WO2010021375A1 - Process for production of protease, protease solution, and solution of pro-form of protease - Google Patents

Abstract

Disclosed is a process for producing a protease.  The process comprises the following steps: a pro-form expression step of introducing DNA encoding a pro-form of the protease into a host cell to induce the expression of the pro-form in the form of an inclusion body; a modification step of modifying the pro-form in the form of an inclusion body to produce a modified pro-form; a refolding step of refolding the modified pro-form in a solution containing a calcium ion and having a pH value of 5 or higher; and a maturation step of maturing the refolded pro-form under alkaline conditions to produce the protease in an active form.  According to the process for producing the protease, the self-degradation of the protease during the production process can be prevented, and a matured form produced from the pro-form can be collected with high efficiency.

Description

Protease production method, protease solution and protease pro-solution

The present invention relates to a method for producing protease, a protease solution produced by the production method, and a pro-body solution of protease.

Protease is a general term for enzymes that catalyze the hydrolysis of peptide bonds, and is widely distributed in microorganisms, animals and plants. Protease is a typical industrial enzyme widely used in detergents, leather processing, food processing, and functional peptide production. In particular, from the viewpoint of preventing secondary infection of medical devices, degradation of infectious protein soil by protease is a difficult alternative technology. The most important aspect of practical use as an industrial enzyme is the stability of the enzyme and its high activity under the conditions of use. In particular, there are many cases where physical and chemical high heat stability is required, and therefore, heat-resistant protease is widely used as industrial protease. Currently, subtilisin family proteases such as Subtilisin Carlsberg and Proteinase K are known as thermostable proteases used in industry. In addition, proteases derived from Alkalifilus transvalensis (Patent Document 1), proteases derived from Pyrococcus horikoshi (Patent Document 2), and proteases derived from Pyrococcus furiosus (Patent Document 3) are known.

In particular, proteases used as enzyme cleaning agents for medical instruments require high detergency, and therefore require high stability and high activity in a high temperature range. It is desired that medical detergents used in machine washing can be sterilized at the same time as washing, and a protease that retains activity even at a sterilization temperature of 93 ° C. is required. Prevention of secondary infection is an important problem in the cleaning of medical devices, and it is known that, in particular, infectious diseases called prion diseases, abnormal prion protein causes secondary infection instead of pathogenic bacteria. Abnormal prion protein cannot inactivate infectivity by normal medical cleaning. At present, medical devices contaminated with abnormal prion protein are treated with harsh physical conditions such as 3% SDS in the presence of 3% SDS at 100 ° C. for 5 minutes to inactivate infectivity. Is going. In addition, Prizyme (Genencor, USA) has been developed as an enzyme cleaning agent capable of decomposing and removing abnormal prion protein, although infectivity cannot be guaranteed.

The present inventors have found a protease belonging to the subtilisin family (hereinafter referred to as “Tk-subtilisin”) derived from Thermococcus kodakaraensis KOD1 strain, which is one of the hyperthermophilic bacteria, and the Tk-subtilisin has a pH of 9.5 and a temperature of 80. It has been reported that it exhibits the highest activity at 100 ° C. to 100 ° C., has the highest thermal stability among known proteases, and has the highest proteolytic activity under alkaline conditions (Non-Patent Documents 1 and 2). reference). Therefore, Tk-subtilisin can be used under conditions of high temperature and high pH advantageous for industrial use, and the activity under the conditions is significantly higher than that of known proteases, so that conventional proteases can be used. It is expected to be applied to new uses.

JP 2006-141259 A JP 2006-149307 A Japanese Patent No. 3516455

When mass-producing Tk-subtilisin, a general method is to use E. coli to express the pro-form as a recombinant protein by gene recombination technology, and to purify and recover through refolding and maturation (non-patented). Reference 2). However, Tk-subtilisin is less active due to its high activity due to autolysis at the refolding and maturation stages, and only about 20% of the refolded pro form is Tk-subtilisin. Could not be recovered. In addition, a Tk-satilysin solution that can be stored in a state in which the stability of the structure is maintained without causing autolysis has not yet been realized. Furthermore, when the concentration of Tk-subtilisin was attempted, an autolysis reaction occurred immediately when the concentration reached a certain level, and a high concentration Tk-subtilisin solution could not be prepared. Thus, Tk-subtilisin has various problems related to production and storage, and it has been necessary to solve these problems in order to use it as an industrial protease.

Therefore, the present invention suppresses self-degradation in the production process, and provides a method for producing a protease capable of recovering a mature form produced from a pro form with high efficiency, and can be stably stored at a high concentration without causing self-degradation. It is an object of the present invention to provide a protease solution and a pro-body solution of protease. Furthermore, it aims at providing the novel industrial use of protease obtained by the said manufacturing method, especially Tk-subtilisin.

The present invention includes the following inventions in order to solve the above problems.
[1] A pro-body expression step in which DNA encoding a pro-form of protease is introduced into a host cell and expressed as an inclusion body, a denaturation step to denature the pro-form in the inclusion body to obtain a denatured pro-form, and calcium ions A refolding step of refolding the denatured pro-form in a solution having a pH of 5 or higher, and a maturation step of maturing the pro-form after refolding to obtain an active protease, A method for producing a protease, wherein 50% or more of the pro-form subjected to the maturation step is recovered as an active protease.
[2] The production method according to [1], further comprising a protease solution preparation step of preparing a protease solution having a pH of 2 to 6 after the maturation step.
[3] The production method according to [2], wherein the protease solution has a protease concentration of 0.1 mg / ml or more.
[4] The above-mentioned [1] to [1], further comprising a pro-form-containing solution preparation step for preparing a solution having a pH of 2 to 6 containing the pro-form after refolding immediately after the refolding step. 3].
[5] The production method according to any one of [1] to [4], wherein the protease comprises the amino acid sequence according to any one of the following (a) to (d).
(A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 [6] The pro-form of the protease is represented by the following (e) or (f): The production method according to any one of [1] to [5] above, which comprises the amino acid sequence described above.
(E) Amino acid sequence shown in SEQ ID NO: 3 (f) Amino acid sequence shown in SEQ ID NO: 3 wherein one or several amino acids are deleted, substituted or added [7] A protease solution comprising a protease comprising the amino acid sequence of any one of d) and having a pH in the range of 2-6.
(A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 [8] The protease concentration is 0.1 mg / ml or more [7] Protease solution.
[9] A pro-form solution of a protease comprising a pro-form having the amino acid sequence described in (e) or (f) below and having a pH in the range of 2 to 6.
(E) Amino acid sequence shown in SEQ ID NO: 3 (f) Amino acid sequence shown in SEQ ID NO: 3 wherein one or several amino acids are deleted, substituted or added [10] A detergent containing a protease comprising the amino acid sequence according to any one of d).
(A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 [11] The detergent according to [10] above, further comprising a surfactant.
[12] The detergent according to [10] or [11], which is used for cleaning medical equipment.
[13] An abnormal prion proteolytic agent containing a protease comprising the amino acid sequence according to any one of (a) to (d) below:
(A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) Amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 [14] A washing object to which an abnormal prion protein is attached; (D) The inactivation method of abnormal prion protein including the process which contacts the protease which consists of an amino acid sequence in any one of.
(A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 [15] The above-mentioned washing object and protease are brought into contact under protein denaturing conditions [14] The method for inactivating an abnormal prion protein according to [14].

According to the present invention, it is possible to provide a method for producing a protease that can suppress autolysis in the production process and can recover a mature form produced from a pro form with significantly higher efficiency than the conventional one. Further, it is possible to provide a protease solution and a pro-prote solution of protease that can be stably stored at a high concentration without causing autolysis. Protease produced by the production method of the present invention has high activity even under high temperature and high alkaline conditions, and is stable against surfactants and protein denaturing agents. Proteins can be decomposed and removed while being physically denatured (infectivity is inactivated), and are very useful as various industrial detergents. Furthermore, since the protease produced by the production method of the present invention can degrade abnormal prion protein, it is very useful as an abnormal prion protein degrading agent.

