Recombinant cocoonase and precursor thereof

Abstract

To solve the problem in which: traditionally, methods such as boiling or alkali treatment have been employed to purify raw silk and remove sericin, which is a glue component, to obtain silk thread; however, these methods failed to completely remove sericin and sometimes damaged the silk thread; silkworm-secreted cocoonase is regarded as a suitable treatment solution for sericin removal, but its natural yield is available only in traces amounts; furthermore, when the cocoonase was prepared by genetic recombination, non-specific cleavage occurs during self-processing for refolding or enzymatic activation reactions, leading to low recovery of the target enzyme.SOLUTION: The recombinant cocoonase obtained from the recombinant prococoonase of SEQ ID NO: 3 has enzymatic activity comparable to that of the wild type, and in addition, during self-processing for refolding or enzymatic activation reactions, avoids undesirable cleavage, achieving high-yield recovery of the target enzyme.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a recombinant cocoonase having suppressed autolytic reaction. [Background technology]

[0002] Silk thread is a traditional thread material obtained from silkworms, and its smooth texture allows it to produce luxurious and highly desirable textiles. Silk thread is usually obtained as follows: The cocoons produced by the silkworms are boiled, and the resulting cocoon threads are collected and wound up in several strands depending on the thickness of the thread required, to produce "raw silk (or "reeled silk")".

[0003] Raw silk is usually made up of around 70% fibroin and 30% sericin. In other words, raw silk is structured in such a way that filaments made of fibroin are bonded together with gelatinous sericin. Therefore, raw silk is treated with hot water or dilute alkali to remove the sericin (refining), resulting in a thread material made up of almost pure fibroin. This is what we call "silk thread."

[0004] To explain the refining process more specifically, known methods include heating in an aqueous sodium carbonate solution at 85°C to 90°C for about 40 minutes (Patent Document 1), and refining using hydrolytic enzymes such as protease and ultrasonic vibrations (Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2000-199121 A [Patent Document 2] Japanese Patent Application Publication No. 53-130397 [Non-patent literature]

[0006] [Non-Patent Document 1] Felsted , RL ; Kramer , KJ ; Law , JH ; Berger, E.; Kafatos, FC Cocoonase. IV. Mechanism of activation of prococoonase from Antheraea polyphemus. J. Biol. Chem. Rev. 1973, 248, 3012–3

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[0007] In order to emerge from the cocoon, silkworms secrete the enzyme cocoonase, which breaks down sericin, a cocoon protein equivalent to glue. Therefore, it is considered most preferable to use this cocoonase when refining silk thread. However, cocoonase is only produced for a short period of time when the silkworm emerges from the cocoon, and the amount of cocoonase produced by a single silkworm is very small. For this reason, it is difficult to obtain enough cocoonase from silkworms to process a large amount of raw silk.

[0008] Therefore, attempts have been made to produce cocoonase industrially by genetic recombination. When genetically recombinant cocoonase is produced from E. coli, it is often produced as an inactive precursor protein with no enzymatic activity. Therefore, in order to obtain enzymatic activity, a three-dimensional structure formation reaction and an enzyme activation reaction are necessary. However, since cocoonase is an enzyme protein, nonspecific autolysis occurs during the three-dimensional structure formation and enzyme activation reactions, resulting in a problem of extremely low recovery rate of active cocoonase. [Means for solving the problem]

[0009] The present invention has been conceived in view of the above problems, and provides a recombinant cocoonase that does not undergo nonspecific autolysis through the formation of a three-dimensional structure and the enzyme activity reaction.

[0010] More specifically, the recombinant cocoonase according to the present invention is characterized in that it is obtained from an inactive cocoonase (precursor protein: prococoonase) having the amino acid sequence of SEQ ID NO: 3. This precursor protein is also included in the present invention. The protein and precursor protein according to the present invention may also be referred to as "recombinant precursor cocoonase" or "recombinant prococoonase", respectively. Effect of the Invention

[0011] The recombinant cocoonase according to the present invention is synthesized as a precursor protein (called "prococoonase") to which a propeptide moiety that inhibits enzyme activity is bound. Since this prococoonase is inhibited from self-decomposing, even if a three-dimensional structure formation reaction (refolding) and an enzyme activation reaction (self-processing) are performed, the effect of inhibiting a decrease in the recovery rate due to a non-specific self-decomposition reaction is achieved. More specifically, when wild-type cocoonase is produced by genetic recombination using E. coli, the recovery rate of cocoonase is about 10% due to self-processing and refolding, but in the case of the recombinant cocoonase according to the present invention, the recovery rate dramatically increases to about 60%.

[0012] The boiling method currently used for refining silk damages silk proteins and reduces the gloss of the final product, so high-yield cocoonase (or prococoonase) is expected to be used industrially as a washing solution in the raw silk refining process. The recombinant cocoonase of the present invention has a high yield and has the same enzymatic activity against sericin as wild-type cocoonase, so it is useful for treating cocoons under conditions much milder than the boiling method and providing undamaged silk thread. [Brief description of the drawings]

