JP2016052302A - Chymotrypsin-like protease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel protease which is derived from a queen bee larva of honeybee, decomposes royal jelly to obtain functional peptide, and a manufacturing method of a product of proteolysis using the enzyme.SOLUTION: Chymotrypsin-like protease consists of (A) polypeptide made up of a specific amino acid sequence, (B) polypeptide which is made up of amino acid sequence in which 1 or several pieces of amino acid out of the specific amino acid sequence are deleted, substituted or added, and has chymotrypsin activity, or (C) polypeptide made up of an amino acid sequence having 90% or more of sequence identity with the specific amino acid sequence, and has chymotrypsin activity. The chymotrypsin-like protease has chymotrypsin activity which is inhibited by phenylmethylsulfonyl fluoride and tosyl-L-phenylalanine chloromethyl ketone, but not inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride.SELECTED DRAWING: None

Description

  The present invention relates to a novel chymotrypsin-like protease expressed in larvae of bees (Apis mellifera).

  A bee is a social insect with several castes. A queen bee or a worker bee is a caste determined by food during the larval stage. Royal Jelly (RJ) is a nutrient for queen bees and larvae, but is secreted from the hypopharyngeal and submandibular glands of worker bees and plays an important role in the differentiation of queen bees. RJ is a protein-rich food (about 40% of dry mass), and it has recently been reported that the 57 kDa protein (royalactin) in RJ is extremely important for queen differentiation (Non-patent Document 1). On the other hand, several functional activities of RJ for human health have also been reported. RJ is commonly used as a health food in many countries. In addition, RJ protein-derived peptides produced by commercially available proteases have an antioxidant action (Non-Patent Documents 2 to 4), an antihypertensive action (Non-Patent Documents 5 and 6), an anti-osteoporosis action, as well as a sarcopenia prevention action. Various properties including non-patent document 7 and cholesterol lowering action (non-patent document 8) are shown. However, it is not yet clear how the queen bee and larvae actually digest RJ.

  Several digestive proteases such as trypsin, chymotrypsin, cysteine protease, aminopeptidase, and carboxypeptidase have been found in insects (Non-patent Document 9). Several reports suggest that trypsin activity or chymotrypsin activity plays an important role in the food digestion of bees. For example, Burgess et al. Reported that oral intake of Kunitz soybean trypsin inhibitor (SBTI) increased worker bee mortality and reduced trypsin activity, chymotrypsin activity, and elastase activity in bee midgut. (Non-Patent Document 10). Correspondingly, the bee larvae that gave 1.0% STBI in the food showed high mortality and low weight even when they became adults (Non-patent Document 11). In addition, Giebel et al. Found four endopeptidases from the midgut of an adult bee worker (12). One of them was similar to bovine trypsin and the other three were similar to bovine chymotrypsin. Only one of these proteases was found in queen and adult bee larvae. The protease found in the queen bee adults and the worker bee larvae is a protease having a molecular weight of about 30 kDa having chymotrypsin-like properties, such as tosyl-L-phenylalanine chloromethyl ketone (TPCK) and phenylmethylsulfonyl fluoride (PMSF). However, trypsin substrates (BAEE and BANA) and chymotrypsin substrates (ATEE, APNE, and GlupNa) could not be digested. No trypsin-like protease obtained from adult worker bees was found in queen bees and larvae. Dahlmann et al. Examined changes in chymotrypsin, trypsin, and aminopeptidyl protease activity during the development of worker bees, for example, in larvae, pupae, and adults before and after capping the nest ( Non-patent document 13). They showed that the three protease activities change at each developmental stage within the same caste. They also showed that chymotrypsin activity during the larval stage was reduced when no more food was needed after capping the nest.

  These prior art reports suggest that chymotrypsin enzymes may play an important role in the digestion of food by bee larvae. There are 44 serine type proteases and 12 serine type protease homologs in the bee genome (Non-patent Documents 14 and 15). However, there is little useful information about the expression and function of the proteases encoded by these honey bee serine protease genes.

