of superoxide dismutase used as a substrate.
- no signal other than the amino acid sequence of the above superoxide dismutase was mixed.
- the amino terminal protecting group releasing enzyme of the present invention exhibits an amino terminal protecting group releasing activity for two or more kinds of protecting groups.
- the present invention also provides a method for removing the amino-terminal protective blockage of a peptide using such an enzyme.
- the enzymes are useful for amino acid sequencing of peptides, especially proteins and peptides where the amino terminus is blocked by an unidentified protecting group.
- 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 releasing enzyme produced by modifying the DNA is not subject to Edman degradation, and particularly, in the amino acid sequence analysis method using Edman degradation, the amino acid sequence information derived from the enzyme is noise. This is useful.
- Sequence type nucleic acid
- Sequence type nucleic acid
- GTAGTTTCTC CTTAAAAGTC TCGCCCAAAA TCCTTATATA ATGAGAAAAT AACACTTAGA 60 TGATCATCTA AATGGGGGGA GGAAGATGGT GGACTATGAG CTTTTAAAAA AGGTAGTAGA 120 GGCTCCGGGA GTTTCAGGAT ATGAGTTCAT GGGAATTAGA GATGTCGTATAGAG
- Sequence type nucleic acid
- Sequence type nucleic acid
- Sequence type Other nucleic acid (synthetic DNA) Sequence:
- Sequence type nucleic acid
- Sequence type Other nucleic acid (synthetic DNA) Sequence:
- Sequence type nucleic acid
- Sequence type Other nucleic acid (synthetic DNA) Sequence:
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU31907/97A AU3190797A (en) | 1996-06-24 | 1997-06-19 | Novel amino-terminal deblocking enzyme |
EP97927398A EP0911411A4 (en) | 1996-06-24 | 1997-06-19 | NEW AMINO TERMINATION RELEASE ENZYME |
US09/202,832 US6194190B1 (en) | 1996-06-24 | 1997-06-19 | Amino-terminal deblocking enzyme |
JP50267398A JP3493400B2 (ja) | 1996-06-24 | 1997-06-19 | 新規なアミノ末端保護基遊離酵素 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP18405096 | 1996-06-24 | ||
JP8/184050 | 1996-06-24 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1997049819A1 true WO1997049819A1 (fr) | 1997-12-31 |
Family
ID=16146504
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP1997/002121 WO1997049819A1 (fr) | 1996-06-24 | 1997-06-19 | Nouvelle enzyme de deblocage des terminaisons amino |
Country Status (5)
Country | Link |
---|---|
US (1) | US6194190B1 (ja) |
EP (1) | EP0911411A4 (ja) |
JP (1) | JP3493400B2 (ja) |
AU (1) | AU3190797A (ja) |
WO (1) | WO1997049819A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000061711A1 (fr) * | 1999-04-07 | 2000-10-19 | Takara Shuzo Co., Ltd. | Composition de decomposition de proteines |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6156535A (en) * | 1995-08-04 | 2000-12-05 | University Of Ottawa | Mammalian IAP gene family, primers, probes, and detection methods |
WO2002022631A2 (en) * | 2000-09-14 | 2002-03-21 | Bayer Aktiengesellschaft | Regulation of human pyroglutamyl peptidase-like enzyme |
AU2002310050A1 (en) * | 2001-05-23 | 2002-12-03 | Diversa Corporation | Peptide synthesis method |
US8546072B2 (en) * | 2008-02-22 | 2013-10-01 | Wako Pure Chemical Industries, Ltd. | Substrate for assaying β-glucan and/or endotoxin and assay method |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH07298881A (ja) * | 1994-05-10 | 1995-11-14 | Takara Shuzo Co Ltd | 耐熱性ピログルタミルペプチダーゼ及びその遺伝子 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP3464810B2 (ja) * | 1993-05-18 | 2003-11-10 | タカラバイオ株式会社 | 超耐熱性アミノペプチダーゼ遺伝子 |