[0013] [Figure 1] The results of SDS-PAGE analysis of the reaction mixtures of the self-processing of the synthesized cocoonase are shown in Fig. 1(a) for wild-type proCCN, Fig. 1(b) for [K63G]·proCCN, Fig. 1(c) for proCCN', and Fig. 1(d) for [K8D]·proCCN'. [Diagram 2] High-performance liquid chromatography profiles are shown in Fig. 2(a) for the reaction mixture for the auto-processing of wild-type recombinant proCCN, and in Fig. 2(b) for the reaction mixture for the auto-processing of [K63G]·proCCN. [Diagram 3] 3 is a graph showing the results of measuring circular dichroism (CD) spectra, in which (a) is a series of CD spectra of proCCN, and (b) is a CD spectrum of the mature CCN protein. [Figure 4] 4 is a graph showing the results of measuring circular dichroism (CD) spectra, in which (a) shows the results for a series of proCCNs, and (b) shows the CD spectrum of a cassette mutant proCCN protein. [Diagram 5] 1 shows the results of SDS-PAGE analysis of the autoprocessing reaction mixtures of [H56A]·proCCN', [D99A]·proCCN', and [S193A]·proCCN' proteins. [Figure 6]The results of SDS-PAGE analysis of the self-processing solution of proCCN' with a mutation at the catalytic active site are shown in Figure 6. (a) shows the results for [H56A]·proCCN', (b) for [D99A]·proCCN', and (c) for [S193A]·proCCN'. [Figure 7] Figure 7(a) shows the results of casein zymography, and Figure 7(b) shows plots of initial velocity versus substrate concentration for trypsin and mutant proteins. Trypsin was used as a standard enzyme in the control experiment of casein zymography with Bz-Arg-OEt. The filled circle, open square, open circle, and open triangle show the data plots of trypsin, [H56A]·proCCN', [D99A]·proCCN', and [S193A]·proCCN', respectively. [Figure 8] 1 is a sequence listing of cDNA encoding proCCN. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The recombinant cocoonase according to the present invention will be described below with reference to drawings and examples. Note that the following description is merely an example of one embodiment of the present invention and an example, and the present invention is not limited to the following description. The following description can be modified without departing from the spirit of the present invention. In addition, in the following amino acid sequence display, numbers are assigned from the N-terminus to the C-terminus. The drawings used in this application are those published in Non-Patent Document 19.

[0015] When referring to an amino acid with a specific number, it is called "amino acid number __", which is the same as "amino acid number __ from the N-terminus". For example, it can be used as "the 8th lysine (K)" or "the 8th amino acid". It can also be used as "K8 residue" or "8th residue". It can also be written as [amino acid before mutation, mutation site, amino acid after mutation]. For example, [K8D] indicates that the 8th lysine (K) has been mutated to aspartic acid (D).

[0016] In the present invention, the enzyme is synthesized as an inactive enzyme precursor protein to which a residue (propeptide portion) that inhibits the enzyme activity is bound. First, the amino acid sequence (SEQ ID NO: 1) of prococcinase (referred to as "proCCN"), which is the precursor of wild-type coccinase, is shown in Table 1. proCCN is expressed as 235 amino acids from threonine 1 (T: Thr) to threonine 238 (L: Leu).

[0017] When the N-terminal 12-residue peptide of prococcusanase is cleaved autocatalytically or enzymatically, it becomes a biologically active mature coccusanase (hereinafter referred to as "CCN"). In other words, the mature coccusanase consists of 226 amino acids from the 13th isoleucine (I:Ile) to the 238th leucine (L:Leu).

[0018] Cocoonase (CCN) is a protein produced by silkworms (Bombyx mori) that is known to specifically digest sericin, a protein that coats the fibroin of silkworm cocoons, and belongs to the trypsin-like protease family. The process by which the N-terminal 12-residue peptide is released is called "autoprocessing." On the other hand, when the propeptide portion is removed by autoprocessing, autocatalytic or enzymatic autolysis occurs. This is also called nonspecific autolysis. When a process called autoprocessing is performed, nonspecific autolysis also occurs, resulting in a decrease in yield.

[0019] [Table 1]

[0020] Prococcinase (proCCN) undergoes self-processing between the 12 K (lysine) residues (enclosed in a box) and the 13 I (isoleusone) residues. Self-processing occurs when the temperature is raised to 37°C. On the other hand, at temperatures below 4°C, folding and other reactions are stable, but self-processing does not occur. Therefore, by keeping the temperature at a level that does not cause self-processing, proCCN can be maintained as an enzyme that is inactive.

[0021] As described above, the T1 to K12 residues are a propeptide sequence that inhibits the enzyme activity. The mutation sites during refolding are K63, K131, and K133 (boxed in Table 1). Based on the knowledge of trypsin, which has high homology, the predicted catalytic residues are H56, D99, and S193 (circled), which was confirmed in the Examples as described below.

[0022] On the other hand, the recombinant coccinase according to the present invention has, in the form of recombinant prococcinase, the amino acid sequence of SEQ ID NO: 2 shown in Table 2. As will be described later, this is called "[K8D]proCCN'".

[0023] [Table 2]

[0024] With reference to Table 2, the recombinant prococcinase according to the present invention, which will be described in the following Examples, has lysines (K:Lys) at positions 8, 63, 131, and 133 of the wild-type prococcinase substituted with aspartic acid (D:Asp), glycine (G:Gly), glycine (G:Gly), and alanine (A:Ala), respectively (enclosed in a box in SEQ ID NO:2). Note that the substitutions of the 63rd, 131st, and 133rd residues are for preventing self-processing, and it is sufficient that the lysines (K:Lys) are converted to glycine (G:Gly) or alanine (A:Ala).

[0025] More generally, residues 8, 63, 131, and 133 may be replaced with amino acids other than arginine (R: Arg) and lysine (K: Lys). This is because cokunase is an enzyme that cleaves peptides after arginine and lysine (C-terminal side). Therefore, the amino acids that can be replaced are alanine (A), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Only residue 8 can be replaced with lysine (K). In this case, the 8th, 63rd, 131st and 133rd residues do not necessarily have to be substituted with the same amino acid.

[0026] Thus, the recombinant prococcusenase of the present invention is more generally represented by SEQ ID NO:3 as shown in Table 3.