  The composition of bee digestive proteases varies depending on caste, stage (larvae or adults), and age, mainly due to food differences. Previous reports on bee digestive proteases include trypsin-like proteases and chymotrypsin-like proteases in the digestion of adult worker bee food (Non-Patent Documents 12, 13, 16). Most of the insect chymotrypsin-like enzymes isolated from the midgut of adults and larvae have an optimum pH within the range of pH 8-9 (Non-patent Document 9). The intestinal pH of adult honeybees is weakly acidic and almost neutral in larvae (Non-patent Document 17), but chymotrypsin-like and trypsin-like enzymes of honeybee adults are also highly active in the range of pH 7-9. (Non-Patent Documents 16 and 18). In addition, ethylenediaminetetraacetic acid (EDTA) is a typical metalloprotease inhibitor. 23), and some chymotrypsin-like or trypsin-like digestive enzymes such as scorpion (24) are reported to be weakly inhibited by EDTA. Moreover, the chymotrypsin-like enzyme of the larvae (Non-patent Document 20) and Kamiari (Non-patent Document 25) of the beetle was not affected by both N-tosyl-L-lysine chloromethyl ketone (TLCK) and TPCK. In addition, the ant chymotrypsin is remarkably similar to the chymotrypsin found in homologs derived from different mammalian systems, but it has also been reported that there are differences (Non-patent Document 26). However, there have been few reports on bee queen bee or larval digestive proteases.

  Recently, the present inventors have reported three protease fractions capable of digesting RJ protein, including carboxypeptidase A-like activity and chymotrypsin-like activity, derived from queen bee larvae of 2-3 days old (non-patented). Reference 27).

Kamakura, Nature, 2011, vol.473, p.478-483. Guo et al., Journal of Nutritional Science and Vitaminology, 2008, vol.54, p.191-195. Guo et al., Food Chemistry, 2009, vol.113, p.238-245. Nagai et al., Journal of Medicinal Food, 2006, vol.9, p.363-367. Tokunaga et al., Biological and pharmaceutical bulletin, 2004, vol.27, p.189-192. Matsui et al., Journal of Nutritional Biochemistry, 2002, vol. 13, p.80-86. Hidaka et al., Evidence-Based Complementary and Alternative Medicine, 2006, vol.3, p.339-348 Nakasa et al., Nippon Shokuhin Kagaku Kogaku Kaishi, 2003, vol.10, p.463-467. Terra et al., Comparative Biochemistry and Physiology, 1994, vol.109B, p.1-62. Burgess et al., Journal of Insect Physiology, 1996, vol.42, p.823-828. Brodsgaard et al., Apidologie, 2003, vol.34, p.139-145. Giebel et al., Comparative Biochemistry and Physiology, 1971, vol.38B, p.197-210. Dahlmann et al., Insect Biochemistry, 1978, vol. 8, p. 203-211. Honeybee Genome Sequencing Consortium, Nature, 2006, vol.443, p.931-949. Zou et al., Insect Biochemistry and Molecular Biology, 2006, vol.15, p.603-614. Moritz et al., Journal of Insect Physiology, 1987, vol.12, p.923-931. Herbert et al., Apidologie, 1983, vol.14, p.191-196. Jimenez et al., Apidologie, 1989, vol. 20, p.287-303. Vinokurov et al., Comparative Biochemistry and Physiology Part B, 2006, vol.145, p.126-137. Elpidina et al., Biochimie, 2005, vol.87, p.771-779. Tsybina et al., Biochemistry (Moscow), 2005, vol.70, p.370-377. Wagner et al., Insect Biochemistry and Molecular Biology, 2002, vol.32, p.803-814. Herrero et al., Insect Biochemistry and Molecular Biology, 2005, vol.35, p.1073-1082. Louati et al., Lipids in Health and Disease, 2011, vol.10, p.121 Whitworth et al., Journal of Biological Chemistry, 1998, vol.273, p.14430-14434. Botos et al., Journal of Molecular Biology, 2000, vol.298, p.895-901. Matsuoka et al., Apidologie, 2012, vol.43, p.685-697. Moore, Journal of Biological Chemistry, 1968, vol.243, p.6281-6283. Shevchenko et al., Analytical Chemistry, 1996, vol.68, p.850-858. Jones et al., Science, 2004, vol.305, p.402-404.

  An object of the present invention is to provide a novel proteolytic enzyme that is derived from a bee queen bee larva and is suitable for degrading royal jelly to obtain a functional peptide, and a method for producing a proteolysate using the enzyme. .

  As a result of diligent research to solve the above-mentioned problems, the present inventors have separated a fraction containing carboxypeptidase-like activity and chymotrypsin-like activity from a honeybee queen larva homogenate, and a chymotrypsin-like protease contained in the fraction The present invention was completed by identifying (HCLPASE) and analyzing its enzyme activity.