It is a graph which shows the result of having examined the temperature dependence of Tk-subtilisin. 3 is a graph showing the results of examining the pH dependence of Tk-satilysin. It is a graph which shows the result of having examined the influence of pH on the structural stability of Tk-subtilisin. 3 is a graph showing the results of comparing and examining the refolding efficiency of pro-Tk-satilysin under various pH conditions. It is a graph which shows the result of having examined the thermal stability of Tk-sachilysin. It is a graph which shows the result of having examined the stability with respect to TritonX-100 of Tk-subtilisin. It is a graph which shows the result of having examined the stability with respect to Tween-20 of Tk-subtilisin. It is a graph which shows the result of having examined the stability with respect to sodium dodecyl sulfate (SDS) of Tk-satilysin. It is a graph which shows the result of having examined the stability with respect to guanidine hydrochloride (Guanidine HCl) of Tk-sachilysin. It is a graph which shows the result of having examined the stability with respect to urea (Urea) of Tk-subtilisin. It is the figure which compared the collection | recovery efficiency of the matured body in the manufacturing method of this invention, and a conventional method. It is a figure which shows the result of having investigated the presence or absence of the autolysis accompanying the concentration of a Tk-subtilisin solution. It is a figure which shows the result of having tried preparation of the pro Tk- subtilisin containing solution which has a native structure. FIG. 4 shows the results of confirming the BSA degradation activity of Tk-subtilisin under reaction conditions of 100 ° C. and 10 minutes in the presence of various surfactants. FIG. 6 is a view showing the results of confirming the BSA decomposition activity of Tk-subtilisin under reaction conditions of 65 ° C. and 10 minutes in the presence of various surfactants. FIG. 4 is a view showing the results of confirming the BSA-decomposing activity of Tk-subtilisin under the reaction conditions of 37 ° C. and 10 minutes in the presence of various surfactants. FIG. 3 is a graph showing the results of confirming the BSA-decomposing activity of Tk-subtilisin under reaction conditions of 25 ° C. and 10 minutes in the presence of various surfactants. FIG. 4 is a view showing the results of confirming the BSA decomposition activity of Tk-subtilisin under reaction conditions of 100 ° C. and 5 minutes in the presence of various surfactants. It is a figure which shows the result of having confirmed the BSA decomposition activity of ProteinaseK on 100 degreeC and reaction conditions for 5 minutes in presence of various surfactant. It is a figure which shows the result of having confirmed the decomposition activity of SubtilisinA on 100 degreeC and reaction conditions for 5 minutes in presence of various surfactant. FIG. 4 is a view showing the results of confirming the residual activity of Tk-satilysin at 100 ° C. in the presence of 3% SDS. FIG. 4 is a view showing the results of confirming the BSA-degrading activity of Tk-subtilisin under reaction conditions of 100 ° C. for 5 minutes in the presence of 1 to 7% SDS. It is a figure which shows the result of having investigated decomposition | disassembly of the abnormal prion protein by Tk-satilysin in alkaline condition. It is a figure which shows the result of having investigated decomposition | disassembly of the abnormal prion protein by Tk-sachilysin on conditions near neutrality. It is a figure which shows the result of having examined the influence of the Tk-sachilysin inactivation after reaction in decomposition | disassembly of abnormal prion protein by Tk-satilysin.

[Protease production method]
The method for producing a protease of the present invention includes the following steps (1) to (4), and any method can be used as long as 50% or more of the pro form subjected to the maturation step is recovered as an active protease. .
(1) A pro-body expression step in which DNA encoding the pro-form of protease is introduced into a host cell and expressed as an inclusion body (2) A denaturation step (3) in which the pro-form in the inclusion body state is denatured to obtain a denatured pro-form A refolding step of refolding a denatured pro-form in a solution containing calcium ions and having a pH of 5 or higher. (4) Maturation of the pro-form after refolding under alkaline conditions to obtain an active protease. Process

The method for producing a protease of the present invention preferably further comprises (5) a protease solution preparation step for preparing a protease solution having a pH of 2 to 6 after the maturation step. By providing the protease solution preparation step, it is possible to prepare a high-concentration protease solution that can be stably stored for a long time without causing autolysis.
In addition, the method for producing a protease of the present invention comprises (6) a pro-form-containing solution for preparing a solution having a pH of 2 to 6 after the refolding process and before the maturation process. A preparation step may be provided. By providing a pro-form-containing solution preparation step, a pro-form having a correctly folded native structure, a complex having a structure in which a pro-sequence and a mature sequence generated by self-cleavage are combined, and a mature form are contained. Can be prepared in a stable manner.

The production method of the present invention comprises a thermophilic bacterium Thermococcus kodakaraensis KOD1 strain (Morikawa Met al. Appl Environ Microbiol, 1994 Dec; 60 (12): 4559-66, hereinafter referred to as “KOD1 strain”). This is a particularly suitable method for manufacturing. However, the protease produced by the present invention is not limited to Tk-subtilisin, and may be a protease having biochemical characteristics similar to those of Tk-subtilisin (especially, biochemical characteristics related to calcium ion, temperature, and pH). Thus, it can be efficiently produced by the production method of the present invention. Tk-subtilisin is a protease belonging to the subtilisin family found by the present inventors as described above (see Non-Patent Document 1), and is expressed as a precursor having a pre-sequence, a pro-sequence and a mature sequence, and is secreted and After maturation, it finally becomes a mature form (Tk-subtilisin having protease activity) having no pre-sequence and pro-sequence. A pre-sequence (also referred to as a signal sequence) is a sequence necessary for secretion of the enzyme out of the microbial cell, and a pro-sequence is a sequence necessary for forming an active three-dimensional structure of the enzyme. The base sequence (SEQ ID NO: 6) and deduced amino acid sequence (SEQ ID NO: 5) of the gene encoding Tk-satilysin precursor are registered in DDBJ, and the accession number is AB056701.

The protease produced by the production method of the present invention is preferably composed of an amino acid sequence described in any of the following (a) to (d).
(A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) Amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of Tk-subtilisin (mature) And corresponds to positions 94 to 422 of the amino acid sequence of the Tk-subtilisin precursor (SEQ ID NO: 5). The amino acid sequence shown in SEQ ID NO: 2 is a Tk-subtilisin (mature) amino acid sequence from which 13 amino acid residues have been deleted from the N-terminus, and the Tk-subtilisin precursor amino acid sequence (SEQ ID NO: 5 No. 107-422. In addition, a protein having any one of 12 to 12 amino acid residues deleted from the N-terminus of the amino acid sequence (SEQ ID NO: 1) of Tk-subtilisin (mature) and having protease activity can also be obtained by the production method of the present invention. It can manufacture suitably.

In addition, the pro-form expressed as a recombinant protein by the production method of the present invention is preferably composed of the amino acid sequence described in (e) or (f) below. In the present specification, the “pro form” means a protease precursor having no pre sequence and including a pro sequence and a mature sequence.
(E) amino acid sequence shown in SEQ ID NO: 3 (f) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 3 The amino acid sequence shown in SEQ ID NO: 3 is This is the amino acid sequence of the pro form of Tk-subtilisin (hereinafter referred to as “pro-Tk-subtilisin”) and corresponds to positions 25 to 422 of the amino acid sequence of the Tk-subtilisin precursor (SEQ ID NO: 5).

“One or several amino acids have been deleted, substituted or added” means that the number can be deleted, substituted or added by a known mutant peptide production method such as site-directed mutagenesis (preferably 10 Or less, more preferably 7 or less, and even more preferably 5 or less) amino acids are deleted, substituted or added. Such a mutant protein is not limited to a protein having a mutation artificially introduced by a known mutant polypeptide production method, and may be a protein obtained by isolating and purifying a naturally occurring protein. It is well known in the art that some amino acids in a protein's amino acid sequence can be easily modified without significantly affecting the structure or function of the protein. Furthermore, it is also well known that there are variants that not only artificially modify, but also do not significantly alter the structure or function of the protein in the native protein.

Preferred variants have conservative or non-conservative amino acid substitutions, deletions, or additions. Silent substitution, addition, and deletion are preferred, and conservative substitution is particularly preferred. These do not alter the polypeptide activity according to the invention. Typically seen as conservative substitutions are substitutions of one amino acid for another in the aliphatic amino acids Ala, Val, Leu, and Ile, exchange of hydroxyl residues Ser and Thr, acidic residues Asp and Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.

The protease and protease precursor of the present invention may contain an additional peptide. Examples of the additional peptide include polyhistidine tag (His-tag), epitope-labeled peptides such as Myc, Flag and the like.

Hereinafter, each step will be described in detail by taking as an example the production of Tk-subtilisin by the production method of the present invention, but the protease produced by the present invention is not limited to Tk-subtilisin, and Tk -Proteases other than subtilisin can be easily produced according to the following explanation.

(1) Pro-body expression step In the pro-body expression step, DNA encoding the pro-form of protease is introduced into a host cell and expressed as inclusion bodies. The DNA encoding the pro-form of the protease may be any DNA that encodes the pro-form of the protease to be produced by the production method of the present invention, and can be easily obtained by using a general genetic engineering technique. Can do. When producing Tk-subtilisin, the following DNA (A) or (B) can be preferably used as DNA encoding pro-Tk-subtilisin.
(A) DNA encoding pro-Tk-subtilisin comprising the amino acid sequence shown in SEQ ID NO: 3
(B) DNA encoding pro-Tk-subtilisin consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 3

In addition, as DNA encoding pro-Tk-subtilisin, the following DNA (C) or (D) is preferably used.
(C) DNA consisting of the base sequence shown in SEQ ID NO: 4
(D) DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 4 and encodes pro-Tk-subtilisin
The base sequence shown in SEQ ID NO: 4 corresponds to the 134th to 1327th positions of the base sequence (SEQ ID NO: 6) of the gene encoding the Tk-subtilisin precursor.

Hybridization is described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed. , Cold Spring Harbor Laboratory (2001). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize) and the more homologous DNA can be obtained. The appropriate hybridization temperature varies depending on the base sequence and the length of the base sequence. For example, when a DNA fragment consisting of 18 bases encoding 6 amino acids is used as a probe, a temperature of 50 ° C. or lower is preferable.

“Hybridization under stringent conditions” means hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × After overnight incubation at 42 ° C. in Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA, wash the filter in 0.1 × SSC at about 65 ° C. Is intended.

The DNA encoding pro-Tk-subtilisin is at least 80% identical to the base sequence complementary to the base sequence shown in SEQ ID NO: 4, more preferably at least 85%, 90%, 92%, 95%, 96%, DNA consisting of a base sequence that is 97%, 98% or 99% identical, and that encodes pro-Tk-subtilisin is preferred.
Any particular DNA is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the base sequence shown in SEQ ID NO: 4 Whether it is a well-known computer program (for example, using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix (registered trademark), Genetic Computer Research Group 75, University Research 75). Can be determined.

Examples of a method for obtaining DNA encoding pro-Tk-satilysin include a method using an amplification means such as PCR. For example, primers are designed based on the 5 ′ side and 3 ′ side sequences (or their complementary sequences) of the base sequence shown in SEQ ID NO: 4, and PCR is performed using these primers as a template for genomic DNA or cDNA. And amplifying the DNA region sandwiched between both primers, a large amount of DNA fragments containing DNA encoding pro-Tk-satilysin can be obtained.