[0027] [Table 3]

[0028] Here, a recombinant prococcinase containing proCCN' described below has X at position 8 as lysine (K:Lys) and X at positions 63, 131, and 133 as substituted with glycine (G:Gly) or alanine (A:Ala) (lysine (K:Lys) in the wild type). More generally, the recombinant prococcinase according to the present invention has X at position 8 as lysine (K:Lys) and X at positions 63, 131, and 133 as substituted with amino acids other than arginine (R:Arg) and lysine (K:Lys).

[0029] Furthermore, the recombinant prococcinase contains [K8D]proCCN' as shown in SEQ ID NO: 2, in which the 8th X is aspartic acid (D:Asp) and the 63rd, 131st, and 133rd Xs are substituted with glycine (G:Gly) or alanine (A:Ala) (K in the wild type). More generally, the recombinant prococcinase according to the present invention is a prococcinase in which the 8th, 63rd, 131st, and 133rd Xs are substituted with amino acids other than arginine (R:Arg) and lysine (K:Lys).

[0030] Furthermore, the cocoonase obtained by removing the propeptide portion from these prococoanases is the recombinant cocoonase according to the present invention.

[0031] In the recombinant prococcinase according to the present invention, the lysine at the mutation site is replaced with another amino acid, so that the propeptide portion is removed by self-processing, and decomposition is suppressed by refolding even when the enzyme activity is expressed. Therefore, the recombinant prococcinase having the enzyme activity of SEQ ID NO: 3 can be recovered in high yield.

[0032] Therefore, in the purification of silk thread, recombinant prococinase (or recombinant cocoonase) having the enzymatic activity capable of removing sericin in the most natural way can be produced industrially in large quantities, and a suitable silk thread washing solution can be obtained. EXAMPLES

[0033] Examples of obtaining the recombinant prococcus enzyme of the present invention will be described below.

[0034] <Determination of decomposition site> To suppress the degradation that occurs during the refolding and activation reactions, we searched for the site on the molecule that causes the degradation. First, wild-type CCN protein purified with an octadecylsilyl (ODS) open column (Nacalai tesque, Kyoto, Japan) was refolded and self-processed by the method previously described (Non-Patent Document 3).

[0035] Figure 1(a) shows the results of SDS-PAGE analysis of the reaction mixtures for the autoprocessing of proCCN, (b) [K63G]·proCCN, (c) proCCN', and (d) [K8D]·proCCN'. The vertical axis in each figure is protein mass (kDa). In Figures 1(a) to 1(c), lanes 1 and 2 show the results at 0 min and 16 h at 4°C, respectively, and lanes 3 to 8 show the results at 0 min, 1 h, 3 h, 24 h, 48 h, and 72 h at 25°C, respectively.

[0036] In Figure 1(d), lanes 1 and 2 show proCCN' after 0 min and 24 h of reaction, respectively, and lanes 3 and 4 show the results for [K8D]·proCCN' after 0 min and 24 h of reaction, respectively. In each graph, the black (B), gray (Gr), and red (R) arrowheads indicate proCCN protein, degradation products, and mature CCN protein, respectively. The blue (Bl) arrowheads indicate degradation products cleaved at the Lys8 residue. Details of the structures of proCCN' and other proteins are shown in Table 4 below.

[0037] Referring to Figure 1(a), several degradation products (arrows marked "Gr"), including an 18 kDa protein, were produced in the self-processing reaction solution. Therefore, to investigate the cleavage site during the reaction, the components of the reaction mixture were separated by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0038] Figure 2(a) shows the high performance liquid chromatography (HPLC) profile of the reaction mixture after the self-processing of proCCN, and Figure 2(b) shows the HPLC profile of the reaction mixture after the self-processing of [K63G]·proCCN. The reaction mixtures in lanes 8 in Figure 1(a) and (b) were purified by RP-HPLC (a,b), respectively. The reaction mixtures were analyzed using HPLC systems from Waters and Hitachi.

[0039] The major peaks 1 and 2 in Figure 2(a) were purified and subjected to mass spectrometry, giving mass values ​​of 25,243 Da and 17,872 Da, respectively. The results indicated that peaks 1 and 2 corresponded to the proCCN protein and the C-terminal fragment (Val64 residue to Thr235 residue: see SEQ ID NO: 1), respectively, and that the proCCN protein was cleaved at the C-terminal peptide bond of the Lys63 residue. The mass value of the peak 2 protein in Figure 2(a) also matched the apparent molecular weight (18 kDa protein) of the degradation product fractionated using SDS-PAGE analysis shown in Figure 1(a).

[0040] To suppress degradation at position 63, we created a recombinant [K63G]·proCCN protein in which the Lys63 residue was replaced with a Gly residue. The amino acid sequence is shown in Table 3 as SEQ ID NO:3. In Table 3, the glycine (G:Gly) at the 63rd residue that was replaced is enclosed in a box.

[0041] [Table 4]

[0042] SDS-PAGE of the reaction mixture of the auto-processing of [K63G]·proCCN showed that the 18 kDa protein was not generated in the refolding and auto-processing reaction of [K63G]·proCCN protein, as shown in Fig. 1(b). However, a new degradation product, a 6 kDa protein, was generated during the activation of [K63G]·proCCN protein (the “Gr” part in lanes 3–8 of Fig. 1(b)).

[0043] Therefore, to further explore the cleavage site of the [K63G]·proCCN protein, the reaction mixture of the [K63G]·proCCN protein was also applied to RP-HPLC, and the degradation products were separated as shown in Figure 2(b).