That is, the present invention provides the following methods for producing chymotrypsin-like protease and protein degradation product.
[1] (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) a polypeptide having an amino acid sequence in which one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 are deleted, substituted or added, and having chymotrypsin activity, or (C) SEQ ID NO: 1 A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented, and having chymotrypsin activity,
A chymotrypsin-like protease having a chymotrypsin catalytic domain consisting of
[2] Chymotrypsin according to the above [1], which has chymotrypsin activity that is inhibited by phenylmethylsulfonyl fluoride and tosyl-L-phenylalanine chloromethyl ketone but not 4- (2-aminoethyl) benzenesulfonyl fluoride. Like protease.
[3] A method for producing a protein degradation product, wherein the protein is degraded at a pH of 9 to 12 using the chymotrypsin-like protease of [1] or [2] as a catalyst.
[4] The method for producing a protein degradation product according to [3], wherein the protein is royal jelly.

  The chymotrypsin-like protease according to the present invention is an enzyme derived from a honey bee queen larva having RJ as a main nutrient source. Therefore, the chymotrypsin-like protease is particularly useful as an RJ degrading enzyme.

In Example 1, it is the figure which showed the result of SDS-PAGE of the fraction (fraction I) which shows carboxypeptidase-like activity from the queen larva of a bee, and the fraction (fraction II) which shows chymotrypsin-like activity. In Example 2, the result of the SDS-PAGE analysis of the recombinant GST tag-chymotrypsin-like protease expressed in E. coli is shown. In Example 2, the result of having investigated the effect of pH and temperature with respect to the chymotrypsin activity of recombinant chymotrypsin-like protease is shown. In Example 2, the measurement result (relative value) of the chymotrypsin activity with respect to each substrate of recombinant chymotrypsin-like protease is shown. In Example 3, it is the figure which showed the result of SDS-PAGE of recombinant HCLPASE expressed with colon_bacillus | E._coli and an insect cell (SF9 cell). In Example 3, the result of having investigated the influence with respect to pH of the enzyme activity of the recombinant HCLPASE expressed in colon_bacillus | E._coli and an insect cell is shown. In Example 3, the result of having investigated the influence with respect to the temperature of the enzyme activity of recombinant HCLPASE expressed in colon_bacillus | E._coli and an insect cell is shown. In Example 3, the measurement result of the specific activity of chymotrypsin activity with respect to each substrate of the recombinant HCLPASE expressed in E. coli and insect cells is shown. In Example 3, the result of investigating the influence of each inhibitor on the chymotrypsin activity of recombinant HCLPASE expressed in Escherichia coli and insect cells is shown. In Example 4, the result of investigating the time-dependent change of degradation of RJ protein by recombinant HCLPASE expressed in Escherichia coli and insect cells is shown. In Example 5, the result of investigating the time-dependent change of degradation of RJ protein by the recombinant HCLPASE expressed in insect cells and the crude queen larvae enzyme is shown. In Example 6, the result of investigating the time-dependent change of RJ protein degradation by recombinant HCLPASE expressed in insect cells in the presence of various concentrations of SDS is shown. In Example 6, the result of investigating the chymotrypsin activity of recombinant HCLPASE expressed in insect cells in the presence of various concentrations of SDS is shown. In Example 7, the result of having investigated the time-dependent change of the body weight of the queen bee larva is shown. In Example 7, the result of having investigated the time-dependent change of the mRNA expression level of HCLPASE in the queen bee larva is shown.

[Chymotrypsin-like protease]
The present inventors thought that the bee queen larva would have an efficient protease that digests RJ to produce a functional peptide. Therefore, as shown in the examples below, the honey bee queen larvae homogenate was subjected to cation exchange column chromatography and gel filtration column chromatography to separate the fraction showing carboxypeptidase activity and the fraction showing chymotrypsin activity. The carboxypeptidase B-like protease and the chymotrypsin-like protease contained in were isolated and identified. The identified carboxypeptidase B-like protease was a protein (XP — 623727) that was estimated to be a carboxypeptidase B-like protease from the nucleotide sequence information revealed by the bee genome analysis. The identified chymotrypsin-like protease was a protein (XP — 394370, gi | 48095159) that was also estimated to be a chymotrypsin-like protease from the nucleotide sequence information of the bee genome. These two types of proteases were first discovered as proteases that are actually expressed and functioning in honey bee queen larvae. The inventors have named the identified chymotrypsin-like protease as honeybee chymotrypsin-like protease (HCLPASE).

That is, the chymotrypsin-like protease according to the present invention is a chymotrypsin-like protease having a chymotrypsin catalytic domain consisting of any one of the following (A) to (C): (A) a polycrystal consisting of an amino acid sequence represented by SEQ ID NO: 1 Peptide (HCLPASE),
(B) a polypeptide having an amino acid sequence in which one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 are deleted, substituted or added, and having chymotrypsin activity, or (C) SEQ ID NO: 1 A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented and having chymotrypsin activity.