-
1997
- 1997-06-19 WO PCT/JP1997/002121 patent/WO1997049819A1/ja active IP Right Grant
- 1997-06-19 JP JP50267398A patent/JP3493400B2/ja not_active Expired - Fee Related
- 1997-06-19 EP EP97927398A patent/EP0911411A4/en not_active Withdrawn
- 1997-06-19 US US09/202,832 patent/US6194190B1/en not_active Expired - Fee Related
- 1997-06-19 AU AU31907/97A patent/AU3190797A/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH07298881A (ja) * | 1994-05-10 | 1995-11-14 | Takara Shuzo Co Ltd | 耐熱性ピログルタミルペプチダーゼ及びその遺伝子 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000061711A1 (fr) * | 1999-04-07 | 2000-10-19 | Takara Shuzo Co., Ltd. | Composition de decomposition de proteines |
Also Published As
Publication number | Publication date |
---|---|
EP0911411A4 (en) | 2004-05-06 |
JP3493400B2 (ja) | 2004-02-03 |
US6194190B1 (en) | 2001-02-27 |
AU3190797A (en) | 1998-01-14 |
EP0911411A1 (en) | 1999-04-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5266265B2 (ja) | ヘモグロビンA1c測定法およびそれに用いる酵素とその製造法 | |
Tsunasawa et al. | Methionine aminopeptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: molecular cloning and overexperssion in Escherichia coli of the gene, and characteristics of the enzyme | |
JPH08500741A (ja) | ストレプトコッカス・ピオゲネスから誘導された組換えdnアーゼb | |
CN113801862A (zh) | 一种海洋链霉菌磷脂酶d突变体及其重组表达菌株的制备方法 | |
EP0739983B1 (en) | Gene encoding lacto-n-biosidase | |
KR100454174B1 (ko) | 엔도글리코세라미데이즈를 암호화하는 유전자 | |
Stojković et al. | Coliphage N4 N-acetylmuramidase defines a new family of murein hydrolases | |
EP0759470B1 (en) | Gene encoding endoglycoceramidase activator | |
KR100543051B1 (ko) | 푸코스 황산 함유 다당 분해 활성을 가지는 폴리펩티드를암호화하는 유전자 | |
WO1997049819A1 (fr) | Nouvelle enzyme de deblocage des terminaisons amino | |
EP0877084A1 (en) | Thermostable diaphorase gene | |
Thapar et al. | Expression, purification, and characterization of the protein repair L-isoaspartyl methyltransferase from Arabidopsis thaliana | |
JP3463951B2 (ja) | 耐熱性ピログルタミルペプチダーゼ及びその遺伝子 | |
KR100496748B1 (ko) | 신규 아미노말단 보호기 유리 효소 | |
CN113913414B (zh) | 高稳定性和高催化效率的双碱基酶Kex2突变体 | |
CA2041875A1 (en) | Peptide amidase and the use thereof | |
JP3512237B2 (ja) | 耐熱性メチオニンアミノペプチダーゼ及びその遺伝子 | |
JP3616203B2 (ja) | エンドグリコセラミダーゼアクチベーター遺伝子 | |
JP3335287B2 (ja) | ヘキソキナーゼ遺伝子 | |
CA1200775A (en) | Expression linkers | |
JP2961143B2 (ja) | アミノペプチダーゼ遺伝子、該遺伝子を含むプラスミドベクターおよび形質転換体 | |
JP2005312459A (ja) | 遺伝子 | |
EP0472651A4 (en) | Endo f-free pngase | |
AU2383088A (en) | Acyl-peptide hydrolase and methods of production and use | |
JPH09191885A (ja) | Nsp7524III制限・修飾系酵素及びその遺伝子 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU BG BR CA CN CZ HU JP KR MX NO NZ PL RO SK US VN AM AZ BY KG KZ MD RU TJ TM |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 09202832 Country of ref document: US |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1019980710524 Country of ref document: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1997927398 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1997927398 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: CA |
|
WWP | Wipo information: published in national office |
Ref document number: 1019980710524 Country of ref document: KR |
|
WWG | Wipo information: grant in national office |
Ref document number: 1019980710524 Country of ref document: KR |