[0044] Peak 3 in Figure 2(b) showed mass values ​​of 5789 Da and 5559 Da by mass spectrometry, indicating that degradation fragments (Thr132 residue to Lys186 residue and Leu134 residue to Lys186 residue) were generated by cleavage at the -Lys131 residue-Thr132 residue- and -Lys133 residue-Leu134 residue- sequences during the refolding and self-processing reactions.

[0045] Cocoonase has high homology with trypsin [Non-Patent Document 2]. It is known that the peptide bond between the Lys138 and Cys139 residues of human trypsin is cleaved during refolding and enzyme activation reactions [Non-Patent Document 4]. This position is relatively similar to the sequence of Lys133 residue-Leu235 residue of cocoonase. Therefore, in order to avoid non-specific cleavage at these sites, the 131st and 133rd lysines (K: Lys) of the [K63G]·proCCN molecule were mutated to glycine (G: Gly) and alanine (A: Ala) to prepare [K63G, K131G, K133A]·proCCN (referred to as "proCCN'"). The residues are shown in Table 5 as SEQ ID NO:5. The mutated residues in Table 5 are boxed.

[0046] This proCCN' is included in the recombinant proCCNase of the present invention (SEQ ID NO: 3 in which the 8th X is lysine (K: Lys) and the 63rd, 131st, and 133rd Xs are replaced with glycine (G: Gly) or alanine (A: Ala)), and mature CCN' obtained by removing the propeptide portion from proCCN' is also included in the recombinant proCCNase of the present invention.

[0047] [Table 5]

[0048] The results of SDS-PAGE of the solution after refolding and self-processing of proCCN' are shown in Figure 1(c). In Figure 1(c) and Figure 1(d), the red (R) arrows indicate the mature CCN' (sequence with K63G, K131G, and K133A mutations and with the propeptide sequence deleted).

[0049] As shown in Fig. 1(c), the arrows of Gr representing the degradation products disappeared during the refolding and autoprocessing reactions, and the amounts of the degradation products were significantly decreased. As a result, the overall yields of mature coccinase obtained by the refolding and autoprocessing reactions of wild-type proCCN, [K63G]·proCCN, and proCCN' proteins were approximately 9%, 13%, and 59%, respectively.

[0050] For example, the refolding recovery rate of trypsin and trypsinogen is known to be extremely low due to the considerable aggregation and degradation that occurs during the process. In fact, the refolding recovery rate of reduced trypsin has been reported to be 12% [Non-Patent Documents 5 and 6].

[0051] In fact, the results of refolding trypsin in an immobilized state or on a resin with immobilized inhibitors have been reported [Non-Patent Document 6]. It has also been reported that the folding recovery rate of trypsinogen was about 50% even in an immobilized state [Non-Patent Document 7].

[0052] Therefore, it can be said that the triple mutation of Lys(K)63, Lys(K)131, and Lys(K)133 residues of cocoonase significantly improves the recovery of mature cocoonase obtained by refolding and autoprocessing reaction without the need for inhibitors.

[0053] Thus, we were able to obtain a relatively high yield of recombinant coccinase with suppressed degradation. However, as shown in lanes 6 and 7 of Fig. 1(c) and lane 1 of Fig. 1(d), a certain amount of degraded protein was still observed between the proCCN' and mature CCN' protein bands in the self-processing reaction, suggesting that the propeptide region was cleaved at the Lys8 residue. To investigate the role of the propeptide region, it is necessary to maintain the entire propeptide sequence during the refolding reaction.

[0054] Considering the influence of the propeptide sequence on the intramolecular function, we prepared [K8D]·proCCN' in which the Lys8 residue was replaced with an Asp residue. This is also a recombinant prococinase according to the present invention and is shown in Table 2 as SEQ ID NO: 2. This is included in SEQ ID NO: 3 in which the 8th X is aspartic acid (D: Asp) and the 63rd, 131st, and 133rd Xs are replaced with glycine (G: Gly) or alanine (A: Ala).

[0055] Trypurinogen, which has high homology with prococcinase, has an enterokinase recognition sequence (DDDDK) at the site corresponding to the 8th to 12th residues (KDEEK) of the wild-type prococcinase propeptide sequence. The substitution of Lys8 residue with Asp residue was based on this sequence.

[0056] Therefore, the [K8D]·proCCN' protein was recombinantly expressed, refolded, and self-processed. The results are shown in lanes 3 and 4 of FIG. 1(d). As shown in FIG. 1(d), the SDS-PAGE analysis showed that no bands corresponding to the decomposition proteins between the proCCN mutant ([K8D]·proCCN') and the mature CCN mutant (CCN') were detected. In this way, prococcinase and coccinase that are not decomposed even by self-processing through folding can be obtained. The degradation-suppressed mutant protein [K8D]·proCCN' was finally obtained. This is the recombinant prococcinase (SEQ ID NO: 2) according to the present invention.

[0057] <Confirmation of protease activity> To confirm the protease activity of the mutant proteins, the protease activity of the recombinant proteins was assayed using Nα-Benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) as a substrate [Non-Patent Documents 6 and 9]. The results are shown in Table 6.

[0058] [Table 6]

[0059] With reference to Table 6, the enzyme activity of wild-type mature CCN was cited from a previous report [Non-Patent Document 1]. Km and Vmax values ​​were calculated by fitting the experimental data points to a Lineweaver-Burk plot. As shown in Table 5, the protease activity of the mutant proteins was compatible with that of the wild-type mature CCN protein, indicating that the triple mutation does not affect the enzyme activity of these proteins. In Table 5, "Wild type CCN" is a wild-type coccinase consisting of residues I13 to L238 of SEQ ID NO: 1, and [K63G]·CCN is a mutant coccinase consisting of residues I13 to L238 of SEQ ID NO: 4.