  In the polypeptide (B), the number of amino acids deleted, substituted or added to the amino acid sequence represented by SEQ ID NO: 1 is preferably 1-20, more preferably 1-10. ~ 5 are more preferred. In each amino acid sequence, the position of the amino acid to be deleted, substituted or added is not particularly limited as long as the polypeptide comprising the deleted amino acid sequence has chymotrypsin activity.

  In the polypeptide (C), the sequence identity with the amino acid sequence represented by SEQ ID NO: 1 is not particularly limited as long as it is 90% or more, but is preferably 93% or more, and more preferably 95% or more. Is more preferable, and it is still more preferable that it is 98% or more. The sequence identity between amino acid sequences can be determined using various homology search software known in the art.

  According to PROSITE (http://prosite.expasy.org/), the enzyme active site of HCLPASE is the 41st histidine residue (His-41), the 87th in the amino acid sequence represented by SEQ ID NO: 1. It is presumed to be an asparagine residue (Asn-87) and a 178th serine residue (Ser-178). For this reason, in the polypeptides (B) and (C), the amino acids that are substituted for the amino acid sequence represented by SEQ ID NO: 1 are other than the 41st, 87th, and 178th amino acids.

  In the present invention, chymotrypsin activity means hydrolase activity using N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide (SAAPFpNA) as a substrate. The chymotrypsin-like protease according to the present invention has a hydrolase activity for SAAPFpNA and also has a hydrolase activity for succinyl-alanyl-alanyl-prolyl-leucine p-nitroanilide (SAAPLpNA). -Has no hydrolase activity on alanyl-alanyl-alanine p-nitroanilide (SAAApNA) and N-benzoyl-DL-arginine p-nitroanilide (BAPA).

  The chymotrypsin-like protease according to the present invention is inhibited by PMSF and TPCK. As the chymotrypsin-like protease according to the present invention, 4- (2-aminoethyl) benzenesulfonyl fluoride (AEBSF), leupeptin (Leupeptin), antipain (Antipain), and pepstatin A (Pepstatin A) inhibit its chymotrypsin activity. Preferably not. PMSF and AEBSF are serine protease inhibitors, TPCK is a chymotrypsin inhibitor, leupeptin and antipain are serine and cysteine protease inhibitors, and pepstatin A is an aspartic protease inhibitor.

The chymotrypsin-like protease according to the present invention preferably exhibits chymotrypsin activity at least at 20 to 50 ° C. and exhibits chymotrypsin activity at 20 to 60 ° C.
The chymotrypsin-like protease according to the present invention preferably exhibits chymotrypsin activity at least at pH 7-9 and exhibits chymotrypsin activity at pH 7-12.

  The chymotrypsin-like protease according to the present invention may be an enzyme consisting only of the chymotrypsin catalytic domain consisting of any of the polypeptides (A) to (C), or may contain other regions. For example, the chymotrypsin-like protease according to the present invention may have other functional domains as long as the chymotrypsin activity derived from the polypeptides (A) to (C) is not impaired.

  For example, the chymotrypsin-like protease according to the present invention may have various tags added to the N-terminus or C-terminus, for example, so that it can be easily purified when produced using an expression system. The tag is widely used in the expression and purification of recombinant proteins such as His tag, SUMO (Small Ubiquitin-related (like) Modifier) tag, HA (hemagglutinin) tag, Myc tag, Flag tag, and GST tag. Can be used. Moreover, you may connect these tags and the chymotrypsin catalytic domain which consists of either of the polypeptide of said (A)-(C) with the peptide linker which can be cut | disconnected by a specific enzyme.

  The chymotrypsin-like protease according to the present invention may be chemically synthesized based on an amino acid sequence, and an expression vector incorporating a polynucleotide containing a base sequence encoding the chymotrypsin-like protease can be used by a protein expression system. May be produced. The protein expression system used may be, for example, an expression system for prokaryotic cells such as Escherichia coli, and is an expression system for eukaryotic cells such as yeast, filamentous fungi, insect cultured cells, mammalian cultured cells, plant cells and the like. May be.

  The polynucleotide encoding the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 in (A) can be obtained, for example, by RT-PCR using mRNA extracted from the queen larvae of bees as a template. In addition, a polynucleotide encoding the polypeptide of (B) or (C) may be artificially synthesized by using a gene recombination technique for introducing a base mutation to the obtained polynucleotide. it can.