The introduction of the obtained DNA into the host cell can be carried out, for example, by constructing an expression vector for expressing the DNA in the host cell and introducing it into the host cell to obtain a transformant. The expression vector is not particularly limited, and a vector that can be expressed in the host cell can be appropriately selected. That is, according to the type of the host cell, an appropriate promoter sequence is selected appropriately for the expression of pro-Tk-subtilisin, and a vector in which this and a DNA encoding pro-Tk-subtilisin are incorporated into various plasmids is used as an expression vector. Use it.

The host cell is not particularly limited, and various conventionally known cells that can be used for expression of the recombinant protein can be suitably used. In particular, it is preferable to use Escherichia coli as a host cell from the viewpoint of low cost and high production efficiency, and industrial mass production is possible. A method for introducing an expression vector into a host cell, that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be suitably used.

By culturing host cells such as Escherichia coli transformed with an expression vector in which a DNA encoding pro-Tk-subtilisin can be expressed, the pro-Tk-subtilisin can be expressed as inclusion bodies. Here, the inclusion body is an aggregate in which the recombinant protein is insolubilized by aggregation in the host cell, and is also referred to as an inclusion body.
When Escherichia coli is used as a host cell, the inclusion body can be collected in a precipitate fraction obtained by collecting the cells after culturing, disrupting the cells by ultrasonication or the like, and then centrifuging.

(2) Denaturation step In the denaturation step, the pro-form in the inclusion body state is denatured to obtain a denatured pro-form. Specifically, the inclusion body-state pro-form recovered as a precipitate in the above-mentioned pro-form expression step is dissolved in a denaturation buffer, whereby the inclusion body-state pro-form can be denatured to obtain a solubilized modified pro-form. . As the protein denaturant used in the denaturing buffer, for example, urea (for example, 2M to 8M), guanidine hydrochloride (for example, 2M to 6M) and the like can be preferably used. Further, a surfactant such as SDS can also be used. In addition, in the case of having a disulfide bond like Tk-subtilisin, it is preferable to add a reducing agent in addition to the modifying agent. As the reducing agent, for example, dithiothreitol (DTT, for example, 1 mM to 10 mM), β-mercaptoethanol (βME, for example, 1% to 2%) can be preferably used.
The buffer serving as the base of the denaturing buffer is not particularly limited. For example, the present inventors use 20 mM Tris-HCl (pH 9.0), 5 mM EDTA, 8M Urea. The reason for selecting this denaturing buffer is that it maintains a pH away from the isoelectric point and facilitates purification by ion exchange column chromatography. Further, EDTA is added for the purpose of preventing the solution from being altered by the action of a metal-requiring enzyme (protease).

In the denaturation step, it is preferable to purify the pro-form in a denatured state in order to remove various host-derived molecules (nucleic acid, lipid, protein, etc.) contained in the inclusion body. The present inventors purify denatured pro-Tk-subtilisin using an anion exchange column. However, the present invention is not limited to this and may be appropriately selected from known protein purification methods.

(3) Refolding step In the refolding step, the modified pro-form is refolded in a solution containing calcium ions and having a pH of 5 or higher. Refolding refers to rewinding an inclusion body solubilized with a denaturant or the like to a native structure. Refolding can be performed by diluting or dialyzing the denatured proform with a refolding buffer to remove the denaturing agent.

The refolding buffer contains calcium ions. In addition, since Tk-subtilisin has two cysteine residues, the refolding buffer contains a reducing agent (DTT, βME, etc.) in order to prevent the formation of inappropriate disulfide bonds between different molecules. In the conventional production method, an inactive intermediate containing a prosequence was once formed by refolding using a refolding buffer not containing calcium ions, and then matured. Autolysis occurred during the conversion process, and the amount of Tk-subtilisin (mature) that could be recovered was very small. Therefore, in the production method of the present invention, by including calcium ions and DTT in the refolding buffer, a native structure pro-Tk-subtilisin is formed without forming an inactive intermediate, and this is used for maturation. As a result, the recovery efficiency of the matured body was remarkably improved, and a large amount of Tk-satilysin (matured body) could be recovered.
The calcium ion concentration in the refolding buffer is preferably 1 mM to 50 mM, more preferably 5 mM to 10 mM. In the range of 1 mM to 50 mM, pro-Tk-subtilisin having a correctly folded native structure can be formed.

A refolding buffer having a pH of 5 or more is used. This is based on the new discovery by the inventors that the refolding efficiency of pro-Tk-satilysin is much worse when the pH of the refolding buffer is lower than 5.
The buffer that is the base of the refolding buffer is not particularly limited. Suitable refolding buffer, e.g., 1 mM DTT, pH 5.2 or more buffer containing 10 mM Ca ²⁺ (solute does not matter) can be mentioned.

(4) Maturation process In the maturation process, the pro-form after refolding is matured to obtain an active protease. Maturation means that the pro sequence is separated from the pro form by self-cleavage and is subsequently digested by autolysis to produce a mature form (protein having protease activity) comprising the mature sequence.
Maturation can proceed by transferring the refolded pro form to a solution environment at a pH at which the mature form can express protease activity. Therefore, when maturating pro-Tk-satilysin, it is preferable to use a maturation buffer having a pH of 7 or higher. In addition, use a maturation buffer with a pH that allows the matured body to express higher protease activity (pH 8 or higher for Tk-subtilisin), and use a maturation buffer so that the pro-form after refolding is as low as possible. Maturation efficiency can be increased by diluting, heating the maturation buffer, and the like.

The buffer used as the base of the maturation buffer is not particularly limited. For example, Tris-HCl, CAPS-NaOH, Glycine-NaOH and the like can be preferably used. In addition, it is preferable to add calcium ions to the maturation buffer in order to maintain the calcium ions bound to Tk-subtilisin. The concentration of the pro-form used for maturation is preferably 300 nM or less at the final concentration. This is because if it exceeds 300 nM, self-decomposition occurs. For example, the present inventors performed maturation by diluting pro-Tk-subtilisin to about 300 nM with 50 mM CAPS-NaOH pH 9.5, 5 mM CaCl ₂ and heat-treating at 80 ° C. for 15 minutes. ing. However, it is not limited to this.

According to the production method of the present invention including the steps (1) to (4) above, 50% or more of the pro form used in the maturation process is recovered as an active protease (mature form). Here, “50% or more of the pro form subjected to the maturation process is recovered as an active protease (mature form)” means that the number of moles of the pro form subjected to the maturation process and the recovered mature form It means that the ratio of the number of moles is 50% or more. Whether or not 50% or more of the pro form subjected to the maturation step was recovered as an active protease (mature form), simply, a certain amount of maturation buffer before and after maturation was subjected to SDS-PEGE, This can be done by comparing the density of the professional band with the density of the mature band using a densitometer or the like. To be precise, by measuring the absorbance of ultraviolet light at a wavelength of 280 nm using a maturation buffer before and after maturation, calculating the protein concentration before and after maturation using the molar extinction coefficient, and comparing this Can be confirmed.
In the production method of the present invention, the ratio of the recovered mature form to the pro form subjected to the maturation step is at least 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, Particularly preferably, it is 90% or more.

(5) Protease solution preparation step In the production method of the present invention, it is preferable to provide a protease solution preparation step of preparing a protease solution having a pH of 2 to 6 after the maturation step. The inventors of the present invention show that the activity of Tk-subtilisin is remarkably low below pH 6, and that the structure of Tk-subtilisin is stable in the range of pH 2 to 12, but the structure cannot be maintained when the pH is lower than 2. Based on this new finding, the inventors succeeded in stably storing Tk-subtilisin in a solution state of pH 2-6.

In the protease solution preparation step, the Tk-subtilisin obtained in the maturation step is transferred to a pH 2-6 buffer. The transfer method is not particularly limited. For example, a method of diluting Tk-subtilisin with a buffer of pH 2 to 6 or a method of dialysis can be used.
In the protease solution preparation step, Tk-satilysin may be transferred to a pH 2-6 buffer and then concentrated. By concentration, a high-concentration Tk-satilysin solution can be prepared. The concentration method is not particularly limited, and a known protein solution concentration method may be used. An example is ultrafiltration. Alternatively, the Tk-subtilisin solution may be lyophilized once and then redissolved in a small volume of buffer.

The obtained protease solution preferably has a Tk-subtilisin concentration of 0.1 mg / ml or more. When Tk-subtilisin produced by a conventional method was concentrated, an autolysis reaction occurred and a Tk-subtilisin solution of 0.1 mg / ml or more could not be prepared. Since the Tk-subtilisin solution obtained by the production method of the present invention is a Tk-subtilisin solution having a pH of 2 to 6 that can suppress the expression of activity while maintaining the stability of the structure, the Tk-subtilisin concentration is 0.1 mg. / Ml or more, it does not cause autolysis and can be stored stably.

(6) Pro-form-containing solution preparation step In the production method of the present invention, immediately after the refolding step, a pro-form-containing solution preparation step for preparing a pH 2-6 solution containing the refolded pro-form is provided. Also good. We have found that the modified pro-Tk-subtilisin can efficiently perform correct folding above pH 5, the activity of Tk-subtilisin is remarkably low below pH 6, and the Tk-subtilisin is in the range of pH 2-12. It was newly found that the structure is stable but the structure cannot be maintained when the pH is lower than 2. Based on this new knowledge, an attempt was made to prepare a solution that contains a pro-form having a correctly folded native structure and can be stably stored. A complex containing a complex having a structure in which a pro sequence and a mature sequence are combined, and a Tk-subtilisin (mature) three types of protein are obtained, and the progress of maturation is suppressed to pH 2-6. Succeeded in stable storage in solution.