[0060] Furthermore, [K63G,K131G.K133A]·CCN is a mutant cocoonase consisting of residues I13 to T235 of SEQ ID NO:5, and “[K8D,K63G,K131G,K133A]·proCCN-derived mature CCN′” is a mature cocoonase derived from SEQ ID NO:2 in which lysine (K) at position 8 has been replaced with aspartic acid (D).

[0061] <Circular dichroism> To further clarify the three-dimensional structural characteristics of the mutant proteins prepared above, circular dichroism (CD) spectra were measured and secondary structure analysis was performed. The results are shown in Figure 3. Referring to Figure 3, Figure 3(a) shows the results of a series of proCCNs, and Figure 3(b) shows the CD spectrum of the mature CCN protein. In both graphs, the horizontal axis is wavelength (nm) and the vertical axis is θ (deg cm 2 dmol -1 ).

[0062] Black (B), blue (Bl), and red (R) show the CD spectra of wild-type proCCN, [K63G,K131G,K133A]·proCCN, [K8D,K63G,K131G,K133A]·proCCN (FIG. 3(a)), and its mature form (FIG. 3(b)). Note that [K63G,K131G,K133A]·proCCN, [K8D,K63G,K131G,K133A]·proCCN, [K63G,K131G,K133A]·CCN, and [K8D,K63G,K131G,K133A]·CCN are called mutant types in comparison with the wild-type. The mutant types are the recombinant proCCNase and recombinant coccinase according to the present invention, respectively.

[0063] As shown in Figure 3, the CD spectra of the wild-type and mutant proteins were completely overlapped, indicating that the above mutations do not affect the secondary / tertiary structure of the coccinase protein. This result indicates that the replacement of the Lys8 residue in the propeptide region with an Asp residue does not affect the conformation of the mutant protein or the enzyme activity after self-processing. This also indicates that the Lys8 residue is not essential for the role of the intramolecular chaperone function of the propeptide region, which promotes the reaction from the molten globule state to the native structure.

[0064] <Determination of catalytic residues> Cocoonase is a protein belonging to the serine protease family and has high homology with trypsin. However, the catalytic residues responsible for the protease activity of this protein had not been determined so far. If the catalytic residues could be accurately identified and mutated to inactive residues to suppress the decomposition of the folding intermediate, it would be useful for investigating the folding of cocoonase via the propeptide. Therefore, in order to determine the three residues responsible for the active center of cocoonase, a recombinant cocoonase was prepared in which the predicted catalytic residues (His56 residue, Asp99 residue, and Ser193 residue) were mutated to Ala residues based on the homology alignment of cocoonase and trypsin [Non-Patent Document 11].

[0065] To avoid non-specific degradation during refolding and processing, the mature CCN' was used as a template protein for mutations. This is a recombinant cocoonase with the mutations [K63G, K131G, K133A] shown in SEQ ID NO:5.

[0066] As a result, recombinant [H56A]·proCCN', [D99A]·proCCN' and [S193A]·proCCN' were designed and expressed in E. coli cells, and good three-dimensional structure formation was confirmed (data not shown).

[0067] [H56A]·proCCN' is shown as SEQ ID NO: 6 in Table 7, [D99A]·proCCN' is shown as SEQ ID NO: 7 in Table 8, and [S193A]·proCCN' is shown as SEQ ID NO: 8 in Table 9. The mutated regions are each enclosed in a box.

[0068] [Table 7]

[0069] [Table 8]

[0070] [Table 9]

[0071] After purifying the [H56A]·proCCN', [D99A]·proCCN' and [S193A]·proCCN' proteins, CD measurements were performed to estimate whether the mutations affected the conformation of these proteins. The results are shown in Figure 4.

[0072] Figure 4(a) shows the results for a series of proCCNs, and Figure 4(b) shows the CD spectrum of the cassette mutant proCCN protein (described later). Black (B), blue (Bl), red (R), and light blue (WB) represent the CD spectra of proCCN', [H56A]·proCCN', [D99A]·proCCN', and [S193A]·proCCN', respectively. In addition, the horizontal axis of each graph is wavelength (nm), and the vertical axis is θ (deg·cm 2 dmol -1 ).

[0073] As shown in Figure 4(a), the CD spectra of all mutant proCCN′ proteins essentially overlapped with the CD spectrum of proCCN′, indicating that the proteins were properly folded, similar to wild-type proCCN and proCCN′.

[0074] Figure 5 shows the results of SDS-PAGE analysis of the reaction mixtures after autoprocessing of [H56A]·proCCN', [D99A]·proCCN', and [S193A]·proCCN' proteins. The odd-numbered and even-numbered lanes show the solutions before and after the reaction, respectively. Lanes 1 and 2, 3 and 4, 5 and 6, and 7 and 8 show the profiles of proCCN', [H56A]·proCCN', [D99A]·proCCN', and [S193A]·proCCN' proteins, respectively. The black (B) and red (R) arrows indicate the precursor (proCCN' type) and mature (CCN' type), respectively. As shown in Figure 5, no products of autoprocessing reaction were observed in these mutant proteins.

[0075] Since no self-processing reaction occurred, the propeptide region was cleaved by trypsin treatment. However, trypsin could not be completely removed from the reaction mixture, even when immobilized trypsin agarose (ProteoChem, Inc., Hurricane, UT, USA) [Non-Patent Document 12] was used or purified by cation exchange chromatography. Therefore, the enzymatic activity of the mature form of the mutant protein after trypsin treatment could not be measured (data not shown).

[0076] Therefore, to obtain a mature form of the mutant protein in which the active site is replaced by an Ala residue, an enterokinase recognition sequence (DDDDK) was introduced into the processing site of the proCCN' mutant protein. The cassette mutation of the -Lys-Asp-Glu-Glu- sequence of cocoonase to the -Asp-Asp-Asp-Asp- sequence was originally introduced and used in the E. coli expression system [Non-Patent Document 13]. The mature cocoonase protein obtained using the enterokinase recognition sequence showed a kcat value similar to that of trypsin, although the Km value was not determined [Non-Patent Document 13]. Thus, the cassette mutation does not essentially affect the catalytic activity of cocoonase.

[0077] Specific cassette mutant cocoons are [K8D,E10D,E11D,H56A]·proCCN', [K8D,E10D,E11D,D99A]·proCCN', and [K8D,E10D,E11D,S193A]·proCCN', which are shown in Tables 10, 11, and 12 as SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively.

[0078] [Table 10]

[0079] [Table 11]

[0080] [Table 12]

[0081] [K8D,E10D,E11D,H56A]·proCCN', [K8D,E10D,E11D,D99A]·proCCN' and [K8D,E10D,E11D,S193A]·proCCN' were designed and expressed in colon cells by recombinant DNA technology. After refolding, the CD spectra were measured.

[0082] The results are shown in Figure 4(b). As shown in Figure 4(b), the molecular structure could be confirmed by CD measurements. The CD spectra of all mutant proteins were almost identical to that of proCCN', indicating that the protein conformations of the mutants were accurately constructed in the same way as proCCN. Thus, mutant proCCN' proteins with cassette mutations introduced into the propeptide region were prepared and further processed into the mature form.

[0083] The mutant proCCN' proteins of SEQ ID NOs: 8, 9, and 10 were treated with enterokinase to obtain the mature mutant proteins [H56A]·proCCN', [D99A]·proCCN', and [S193A]·proCCN'. The results of SDS-PAGE of the enterokinase-treated solutions of these proteins are shown in Figure 6.

[0084] Referring to Figure 6, Figure 6(a) shows the results of the cassette mutation [H56A]·proCCN', Figure 6(b) shows the results of the cassette mutation [D99A]·proCCN', and Figure 6(c) shows the results of the cassette mutation [S193A]·proCCN'. Lanes 1, 2, and 3 show purified proCCN', its enterokinase treatment, and its subsequent treatment with EKaptureTM agarose, respectively. Black (B) and red (R) arrows indicate proCCN' (in plain terms, [H56A]·proCCN', [D99A]·proCCN', and [S193A]·proCCN') and CCN' (in plain terms, [H56A]·CCN', [D99A]·CCN', and [S193A]·CCN') proteins, respectively.

[0085] The mature CCN' mutant proteins were then treated with an enterokinase capture kit according to the manufacturer's protocol.

[0086] The results of casein zymography are shown in Figure 7. Figure 7(a) shows the results of casein zymography for proCCN', [K8D]·proCCN', cassette mutant [H56A]·proCCN', [D99A]·proCCN', and [S193A]·proCCN' proteins, and Figure 7(b) shows plots of the initial velocity of trypsin and the mutant proteins versus substrate concentration.

[0087] In Figure 7(b), the horizontal axis is the substrate concentration S (μM), and the vertical axis is the initial velocity V (× 10 -4 The plots of trypsin, [H56A]·proCCN', [D99A]·proCCN', and [S193A]·proCCN' are shown in the solid circle, open square, open circle, and open triangle, respectively. Note that [H56A]·proCCN' (open square), [D99A]·proCCN' (open circle), and [S193A]·proCCN' (open triangle) are superimposed on the horizontal axis.

[0088] Lanes 1 and 2 for proCCN' and [K8D]·proCCN' correspond to the solutions before and after autoprocessing, respectively (Fig. 1(d)). Lanes 1, 2, and 3 for the cassette mutant [H56A]·proCCN', [D99A]·proCCN', and [S193A]·proCCN' proteins correspond to enterokinase-treated purified proCCN followed by EKapture™ agarose digestion, respectively. Black (B) and red (R) arrows indicate proCCN and mature CCN, respectively.

[0089] The obtained [H56A]·CCN', [D99A]·CCN', and [S193A]·CCN' did not show protease activity in casein zymography assays. However, the CCN' proteins autoprocessed from proCCN' or [K8D]·proCCN' showed active protease bands (white bands) on gels, as shown in Figure 7(a). The active protease band of the CCN' protein in lane 1 (top row, left column) of Figure 7(a) was observed even before the autoprocessing reaction, because a trace amount of the mature form was already generated before the autoprocessing reaction.

[0090] In addition, as shown in Figure 7(b), an enzyme assay using Bz-Arg-OEt clearly demonstrated that the mutant CCN' proteins with mutated catalytic residues ([H56A]·proCCN', [D99A]·proCCN', and [S193A]·proCCN') overlap on the horizontal axis and have no protease activity. In addition, in silico analysis of coccinase using Alphafold2, the predicted residues were thought to form a catalytic tridentate at relatively the same positions as trypsin (PDB ID: 5T3H) [Non-Patent Document 14]. Therefore, we concluded that His56, Asp99, and Ser193 are the actual effective residues of the catalytic tridentate.

[0091] The recombinant coccinase according to the present invention retains these three catalytic residues and can be said to maintain its enzymatic activity, similar to that of wild-type CCN.

[0092] The materials, equipment and methods used in the above examples are mentioned below.