In the pro-form-containing solution preparation step, the mixture containing the pro-form having a native structure obtained in the refolding step is transferred to a pH 2-6 buffer. Further, the concentration may be continued. The method described in the above-mentioned protease solution preparation step can be used for shifting to pH 2-6 buffer and concentration. When the mixture of the pro-form and the complex in the obtained pro-form-containing solution is subjected to a maturation step, a mature form (Tk-satilysin) having protease activity can be obtained.

[Protease solution]
The protease solution of the present invention is a protease solution containing a protease having the amino acid sequence described in any of the following (a) to (d) and having a pH in the range of 2 to 6.
(A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, that is, the protease solution of the present invention is a protease solution preparation step of the production method of the present invention. The Tk-subtilisin solution obtained in This Tk-subtilisin solution can be stored stably for a long period of time without Tk-subtilisin self-degrading. Further, it is possible to provide a high concentration Tk-satilysin solution of 0.1 mg / ml or more that could not be realized conventionally.

Since the protease solution of the present invention can contain a high concentration of Tk-satilysin, it is possible to keep storage costs and transportation costs low, and improve the economics of distribution. Moreover, it can utilize as a solution for business preparation in the use which adds and uses protease at the time of use.

[Pro-form-containing solution]
The pro-form solution of the present invention is a pro-form solution of a protease containing a pro-form having an amino acid sequence described in (e) or (f) below and having a pH in the range of 2-6.
(E) an amino acid sequence represented by SEQ ID NO: 3 (f) an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 3, that is, the above-mentioned production method of the present invention A pro-Tk-subtilisin-containing solution prepared in the body-containing solution preparation step. This pro-Tk-subtilisin-containing solution contains a pro-form having a correctly folded native structure, a complex having a structure in which a pro-sequence generated by self-cleavage of the pro-form and a mature sequence are combined, and Tk-subtilisin (mature form). ) And a mixture solution containing three kinds of proteins, and can be stably stored while suppressing the progress of maturation.

The pro-form-containing solution of the present invention supplies an intermediate in the production of Tk-subtilisin. That is, since an intermediate for producing Tk-subtilisin can be distributed, it is possible to produce the target product Tk-subtilisin at the site where the final product containing Tk-subtilisin is produced. Further, the pro-form-containing solution of the present invention is a solution having high storage stability, but by checking the amount of the pro-form in the solution, it can be easily confirmed that self-decomposition has not progressed. That is, there is an advantage that quality control during distribution process and storage can be easily performed.

[Usage of Tk-Sachilysin]
Hereinafter, the use of Tk-subtilisin will be described as a representative example of the protease obtained by the production method of the present invention. However, the protease obtained by the production method of the present invention is not limited to Tk-subtilisin. Other proteases obtained by the method can also be used for similar applications. Needless to say, Tk-subtilisin produced by a method other than the production method of the present invention can also be used for the same purpose. Tk-subtilisin has a significantly higher activity in the high temperature range (70 ° C. to 100 ° C.) than Subtilisin Carlsberg and Proteinase K, which have already been put to practical use, and is higher than other proteases in the lower temperature range. It is a protease with activity. This time, the present inventors have shown that Tk-satilysin has very high activity at pH 8-12, has significantly higher thermal stability compared to Subtilisin Carlsberg and Proteinase K, and has been modified with surfactants and proteins. It was newly found that the stability to the agent is high. Therefore, Tk-subtilisin decomposes persistent industrial waste (including keratin such as feathers and animal hair), bioactive peptide production, fiber processing, wool processing, leather processing, food processing (fish oil processing, meat processing, etc.) ), Feed processing, nucleic acid purification, contact lens cleaning, piping cleaning, and the like. In addition, Tk-sachilysin exhibits high activity in a wide range from room temperature to high temperature, and therefore can be used in accordance with the temperature conditions optimum for the material to be decomposed and the purpose of use. Furthermore, since the reactivity is high, an effect equivalent to that of existing products can be obtained with a small amount. Especially for new applications (cleaning of medical equipment, high-efficiency decomposition of difficult-to-decompose products) in fields where enzymes were not available until now, requiring high alkalis and high-temperature conditions (for example, pH 12, 80 ° C., etc.) Available. Therefore, Tk-subtilisin is used under high temperature and high alkali conditions, and can be formulated into a detergent containing a surfactant to enhance the detergency. In particular, it has significantly higher activity compared to industrial proteases that have been used in the past, so it is possible to powerfully decompose and clean infectious protein stains on medical devices where secondary infection is a problem. And has high utility as a detergent for medical devices. Moreover, it can use suitably also for various detergents, such as a detergent for dishwashers and a laundry detergent.

(1) Detergent The present invention provides a detergent (cleaning composition) containing Tk-sachilysin. Although the content of Tk-subtilisin in the detergent is not particularly limited, it has a high activity as compared with other proteases, so that a high detergency can be exhibited with a small addition. A preferable content is, for example, 0.1 to 10% by weight. If the content is too small, a sufficient cleaning effect cannot be obtained. On the other hand, if the content is too large, an improvement in the cleaning effect relative to the content cannot be obtained, which is not preferable in terms of economy. Tk-subtilisin can be formulated into any known detergent without any change in the composition of the detergent. Further, there are no particular limitations on the components of the detergent containing Tk-subtilisin. Typical examples of such detergents are 10-50 wt% surfactant, 0-50 wt% builder, 1-50 wt% alkaline agent or inorganic electrolyte, 0.1-5 wt% per detergent weight. % Detergents comprising at least one compounding ingredient selected from the group consisting of anti-staining agents, enzymes, bleaches, fluorescent dyes, anti-caking agents and antioxidants.

It is preferable that the detergent containing Tk-subtilisin further contains a surfactant. The surfactant contained in the detergent is not particularly limited, and is an anionic surfactant, a nonionic surfactant (for example, ether type nonionic surfactant, fatty acid ester type nonionic surfactant, ethylene oxide addition) Any of fatty amines, cationic surfactants, and amphoteric surfactants can be suitably used. The present inventors have confirmed that Tk-subtilisin can maintain protease activity in the presence of at least 19 kinds of surfactants (see Example 5 and Table 1). It can be said that it is particularly suitable for blending and using the contained detergent. The content of the surfactant is not particularly limited, but it is preferable that the surfactant is contained in such an amount that the protein present in the washing object is denatured. Therefore, it is preferable to select and use an appropriate content according to the surfactant to be used. For example, it is preferably about 1 to 50% by weight, more preferably about 1 to 30% by weight, and further preferably about 3 to 10% by weight.

Protease derived from Alkaline Filus transvallensis, which is an existing alkaline protease (Patent Document 1), has characteristic properties such as high alkali activity, surfactant resistance, and calcium-independent heat resistance. Since the optimum temperature is 70 ° C., it is not suitable for higher temperature cleaning. Pyrococcus horikoshii-derived protease (Patent Document 2) is characterized by an optimum temperature of about 98 ° C. or more and an optimum pH of about 5 to about 6, but it is active under neutral and alkaline conditions. It is low and there is no description about resistance to surfactants. On the other hand, a protease derived from Pyrococcus furiosus (Patent Document 3) has resistance to acetonitrile, urea, and SDS. As for resistance to SDS, it has an activity of about 80% after treatment at 95 ° C. for 3 hours in the presence of SDS at a final concentration of 1%, but degradation at a concentration of SDS of 1% or more under high temperature conditions. There is no description of activity, and there is no description of resistance to surfactants other than SDS.

Since the detergent of the present invention has much higher detergency and proteolytic ability than existing detergents, it is highly useful as a medical instrument detergent as described above. In particular, enzyme detergents can be washed in a state containing a surfactant at a high temperature that could not be used so far, so that washing and sterilization can be performed in a short time. In addition, it has an activity of 80% or more at 100 ° C. for 3 minutes in the presence of 3% SDS, which is an inactivation condition of abnormal prion protein, and is capable of decomposing and removing abnormal prion protein in preventing secondary infection of prion disease. A detergent that can be inactivated at the same time can be provided. Similarly, it is highly useful for spraying and heating cleaning using a washer disinfector. That is, the detergent of the present invention is very useful in that existing equipment such as a watcher disinfector can be used as it is. When used under high temperature conditions, the amount of enzyme can be reduced, so that the protease can be easily removed after prion inactivation. Since it can be used neutrally, it is safer for workers than existing alkaline detergents, and it can also be used for products such as endoscopes that are vulnerable to alkaline cleaning. Although there is still no effective prion inactivation method for endoscopes that are highly likely to be contaminated with prions and difficult to dispose of, this cleaning agent makes it possible. Furthermore, it is highly useful as a detergent for cleaning contact lenses.

(2) Abnormal Prion Proteolytic Agent The present inventors have found that Tk-satilysin can degrade abnormal prion protein. Therefore, the present invention provides an abnormal prion proteolytic agent containing Tk-satilysin. The abnormal prion proteolytic agent of the present invention is not particularly limited as long as it contains Tk-subtilisin. Preferably it contains a surfactant. The abnormal prion proteolytic agent of the present invention can be produced according to the detergent of the present invention. Moreover, it can be used according to the “inactivation method of abnormal prion protein” described later.

(3) Method for Inactivating Abnormal Prion Protein The method for inactivating the abnormal prion protein of the present invention may be any method as long as it includes a step of bringing the object to be cleaned to which the abnormal prion protein is attached into contact with Tk-satilysin. The “contact” is not particularly limited, and the article to be cleaned may be immersed in a solution containing Tk-satilysin, and a solution containing Tk-satilysin may be applied, sprayed, sprayed, or the like. Note that after the contacting step, it is preferable to perform a step of sufficiently rinsing the object to be cleaned with water or the like so that no Tk-satilysin remains on the object to be cleaned. In addition, by using a Tk-satilysin solution containing a surfactant, it is possible to simultaneously inactivate abnormal prion protein in an object to be cleaned and to wash the object to be cleaned. In addition, Tk-satilysin can effectively degrade abnormal prion protein under the condition of 5 minutes in the presence of 3% SDS surfactant at 100 ° C., so that the abnormal prion protein can be decomposed and removed at the same time. In this respect, it is essential that the “priozyme (registered trademark)”, which is a decontamination agent for abnormal prion protein on the market, immerse the object to be cleaned in a solution in which the preozyme is dissolved after the object is cleaned. In comparison, it is extremely convenient.

In addition, it is necessary to immerse an object to be washed in an alkaline solution for 1 hour at 60 ° C., but Tk-satilysin used in the present invention sufficiently inactivates (decomposes) abnormal prion protein even at a pH near neutral. Can do. Tk-subtilisin has a high activity at 25 ° C. to 100 ° C., and therefore can be used as an abnormal prion proteolytic agent in a wide range from a low temperature range to a high temperature range. Therefore, use in a high temperature region increases decomposition activity, so that abnormal prion protein can be decomposed and removed in a short time. Although preozyme can decompose and remove abnormal prion protein, it is not guaranteed until infectious removal or inactivation. On the other hand, Tk-subtilisin has a degrading activity under inactivation conditions (3% SDS, 100 ° C., 3 minutes) that can remove and infect abnormal prion protein, so that it is impossible to decompose and inactivate abnormal prion protein. Therefore, the method for inactivating abnormal prion protein of the present invention is extremely excellent.

In the method for inactivating an abnormal prion protein of the present invention, it is preferable to contact a material to be washed with Tk-satilysin under protein denaturing conditions. For example, it is known that abnormal prion protein is completely infectious when treated with 3% SDS solution at 100 ° C. for 3 minutes (Reference: Ministry of Health, Labor and Welfare, Ministry of Health, Labor and Welfare Late Virus Infection Research Team, Kreuzfeld-Jacob disease medical care manual revised edition ", pp. 48-49, 2002). Other protein denaturation conditions that eliminate the infectivity of abnormal prion protein include, for example, 7M guanidine hydrochloride 2 hour treatment, 3M guanidine thiocyanate 2 hour treatment, 3M trichloroacetate 2 hour treatment, 50% or more phenol 2 hour treatment, etc. Can be mentioned.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example 1: Biochemical characteristics of Tk-subtilisin]
(1) Expression and purification of pro-Tk-subtilisin A primer pair for amplifying a partial sequence encoding pro-Tk-subtilisin based on the base sequence encoding the Tk-subtilisin precursor (ACCESSION: AB056701, SEQ ID NO: 6) Designed. That is, a forward primer (5′-AGTCCCTGCACATATGGGAGAGCAGAATACAATA-3 ′ (SEQ ID NO: 7)) containing an NdeI site and a reverse primer (5′-AGTGGATCCAATCAGCCCAGGGC-3 ′ (SEQ ID NO: 8)) containing a BamHI site. Using the genomic DNA of Thermococcus kodakaraensis KOD1 strain as a template, PCR was performed using the above primer pairs to amplify the DNA fragment. The obtained DNA fragment was digested with NdeI and BamHI, and this DNA fragment was inserted into the NdeI / BamHI site of pET25b (Novagen). This plasmid was used to transform E. coli BL21 (DE3) CodonPlus.

The obtained strain was cultured at 37 ° C. using NZCYM medium containing 50 μg / ml ampicillin. When OD ₆₆₀ reached 0.5, IPTG having a final concentration of 0.5 mM was added, and the culture was further continued for 4 hours, whereby pro-Tk-subtilisin was expressed in large amounts as inclusion bodies. The cells were collected, suspended in 20 mM Tris-HCl (pH 9.0), subjected to ultrasonic disruption, and the precipitate fraction was collected by centrifugation (15,000 × g, 30 min). This precipitate was dissolved in a denaturing buffer (20 mM Tris-HCl (pH 9.0), 5 mM EDTA, 8 M Urea), centrifuged (15,000 × g, 30 min) to obtain a supernatant, and an anion exchange column Hitrap Q ( GE Healthcare) was used to purify the denatured pro-Tk-subtilisin. The degree of purification was confirmed using SDS-PAGE and Coomassie staining.

(2) Refolding of pro-Tk-subtilisin and preparation of Tk-subtilisin (mature) After purification with Hitrap Q, denatured pro-Tk-subtilisin (1.0 mg / ml, 10 ml) was added to 1 L of refolding buffer (20 mM Tris -Refolding was performed by dialysis twice with HCl pH 9.0 (first time; 2 hours, second time; 12 hours) and removing Urea. Thereafter, 13 μl of refolded pro-Tk-subtilisin is diluted with 987 μl of maturation buffer (50 mM CAPS-NaOH pH 9.5, 5 mM CaCl ₂ ) to a final concentration of 300 nM (about 0.0124 mg / ml). By heat treatment at 80 ° C. for 15 minutes, Tk-subtilisin (mature) was obtained through autoprocessing of propeptide (Autoprocessing) and degradation of propeptide (Degradation).

(3) Activity measurement method (3-1) When azocasein is used as a substrate Add 30 μl of Tk-subtilisin (100 nM) to 270 μl of reaction buffer (50 mM CAPS-NaOH pH 9.5, 5 mM CaCl ₂ , 2% azocasein). Incubated for 20 minutes under each temperature condition. The enzyme reaction was stopped by adding 200 μl of 15% TCA and allowing to stand for 15 minutes in ice. Centrifugation was performed at 15,000 × g for 15 minutes, 20 μl of 2M NaOH was added to the obtained 80 μl supernatant, and the absorbance at 440 nm was measured. At this time, the amount of enzyme required to increase the absorbance at 440 nm of 0.3 ml of the reaction solution by 1 per minute was defined as “1 unit”. For comparison subtilisin Carlsberg and proteinase K, 50 mM CAPS-NaOH pH 8.0, 5 mM CaCl ₂ , 2% azocasein was used as a reaction buffer.

(3-2) Synthesis substrates Tk- subtilisin when 300nM using Suc-AAPF-pNA, to a final concentration of 6nM enzyme activity assay buffer (20mM Tris-HCl (pH 8.0 ), 1mM CaCl 2, The solution was diluted 50-fold with 1 mM Suc-AAPF-pNA) and incubated at 20 ° C. for 4 minutes. The enzyme reaction was stopped by adding 10 μl of 100% acetic acid, and the change in absorbance at a wavelength of 410 nm was measured. In this measurement system, the amount of p-nitroaniline produced from Suc-AAPF-pNA is determined using an extinction coefficient of 8900 M ⁻¹ cm ⁻¹ to produce 1 μmol of paranitroaniline per minute. The amount of enzyme to perform was defined as “1 unit”. The same reaction buffer was also used for the comparison controls Subtilisin Carlsberg and ProteinaseK.

(4) Temperature Dependence of Activity According to the activity measurement method described in (3-1) above, the enzyme activity of Tk-subtilisin at each temperature from 20 ° C. to 100 ° C. was measured. In addition, similar experiments were performed using Subtilisin Carlsberg (Sigma) and Proteinase K (Wako) as a comparative control.
The results are shown in FIG. As is clear from FIG. 1, Tk-satilysin showed the highest activity at 90 ° C., and also had significantly higher activity at 100 ° C. On the other hand, Subtilisin Carlsberg as a comparative control had the highest activity at 60 ° C. and Proteinase K at 65 ° C., but Tk-satilysin was 2 to 3 times higher at these temperatures.

(5) pH Dependency of Activity According to the activity measurement method described in (3-2) above, enzyme activity was measured under each pH condition. The buffers used are as follows.
pH 4.0 to 5.6: 50 mM sodium acetate
pH 5.5-7.0: 50 mM MES-NaOH
pH 7.0 to 7.5: 50 mM HEPES-NaOH
pH 7.0 to 9.0: 50 mM Tris-HCl
pH 8.5 to 10.0: 50 mM CAPS-NaOH
pH 9.0 to 11.5: 50 mM Glycine-NaOH
The results are shown in FIG. As can be seen from FIG. 2, the optimum pH range of Tk-satilysin is pH 8-12, which proves that the enzyme is suitable for use in a highly alkaline environment.

(6) Effect of pH on structural stability 300 nM Tk-subtilisin was incubated overnight at 30 ° C. in a buffer under each pH condition, and the residual activity was measured according to the activity measurement method described in (3-2) above. . The buffers used are as follows.
pH 1.0-1.5: 50 mM KCl-HCl
pH 2.0 to 3.0: 50 mM Glycine-HCl
pH 4.0 to 5.0: 50 mM sodium acetate
pH 6.0: 50 mM MES-NaOH
pH 7.0: 50 mM HEPES-NaOH
pH 8.0-9.0: 50 mM Tris-HCl
pH 10.0-11.0: 50 mM Glycine-NaOH
pH 12.0-13.0: 50 mM KCl—NaOH
The results are shown in FIG. As is apparent from FIG. 3, TK-satilysin was shown to have structural stability in the pH range of 2-12.