[0093] <Material> Glutathione and Bz-Arg-OEt were purchased from Peptide Institute Co., Ltd. (Osaka, Japan). Casein was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All chemicals and solvents used were of special grade. cDNA encoding proCCN (Figure 8) was prepared by Eurofins Japan (Tokyo, Japan), and its codon bias was optimized for the E. coli expression system using the manufacturer's program GENEius (http: / / www.geneius.de / ; accessed May 13, 2017). The protein solution and E. coli cells were centrifuged using CF15R XII (HITACHI, Tokyo, Japan). Note that in Figure 8, the cDNA (SEQ ID NO: 32) and its translated amino acid (SEQ ID NO: 33) are listed together.

[0094] <Construction of expression vector for recombinant mutant protein in E. coli cells> The primer sequences used in this study are summarized in Table 13 as SEQ ID NO: 12 to SEQ ID NO: 31. cDNAs of the proCCN mutant proteins [K63G]·proCCN, [K63G,K131G,K133A]·proCCN, and [K8D,K63G,K131G,K133A]·proCCN were generated by PCR using synthetic cDNA encoding proCCN as a template [Non-Patent Document 3]. PCR reactions were performed using Platinum Pfx DNA polymerase (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, CA, USA).

[0095] The amplified cDNAs were subcloned into pET-17b expression vectors (Novagen, Glendale, CA, USA) after introducing NdeI and XhoI sites at the 5' and 3' ends, respectively. The resulting expression vectors (designated pMT1, pMT2, and pNS1) contained the cDNAs of [K63G]-proCCN, [K63G,K131G,K133A]-proCCN, and [K8D,K63G,K131G,K133A]-proCCN, respectively. The cDNA sequences of the vectors were confirmed by Eurofins Japan DNA sequencing service (Tokyo, Japan). In addition, cDNAs of mutant proteins in which the residues considered to be catalytic were mutated to Ala or Gly residues were also prepared by PCR using the pMT2 vector as a template. The vectors were constructed in the same manner as described above. The resulting expression vectors (designated pAO4, pAO5, pAO6, and pNS1) contained the cDNAs of [H56A,K63G,K131G,K133A]-proCCN, [K63G,D99A,K131G,K133A]-proCCN, and [K63G,K131G,K133A,S193A]-proCCN.

[0096] In addition, the cDNA sequences of the cassette mutant proteins in which the propeptide sequence (KDEEK) was cassette-mutated to the DDDDK sequence were prepared by PCR using the pAO4, pAO5, and pAO6 vectors as templates. The construction of the vectors was performed in the same manner as described above. The resulting expression vectors (referred to as pAO7, pAO9, and pAO10) contain the cDNAs of [K8D, E10D, E11D, H56A, K63G, K131G]·K133A]·proCCN, [K8D, E10D, E11D, K63G, D99A, K131G, K133A]·proCCN, and [K8D, E10D, E11D, K63G, K131G, K133A, S193A]·proCCN, respectively. The cDNA sequences of all the vectors constructed in this example were confirmed by analysis using the dideoxy method.

[0097] [Table 13]

[0098] <Protein expression and recombinant protein purification> Protein expression was performed according to previously reported methods [Non-Patent Document 15, Non-Patent Document 16]. All mutant proteins were obtained as inclusion bodies in E. coli cells and had a Met residue at the N-terminus of the proCCN mutant protein derived from the NdeI site during subcloning. After sonication of the cells, the mixture was centrifuged at 15,000 rpm for 15 min. The residue was then washed with 0.5% Triton X-100, water, and 80% CH3CN / 0.05% TFA. The final product was resuspended in Milli Q water, and the protein expression amount was estimated by SDS-PAGE analysis using BSA as a standard protein.

[0099] <Refolding reaction of recombinant protein and enzyme activation reaction of proCCN> The reduced refolding reaction of the recombinant mutant proCCN protein was carried out by a slightly modified method described previously [Non-Patent Document 3]. Briefly, the recombinant protein was solubilized by treatment with 6 M Gu / HCl in 20 mM Tris / HCl (pH 8.0) containing 10 mM dithiothreitol (DTT) at 50°C for 30 min. After centrifugation, the protein solution was dialyzed twice against 4 M urea / 0.05% TFA containing 0.5 M NaCl to completely remove DTT, and the resulting solution was centrifuged at 15000 rpm for 15 min.

[0100] The supernatant was mixed with an equal volume of 0.2 M Tris / HCl containing 0.5 M NaCl, 4 mM diisopropylaminoethanethiol, and 0.4 mM oxidized glutathione and incubated at 4° C. for 2 h. The reaction mixture was then diluted with an equal volume of 0.2 M Tris / HCl containing 0.5 M NaCl (final concentration of urea, 1 M) and incubated at 4° C. for 2 days. The refolded proCCN was dialyzed twice against 50 mM sodium phosphate buffer (100 mL, pH 7.0) containing 20 mM NaCl. The resulting solution was then subjected to cation exchange chromatography as described below.

[0101] <Cation exchange chromatography> ProCCN and CCN mutant proteins were purified by cation exchange chromatography using an AKTA purifier (GE Healthcare Japan, Tokyo, Japan). Briefly, the refolded proteins were dialyzed against 50 mM phosphate buffer (pH 7.0) containing 20 mM NaCl. After dialysis, the enzyme solution was applied to a HiTrap SP HP column (5 mL, GE Healthcare Japan, Tokyo, Japan) pre-equilibrated with buffer A (50 mM phosphate buffer, pH 7.0) and eluted with buffer B (50 mM phosphate buffer, containing 1 M NaCl, pH 7.0). The concentration of the eluted proteins was measured by the Bradford method or UV absorbance.