(7) Comparison of refolding efficiency under each pH condition Pro-Tk-satilysin (2.0 mg / ml) denatured in the presence of 4M guanidine hydrochloride contains a buffer (10 mM CaCl ₂ , 1 mM DTT) at each pH condition. ) And refolded by 100-fold dilution, and the efficiency was analyzed by circular dichroism (CD) spectrum measurement. The buffers used are as follows.
pH 3.0: 50 mM Glycine-HCl
pH 4.0 to 5.6: 50 mM sodium acetate
pH 5.5-6.0: 50 mM MES-NaOH
pH 7.0: 50 mM HEPES-NaOH
pH 8.0-9.0: 50 mM Tris-HCl
The results are shown in FIG. As is clear from FIG. 4, it was shown that pro-Tk-subtilisin efficiently refolds under pH conditions exceeding pH 5.

(8) Thermal stability 300 nM of Tk-satilysin is heat-treated at a temperature of 20 ° C. to 110 ° C. for 10 minutes under a buffer condition of 20 mM Tris-HCl (pH 8.0) and 1 mM CaCl ₂ . Residual activity was measured according to the activity measurement method of (3-2) above. In addition, similar experiments were performed using Subtilisin Carlsberg (Sigma) and Proteinase K (Wako) as a comparative control.
The results are shown in FIG. As is apparent from FIG. 5, no decrease in activity of Tk-satilysin was observed even after heat treatment at 110 ° C. for 10 minutes. On the other hand, Subtilisin Carlsberg for comparison was inactivated at 70 ° C. and Proteinase K at 80 ° C. From this result, it was revealed that Tk-satilysin is an enzyme with extremely high thermostability.

(9) Stability against surfactants and protein denaturants 300 nM Tk-satilysin was added to various concentrations of surfactant (Triton X-100, Tween-20, sodium dodecyl sulfate (SDS)) and protein denaturant (guanidine hydrochloride). Incubation was carried out at 55 ° C. in the presence of (Guanidine HCl) and urea (Urea), and the residual activity was measured at each time interval according to the activity measurement method of (3-2) above. In addition, similar experiments were performed using Subtilisin Carlsberg (Sigma) and Proteinase K (Wako) as a comparative control.

The results of Triton X-100 are shown in FIG. In FIG. 6, (a) is the result of Tk-subtilisin, (b) is the result of Subtilisin Carlsberg, and (c) is the result of ProteinaseK. As is clear from FIG. 6, no activity was observed for any of the enzymes even when treated with 10% Triton X-100 for 60 minutes.
The results of Tween-20 are shown in FIG. In FIG. 7, (a) is the result of Tk-subtilisin, (b) is the result of Subtilisin Carlsberg, and (c) is the result of ProteinaseK. As can be seen from FIG. 7, no decrease in activity was observed for Tk-satilysin even after treatment with 10% Tween-20 for 60 minutes. On the other hand, Subtilisin Carlsberg and Proteinase K showed a decrease in activity.
The results for sodium dodecyl sulfate (SDS) are shown in FIG. In FIG. 8, (a) is the result of Tk-satilysin, (b) is the result of Subtilisin Carlsberg, and (c) is the result of ProteinaseK. As is apparent from FIG. 8, no decrease in activity was observed for Tk-satilysin even when treated with 5% SDS for 60 minutes. On the other hand, Subtilisin Carlsberg and Proteinase K showed a decrease in activity.
The result of guanidine hydrochloride (Guanidine HCl) is shown in FIG. In FIG. 9, (a) is the result of Tk-subtilisin, (b) is the result of Subtilisin Carlsberg, and (c) is the result of ProteinaseK. As is clear from FIG. 9, no decrease in activity was observed for Tk-satilysin even when treated with 6M guanidine hydrochloride for 60 minutes. On the other hand, Subtilisin Carlsberg and Proteinase K showed a decrease in activity.
The result of urea (Urea) is shown in FIG. In FIG. 10, (a) is the result of Tk-satilysin, (b) is the result of Subtilisin Carlsberg, and (c) is the result of ProteinaseK. As is clear from FIG. 10, Tk-satilysin did not show a decrease in activity even after treatment with 8% urea for 60 minutes. On the other hand, Subtilisin Carlsberg and Proteinase K showed a decrease in activity.
From the above results, it was revealed that Tk-satilysin has high stability against various surfactants and protein denaturants.

[Example 2: High-efficiency production method of Tk-subtilisin (mature)]
The amount of Tk-subtilisin (mature) obtained by the method of preparing Tk-subtilisin (mature) by the method of Example 1 (1) and (2) (conventional method) is the amount of pro-Tk-subtilisin subjected to maturation. It was about 20% of the amount, and the recovery efficiency of matured bodies was very low. Therefore, an attempt was made to improve the recovery efficiency of matured bodies.

Pro-Tk-subtilisin modified with 8M Urea was purified in the same manner as in Example 1 (1) above. The refolding buffer used was different from that in Example 1 and was used with the addition of DTT and calcium ions. Specifically, denatured pro-Tk-subtilisin (1.0 mg / ml, 10 ml) was dialyzed twice with 1 L of refolding buffer (20 mM Tris-HCl pH 7.0, 1 mM DTT, 10 mM CaCl ₂ ) (first time; 2 Time, second time; 12 hours), and refolding was performed by removing Urea. Thereafter, 13 μl of the refolded pro-Tk-subtilisin is diluted with 987 μl of maturation buffer (50 mM CAPS-NaOH pH 9.5, 5 mM CaCl ₂ ) to a final concentration of 300 nM (about 0.0124 mg / ml). By heat treatment at 80 ° C. for 15 minutes, Tk-subtilisin (mature) was obtained through autoprocessing of propeptide (Autoprocessing) and degradation of propeptide (Degradation).

Samples were taken from the pre-heating maturation buffer containing pro-Tk-subtilisin and the post-heating (post-maturation) maturation buffer and subjected to SDS-PAGE. For comparison, a sample obtained by the method of Example 1 (conventional method) was also subjected to SDS-PAGE at the same time.
The results are shown in FIG. Lane 1 is a sample before heating by the conventional method, Lane 2 is a sample after heating by the conventional method, Lane 3 is a sample before heating by the manufacturing method of the present invention, and Lane 4 is a sample after heating by the manufacturing method of the present invention. As apparent from lanes 3 and 4, according to the production method of the present invention, about 90% of the amount of pro-Tk-subtilisin before heating could be recovered as Tk-subtilisin (mature) (mature recovery efficiency of about 90%). ). On the other hand, in the conventional method from lanes 1 and 2, the matured body recovery efficiency was about 20%. From this result, it became clear that according to the method of the present invention, the recovery efficiency of matured bodies was remarkably improved.

[Example 3: Concentration and storage of Tk-subtilisin (mature)]
When Tk-subtilisin (mature) prepared by the method of Examples 1 (1) and (2) above (conventional method) was concentrated using Centriplus (Millipore), 0.1 mg / ml When the above concentration conditions were reached, an autolysis reaction occurred immediately. Therefore, it has not been possible to prepare a Tk-subtilisin (mature) solution with a high concentration until now. Therefore, the novel findings obtained by the above Examples 1 (5) and (6), that is, the activity of Tk-subtilisin is remarkably low at pH 6 or lower (see FIG. 2), and Tk-subtilisin is pH 2 or higher. Thus, using the stable structure (see FIG. 3), it was attempted to concentrate and store Tk-satilysin under pH 2-6 conditions.

The Tk-subtilisin (mature body) produced by the method of Example 2 was dialyzed with a buffer (50 mM sodium acetate (pH 4.6), 10 mM Ca (OAc) ₂ ) to obtain sample A (concentration: 0.0115 mg). / ml). Sample A was concentrated 10 times using Centriplus (Millipore) to prepare Sample B (0.115 mg / ml). Similarly, sample A was concentrated 100 times to prepare sample C (1.15 mg / ml). The obtained samples A, B and C were each left overnight at 4 ° C. Add the above buffer (50 mM Sodium Acetate (pH 4.6), 10 mM Ca (OAc) ₂ ) to Sample B and Sample C, and dilute them 10 and 100 times, respectively. Sample A and Samples B and C after dilution 1.0 ml of each was taken, 112 μl of 100% TCA was added to recover the protein as a precipitate, and the resulting precipitate was dissolved in 20 μl of SDS sample buffer. 10 μl of this was subjected to SDS-PAGE, and the presence or absence of autolysis accompanying the concentration of Tk-subtilisin was examined.

The results are shown in FIG. As can be seen from FIG. 12, it was revealed that there was no autolysis due to concentration and no change in protein amount.
The buffer used for concentration and storage is not limited to the above-mentioned sodium acetate as long as it can be adjusted to an acidic pH (2.0 to 6.0). For example, Glycine-HCl can be preferably used. For the concentration, a centrifugal ultrafiltration unit represented by the above-mentioned Centriplus can be suitably used. Further, since the isoelectric point of Tk-subtilisin (mature) is 4.42, it is bound to a cation exchange column Hitrap SP (GE Healthcare) or the like after dialysis with a buffer of pH 4.0 or lower, After dialysis with a buffer of pH 5.0 or higher, it can be bound to an anion exchange column Hitrap Q (GE Healthcare) or the like, and both can be concentrated by elution with sodium chloride.

[Example 4: Production of pro-form-containing solution of Tk-subtilisin]
According to Examples 1 (5) and (7) above, the modified pro-Tk-satilysin can be correctly refolded in the range of pH 5.2 to 6.0 (see FIG. 4), but Tk-satilysin is in this pH range. Activity was remarkably low (see FIG. 2). From these results, if refolding is performed in the range of pH 5.2 to 6.0, the activity of Tk-subtilisin is suppressed and autolysis does not occur under the pH environment even when the obtained pro-form matures. It was considered a thing. In order to confirm this, the following experiment was conducted.

Pro-Tk-subtilisin (1.24 mg / ml) denatured in the presence of 4M guanidine hydrochloride was refolded by diluting 100-fold with a buffer (containing 10 mM CaCl ₂ and 1 mM DTT) under various pH conditions. Alternatively, the propeptide cleavage (autoprocessing) efficiency after standing at room temperature for 12 hours was analyzed by 15% SDS-PAGE. The buffers used are as follows.
pH 4.0 to 5.6: 50 mM sodium acetate
pH 6.0: 50 mM MES-NaOH
pH 7.0: 50 mM HEPES-NaOH
pH 8.0: 50 mM Tris-HCl

The results are shown in FIG. In FIG. 13, the number at the top of each lane represents the pH of the buffer used, C represents a control using a buffer not containing calcium ions, and LMW represents a molecular weight marker. As is clear from FIG. 13, after standing for 12 hours in the refolding buffer, the pro-form having a native structure, and the composite having a structure in which the pro-sequence and the mature sequence generated by self-cleavage of the pro-form are combined. And three types of proteins, Tk-satilysin (mature). Moreover, the ratio of the pro form, the complex form, and the mature form did not change at any pH. This result shows that self-cleavage proceeds even at a pH of 6 or less, where the activity of Tk-subtilisin is remarkably low, which is a very unexpected result. By setting the pH of the solution containing the three types of proteins, the pro-form, the complex, and the mature form (pro-form-containing solution) to 2 to 6, the activity of Tk-satilysin is suppressed and the maturation is achieved. However, it was almost unproblematic and could be stored stably. In use, dilute with a buffer under alkaline pH conditions so that the final concentration of the protein in the pro-form-containing solution is 300 nM, and heat-treat at 80 ° C. or higher (boiling is acceptable) for 10 minutes. About 300 nM of Tk-satilysin (mature) can be prepared.

[Example 5: Bk degradation activity of Tk-subtilisin in the presence of a surfactant]
Tk-subtilisin (mature) solution produced and prepared by the methods of Examples 2 and 3 was used in this example. Moreover, 19 types shown in Table 1 were used for the surfactant. A 1% surfactant solution was prepared according to the content of the surfactant in each product. As the solvent, 100 mM Tris-HCl (pH 8.0) and 1 mM CaCl ₂ solution were used. Neoperex GS was adjusted to a neutral pH by adding 1/50 amount of 6M NaOH. Since Rheidol SP-010V, Emanon 3299V and Acetamine 86 are not water soluble, they were prepared by changing the water to 50% ethanol. Emanon 3299V was prepared after melting the solid at 80 ° C. and suspending in ethanol.

(1) Confirmation of BSA decomposition activity by reaction temperature 44 μl of 1% surfactant solution and 5 μl of 50 mg / ml BSA solution were added to a 1.5 ml tube. As a control, two tubes containing 44 μl of buffer alone and 5 μl of 50 mg / ml BSA solution were prepared. Add 1 μl of water to one of the controls, add 1 μl of Tk-subtilisin solution (25 mg / ml) to the other tubes, cap, and immediately at 100 ° C., 65 ° C., 37 ° C. or 25 ° C. for 10 minutes. Incubated. Ten minutes later, 1 μl of 0.5 M EDTA was added to stop the reaction. To the reaction solution, 16 μl of 4 × SDS sample buffer and 66 μl of 1 × SDS sample buffer were added, boiling for 5 minutes, and subjected to SDS-PAGE (using 12% acrylamide gel).

The results at a reaction temperature of 100 ° C. are shown in FIG. The results at a reaction temperature of 65 ° C. are shown in FIG. The results at a reaction temperature of 37 ° C. are shown in FIG. The results at a reaction temperature of 25 ° C. are shown in FIG. In FIGS. 14-17, Marker is the molecular weight marker, C1 is control 1 (no surfactant, with Tk-subtilisin), C2 is control 2 (with surfactant, no Tk-subtilisin), and the numbers in each lane are The surfactants corresponding to the lane numbers in Table 1 are respectively represented.

At a reaction temperature of 100 ° C., BSA was almost completely decomposed in 10 minutes except for lane 3 (dodecylbenzenesulfonic acid) (see FIG. 14). At the reaction temperature of 65 ° C., although the reaction rate was different, it was shown that all the surfactants used did not inhibit the BSA degradation activity of Tk-subtilisin (see FIG. 15). Similarly, at the reaction temperatures of 37 ° C. and 25 ° C., although the reaction rate was different, all the surfactants used were shown not to inhibit the BSA degradation activity of Tk-subtilisin (FIGS. 16 and 17). reference).
From these results, it was shown that Tk-subtilisin can maintain activity from room temperature to high temperature (100 ° C.) even in the presence of a surfactant. It was also found that the reaction rate was faster at higher temperatures.

(2) BSA degradation activity under reaction conditions at 100 ° C. for 5 minutes (comparison with other proteases)
To a 1.5 ml tube, 88 μl of 1% surfactant solution and 10 μl of 50 mg / ml BSA solution were added. As a control, two tubes containing 88 μl of buffer alone and 10 μl of 50 mg / ml BSA solution were prepared. Preincubation for 5 minutes in a 100 ° C. heat block. 2 μl of water was added to one of the controls, 2 μl of Tk-subtilisin solution (25 mg / ml) was added to the other tube, the lid was closed, and immediately incubated at 100 ° C. for 10 minutes. Ten minutes later, 100 μl of a mixture of 2 μl of 0.5 M EDTA and 98 μl of 2 × SDS sample buffer was added, and the mixture was boiling for 5 minutes and subjected to SDS-PAGE (using a 12% acrylamide gel).
A similar experiment was performed using a 25 mg / ml aqueous solution of Proteinase K (Wako) or Subtilisin A (Sigma) instead of Tk-subtilisin.

FIG. 18 shows the results of Tk-subtilisin, FIG. 19 shows the results of Proteinase K, and FIG. 20 shows the results of Subtilisin A. 18-20, Marker is the molecular weight marker, C1 is control 1 (no surfactant, with protease), C2 is control 2 (with surfactant, no protease), and the numbers in each lane are the lanes in Table 1. Each of the surfactants corresponding to the number is represented.

Tk-subtilisin almost completely decomposed BSA at 100 ° C. for 5 minutes except for lane 3 (dodecylbenzenesulfonic acid) and lane 4 (semi-cured beef tallow fatty acid potassium soap) (see FIG. 18). On the other hand, in Proteinase K, BSA was decomposed only in Lane 5 (polyoxyethylene lauryl ether), but BSA was not decomposed in other cases (see FIG. 19). Subtilisin A partially decomposes BSA in lane 8 (polyoxyethylene (20) sorbitan monooleate), lane 10 (polyoxyethylene (40) sorbitol tetraoleate) and lane 14 (cetyltrimethylammonium chloride). However, other than these, BSA was not decomposed (see FIG. 20).
From these results, it was revealed that Tk-satilysin is extremely high in activity at high temperature in the presence of a surfactant as compared with other proteases.

(3) Confirmation of residual activity of Tk-subtilisin at 100 ° C. in the presence of 3% SDS 3% SDS was prepared using a buffer (100 mM Tris-HCl (pH 8.0), 1 mM CaCl ₂ ). 49 μl of this was dispensed into six 1.5 ml tubes. Separately, 49 μl of buffer alone (0% SDS) was dispensed into three 1.5 ml tubes. The 3% SDS group was further provided with a group with preincubation (3) and a group without preincubation (3). Three tubes in each group were used for reaction times of 0 minutes, 5 minutes, and 10 minutes, respectively. The 5 minute and 10 minute tubes of the preincubated group were preincubated for 2 minutes at 100 ° C.
The Tk-subtilisin solution was diluted with 200 mM Tris-HCl (pH 8.0) and 2 mM CaCl ₂ to a final concentration of 0.5 mg / ml, and 1 μl was added to each tube. The 0-minute tube was left as it was, and the 5-minute and 10-minute tubes were heated at 100 ° C. for 5 and 10 minutes, respectively, and then placed on ice for 30 seconds.
1 μl of each reaction solution was added to 99 μl of enzyme activity measurement solution (50 mM Tris-HCl (pH 8.0), 1 mM Suc-AAPF-pNA), and incubated at 30 ° C. for 20 minutes. The reaction was stopped by adding 5 μl of acetic acid, and the absorbance was measured at a wavelength of 410 nm. The absorbance of the enzyme activity measurement solution was measured as a blank.

The results are shown in FIG. As is clear from FIG. 21, Tk-satilysin has a residual activity of about 35% to about 50% even under the harsh conditions of 100 ° C. and 10 minutes in the presence of 3% SDS, which is considered to cause protein denaturation. It has been shown.

(4) Confirmation of BSA degradation activity of Tk-subtilisin under protein denaturing conditions Buffer (125 mM Tris-HCl (pH 6.8), 5% 2-mercaptoethanol, 1-7% SDS) and 240 μg in a 1.5 ml tube Of BSA was added to a volume of 39 μl, heated at 100 ° C. for 5 minutes, and then 1 μl of a 1.4 mg / ml protease solution (Tk-subtilisin, Subtilisin Carlsberg, Proteinase K, or buffer only) was added. Heated at 100 ° C. for 5 minutes and subjected to SDS-PAGE (using 12% acrylamide gel).