[0102] Activation of recombinant prococcusanase by treatment with enterokinase The fraction of proCCN mutant protein obtained by cation exchange chromatography was concentrated to a solution containing approximately 1 mg / mL protein at 4°C using an Amicon Ultra-15 (10K, Merck Millipore, Darmstadt, Germany). The temperature was shifted to 25°C or 37°C for autoprocessing to activate the enzyme, and the reaction was stopped by freezing at each reaction time [Non-Patent Document 3]. The reaction solution was then analyzed by SDS-PAGE.

[0103] In the case of treatment with enterokinase, the proCCN mutant protein (0.1 mg of protein) was treated with enterokinase solution (1 mg / mL, 15 μL) overnight at 25 °C. The reaction mixture was further treated with EKaptureTM agarose (50 μL) pre-equilibrated with 0.2 M Tris / HCl (pH 7.4), 20 mM CaCl2, and 0.5 M NaCl, and centrifuged at 10,000 rpm for 1 minute at 4 °C. The supernatant was analyzed by SDS-PAGE. Furthermore, the prepared protein was identified by mass spectrometry and amino acid analysis (data not shown). The protein solution was aliquoted and lyophilized, and stored at 4 °C until use.

[0104] <Casein zymography> The protease activity of the recombinant protein was measured by casein zymography [Non-Patent Document 17]. Briefly, the refolded protein was electrophoresed at 4 °C using a 15% polyacrylamide gel containing SDS and casein (1 mg / mL). The sample was not boiled, mixed with the same amount of SDS sample buffer (50 mM Tris / HCl, 4.5% SDS, 9% glycerol, 0.2% bromophenol blue, pH 7.0), and loaded onto the stacking gel for separation. After electrophoresis, the gel was shaken twice with 2.5% Triton X-100 for 30 minutes to remove SDS, further shaken with 50 mM Tris / HCl (pH 8.0) for 90 minutes, and the enzyme reaction was carried out at room temperature. The gel was stained with Coomassie Brilliant Blue R250 (60 mg / L) in 10% acetic acid containing 10% isopropanol and destained with water.

[0105] <Measurement of enzyme activity using Bz-Arg-OEt> The enzyme activity of the recombinant enzyme was estimated according to a previously published method [Non-Patent Document 8, Non-Patent Document 9]. Briefly, the enzyme reaction was carried out in 50 mM Tris / HCl (1.0 mL, pH 8.0) containing 25 mM NaCl and 1 mM EDTA as follows: purified enzyme (approximately 1 μmol / 10 μL) was mixed with 990 μL of Bz-Arg-OEt (final concentration: 25-100 μM) and then incubated at 25°C for 5 min. The absorbance at 253 nm was measured every 5 s for 5 min. The initial velocity was plotted as a function of substrate concentration, and the apparent kinetic parameters Km and Vmax were calculated by fitting the experimental points to a Lineweaver-Burk plot

[26] .

[0106] <Reversed-phase high-performance liquid chromatography (RP-HPLC)> The high performance liquid chromatography (HPLC) equipment was a Hitachi ELITE LaChrom system (L2130, Hitachi, Tokyo, Japan) or a Waters M600 multisolvent delivery system (Waters, Milford, MA, USA) equipped with a Hitachi L-3000 detector and a D-2500 chromatographic integrator. Peptides and proteins were separated by RP-HPLC using a Cosmosil 5C18-AR-II column (4.6 × 150 mm, Nacalai tesque, Inc., Kyoto, Japan) and confirmed by MALDI-TOF / MS analysis [Non-Patent Document 18].

[0107] <Matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF / MS)> The molecular weights of the peptides and proteins were measured in positive ion mode using an AXIMA confidence spectrometer (SHIMADZU Co., Kyoto, Japan) [Non-Patent Document 18]. Mass spectrometry of the proteins and peptides was carried out in linear mode using 3,5-dimethoxy-4-hydroxycinnamic acid (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) as a matrix. In a typical measurement, the lyophilized sample (about 0.1 nmol) was dissolved in 0.05% TFAaq / 50% CH3CN (1 μL), mixed with 1 μL of the matrix solution (10 mg / mL), air-dried on a sample plate, and used for MALDI-TOF / MS.

[0108] <Measurement of CD> CD spectra were recorded at 25 °C using a JASCO J720 spectrometer (JASCO Corporation, Tokyo, Japan). The protein purified by cation exchange chromatography was dialyzed against 20 mM sodium phosphate buffer (pH 7.4) containing 20 mM NaCl, and its concentration was measured by ultraviolet absorbance [Non-Patent Document 3].

Industrial Applicability

[0109] In the in silico structural analysis of trypsin, the interaction between the propeptide region and the catalytic region was detected [Non-Patent Document 10]. Therefore, since cocnase has high homology with trypsin, the degradation-inhibited mutant cocnase prepared in the process leading to the recombinant cocnase according to the present invention can be used to examine the folding pathway by the propeptides of proteins not only of cocnase but also of other trypsin-like serine protease family.

Claims

1. A recombinant prococcus nodules in which the 8th X from the N-terminus in the amino acid sequence of SEQ ID NO:3 is lysine (K), and the 63rd, 131st, and 133rd Xs are replaced with amino acids other than arginine (R: Arg) and lysine (K: Lys).

2. 2. The recombinant prococcusase according to claim 1, wherein the 8th X from the N-terminus is substituted with an amino acid other than arginine (R: Arg) and lysine (K: Lys).

3. A recombinant cocoonase obtained from the recombinant prococoonase according to claim 1.

4. A recombinant cocoonase obtained from the recombinant prococoonase according to claim 2.

5. A washing solution for raw silk containing the recombinant prococcus enzyme according to claim 1 or 2.

6. A washing solution for raw silk containing the recombinant cocoonase according to claim 3 or 4.

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