The results are shown in FIG. As is clear from FIG. 22, it was shown that Tk-satilysin can degrade BSA at 100 ° C. for 5 minutes even in the presence of 7% SDS. From this result, it was revealed that Tk-subtilisin is effective for proteolysis under protein denaturing conditions.

[Example 6: Degradation of abnormal prion protein by Tk-subtilisin]
The Tk-subtilisin (mature body) solution produced and prepared by the methods of Examples 2 and 3 above was applied to this example, and the concentration was adjusted using buffer A [10 mM Sodium Acetate (pH 5.0), 10 mM CaCl ₂ ]. Prepared.
Abnormal prion protein samples were prepared by collecting brains from mice infected with abnormal prion proteins (mouse adapto scrapie Handler strain) and diluting brain homogenates with PBS. The protein concentration was measured using a DC protein assay (Bio-Rad). In this example, only the results for the Chandler strain are shown, but the present inventors have obtained the same results for the Obihiro strain.

Western blotting was performed by the following method.
Blotting: The voltage was set to 50 V and the current was 140 mA, and blotting was performed for 90 minutes.
Blocking: Blocking was performed overnight at 4 ° C. with 5% skim milk (in PBS-T).
Primary antibody treatment: SAF83 (IgG) was diluted 1000 times with 0.5% skim milk (total amount 4 ml) and treated at room temperature for 1 hour.
Wash: Wash with PBS-T for 10 minutes 3 times.
Secondary antibody treatment: α-mouse-HRP was diluted 10,000 times with 0.5% skim milk (total amount: 4 ml) and treated at room temperature for 1 hour.
Wash: Wash with PBS-T for 10 minutes 3 times.
Photosensitivity: An HRP enzyme reaction was performed using an ECL kit, and the film was exposed to light.

(1) Experiment 1
To the tube, 20 μl of 0.5 M KCl—NaOH (pH 12.0), 7.5 μl of mouse brain homogenate (8 mg / ml) and 17.5 μl of water were added and mixed, followed by heating at 65 ° C. for 5 minutes. To this was added 5 μl of water or Tk-subtilisin solution (35.6 mg / ml) (final concentration 3.56 mg / ml), and the mixture was heated at 65 ° C. for 30 minutes. The reaction was stopped by adding 5 μL of 0.5 M EDTA and transferred to ice.
Each sample is dispensed in equal amounts and divided into “samples that are subjected to Proteinase K treatment” or “samples that are not subjected to Proteinase K treatment”. For “Samples that are subject to Proteinase K treatment”, a Proteinase K solution (200 μg / ml) is used as a sample amount. One minute was added and heated at 37 ° C. for 1 hour. 4 × SDS sample buffer was added to each sample, and boiling was performed at 100 ° C. for 5 minutes, followed by SDS-PAGE (using 15% acrylamide gel), electrophoresis, and Western blotting by the above method.

The results are shown in FIG. Since Proteinase K cannot degrade the abnormal prion protein, the band of the abnormal prion protein remains in the lane of the sample subjected to only the Proteinase K treatment. On the other hand, in the sample that had been treated with Tk-subtilisin, all proteins including abnormal prion protein were degraded regardless of the presence or absence of Proteinase K treatment. That is, it has been clarified that Tk-subtilisin can degrade abnormal prion protein. The reaction conditions of this experiment 1 were compared with previous studies with reference to literature (Proteolytic inactivation of the bovine spongiform encephalopathy agent, Biochem Biophys Res Commun., 2004 May 14; 317 (4): 1165-70.) It is set to be easy to do.

(2) Experiment 2
The experiment was conducted by changing the pH of the reaction solution to 8.0. That is, 20 μl of 0.5 M Tris-HCl (pH 8.0), 7.5 μl of mouse brain homogenate (8 mg / ml), and 17.5 μl of water were mixed in 4 tubes, and heated at 65 ° C. for 5 minutes. did. To this, 5 μl of buffer A or Tk-subtilisin solution (20.0 mg / ml) was added to each of two tubes (final concentration 2 mg / ml) and heated at 65 ° C. for 30 minutes. The reaction was stopped by adding 5 μL of 0.5 M EDTA and transferred to ice. Add 1/10 of the amount of Proteinase K solution (200 μg / ml) to each sample with and without Tk-subtilisin, and add the same amount of PBS to each remaining sample. In addition, the mixture was heated at 37 ° C. for 1 hour. 4 × SDS sample buffer was added to each sample, and boiling was performed at 100 ° C. for 5 minutes, followed by SDS-PAGE (using 15% acrylamide gel), electrophoresis, and Western blotting by the above method.

The results are shown in FIG. As in Experiment 1, an abnormal prion protein band remained in the sample treated with only Proteinase K (lane 2), but the sample treated with Tk-satilysin was treated with the abnormal prion protein regardless of the presence of Proteinase K treatment. All proteins including were degraded (lanes 3 and 4). That is, it has been clarified that Tk-subtilisin can sufficiently degrade abnormal prion protein even at a pH near neutral. Therefore, the abnormal prion degrading agent containing Tk-satilysin is more convenient in terms of handling and disposal than the preozyme (registered trademark, abnormal prion protein decontamination agent), which must be immersed in an alkaline solution. It was shown to be expensive.

(3) Experiment 3
Four tubes were mixed with 20 μl of 0.5 M Tris-HCl (pH 8.0), 7.5 μl of mouse brain homogenate (8 mg / ml) and 17.5 μl of water, and heated at 65 ° C. for 5 minutes. To this, 5 μl of buffer A or Tk-subtilisin solution (20.0 mg / ml) was added to each of two tubes (final concentration 2 mg / ml) and heated at 65 ° C. for 30 minutes. The reaction was stopped by adding 5 μL of 0.5 M EDTA and transferred to ice. 5 μl of 0.5 M DFP (diisopropyl fluorophosphate) was added to each of the sample to which Tk-satilysin had been added and the sample to which Tk-subtilisin had not been added, and treated at room temperature for 15 minutes. To each of the remaining ones, 5 μl of 2-propanol as a solvent was added as a control. 4 × SDS sample buffer was added to each sample, and boiling was performed at 100 ° C. for 5 minutes, followed by SDS-PAGE (using 15% acrylamide gel), electrophoresis, and Western blotting by the above method. At this time, the final concentration of SDS in the sample was adjusted to 3%, which is said to be easily inactivated by prions.

The results are shown in FIG. As is clear from FIG. 24, all the proteins including abnormal prion protein were degraded in lane 3 (Tk-subtilisin, without DFP treatment), but some proteins were degraded in lane 4 (with Tk-subtilisin, DFP treatment). Was not decomposed. This result shows that not all proteins were completely degraded by treatment with Tk-satilysin at 65 ° C for 30 minutes, and then the sample was completely degraded by adding SDS sample buffer and treating at 100 ° C for 5 minutes. It is shown that it is done. That is, it was revealed that Tk-subtilisin exhibits protease activity under the severe conditions of prion denaturation at 100 ° C. in the presence of 3% SDS and can completely decompose abnormal prions.

The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

Claims (15)

1. A pro-body expression step of introducing a DNA encoding a pro-form of protease into a host cell and expressing it as an inclusion body;
   A denaturing step to denature the pro-form in the inclusion body state to obtain a denatured pro-form,
   A refolding step of refolding the denatured pro-form in a solution containing calcium ions and having a pH of 5 or higher;
   A maturation step of maturing the pro-form after refolding to obtain an active protease,
   A method for producing a protease, wherein 50% or more of the pro form subjected to the maturation step is recovered as an active protease.
2. The method according to claim 1, further comprising a protease solution preparation step of preparing a protease solution having a pH of 2 to 6 after the maturation step.
3. The production method according to claim 2, wherein the protease solution has a protease concentration of 0.1 mg / ml or more.
4. The pro-form-containing solution preparation step of preparing a solution having a pH of 2 to 6 and containing a pro-form after refolding immediately after the refolding step is further included. The manufacturing method as described.
5. The production method according to any one of claims 1 to 4, wherein the protease comprises an amino acid sequence according to any one of the following (a) to (d).
   (A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2
6. The production method according to any one of claims 1 to 5, wherein the pro-form of the protease comprises an amino acid sequence described in (e) or (f) below.
   (E) Amino acid sequence shown in SEQ ID NO: 3 (f) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 3
7. A protease solution comprising a protease comprising the amino acid sequence described in any of the following (a) to (d) and having a pH in the range of 2 to 6.
   (A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2
8. The protease solution according to claim 7, wherein the protease concentration is 0.1 mg / ml or more.
9. A pro-former solution of a protease comprising the pro-form comprising the amino acid sequence described in (e) or (f) below and having a pH in the range of 2 to 6.
   (E) Amino acid sequence shown in SEQ ID NO: 3 (f) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 3
10. A detergent containing a protease comprising the amino acid sequence described in any of (a) to (d) below.
    (A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2
11. Furthermore, the detergent of Claim 10 containing surfactant.
12. The detergent according to claim 10 or 11, which is used for cleaning medical equipment.
13. An abnormal prion proteolytic agent comprising a protease comprising the amino acid sequence according to any of the following (a) to (d):
    (A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2
14. A method for inactivating an abnormal prion protein, comprising a step of contacting an object to be cleaned to which an abnormal prion protein is attached and a protease having an amino acid sequence described in any of the following (a) to (d):
    (A) amino acid sequence shown in SEQ ID NO: 1 (b) amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 (c) amino acid shown in SEQ ID NO: 2 Sequence (d) Amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2
15. The method for inactivating an abnormal prion protein according to claim 14, wherein the object to be cleaned and the protease are contacted under protein denaturing conditions.

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