  As an expression vector to be introduced into the protein expression system, any expression vector that is usually used depending on the type of the protein expression system to be used can be used. In addition, incorporation of a polynucleotide encoding a chymotrypsin-like protease into an expression vector can be performed by using a well-known gene recombination technique. Moreover, you may use a commercially available expression vector preparation kit.

  The method for producing a transformant using an expression vector is not particularly limited, and can be carried out by a method usually used for producing a transformant. Examples of the method include a PEG (polyethylene glycol) -calcium method, an electroporation method, and a particle gun method.

  The chymotrypsin-like protease according to the present invention does not require refolding after being recovered from the protein expression system, and can efficiently synthesize an enzyme having a higher specific activity, so that the above (A) to (C) A peptide linker in which a His tag or SUMO tag is added to any polypeptide is preferred, and the polypeptide of any one of (A) to (C) and the His tag or SUMO tag can be cleaved by a specific enzyme More preferred are those linked by. For example, an expression vector incorporating a polynucleotide encoding a chymotrypsin-like protease in which any of the polypeptides (A) to (C) and a His tag or a SUMO tag are linked with a cleavable peptide linker, A transformant is produced by introducing it into a host cell of an expression system such as an insect cell or a mammalian cell. The transformant can be cultured and the expressed chymotrypsin-like protease can be recovered. The recovered chymotrypsin-like protease may be used as it is as a proteolytic enzyme or may be used after cleaving the tag.

  The chymotrypsin-like protease according to the present invention can be used as a proteolytic enzyme like other chymotrypsins. The temperature and pH of the enzyme reaction may be within a range in which the chymotrypsin-like protease according to the present invention can exhibit chymotrypsin activity. For example, at pH 9-12, the protein can be degraded by contacting the protein with the chymotrypsin-like protease according to the present invention as a substrate to produce a protein degradation product. The temperature of the proteolytic reaction may be performed at 20 to 40 ° C., but is preferably performed at 40 to 60 ° C. because the chymotrypsin-like activity of the chymotrypsin-like protease according to the present invention is higher.

  The chymotrypsin-like protease according to the present invention is a digestive enzyme expressed in honey bee queen larvae, and can be expected to be an efficient protease that digests RJ to produce a functional peptide. For this reason, the chymotrypsin-like protease according to the present invention is particularly preferably used for the degradation of RJ. In the degradation of RJ, the chymotrypsin-like activity of the chymotrypsin-like protease according to the present invention can be further enhanced. Therefore, it is preferably performed in the presence of a low concentration of an anionic surfactant. ) In the presence of 0.01 to 0.05% of SDS.

  It is not yet clear how the bee queen and larvae digest RJ and use it for its development and physiology. The chymotrypsin-like protease according to the present invention is a useful tool in analyzing the development and differentiation of bees. In addition, the chymotrypsin-like protease according to the present invention is useful for clarifying the mechanism of RJ function as a health food and for developing functional value.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
A protease derived from a queen bee larvae, specifically, a protease in a protease fraction containing chymotrypsin activity and carboxypeptidase activity obtained from queen bee larvae disclosed in Non-patent Document 27 was isolated and identified.
For mass analysis of honey bee larvae protease, as described in Non-Patent Document 27, the protein of honey bee queen larvae was first separated by cation exchange and gel filtration chromatography. The activity of the fraction containing the fraction containing chymotrypsin activity (Fraction I) and the fraction containing carboxypeptidyl activity (Fraction II) was determined using SAAPFpNa or N-carbobenzooxy-glycyl-L-phenylalanine (Z-Gly-Phe), respectively. Determined as substrate. Fractions were separated by SDS-PAGE. Details are shown below.

<Sample preparation>
2-3 days old bee queen larvae were obtained from Akitaya Honten Co., Ltd. (Gifu Prefecture, Japan). The bee queen larva was homogenized and filtered using a nylon mesh. The homogenate was diluted with an equal volume of 50 mM phosphate buffer (pH 7.0), and then centrifuged at 10000 × g and 4 ° C. for 20 minutes. The transparent yellowish intermediate layer was collected, filtered through a glass fiber filter paper (manufactured by Advantec, Tokyo (Japan)), and then a 0.20 μm cellulose acetate filter (manufactured by Advantec), and the crude enzyme solution (LC) was obtained.

<Column chromatography>
The protease was purified by cation exchange column chromatography and gel filtration column chromatography. The pH value of the cation exchange column chromatography was examined by a preliminary experiment in order to recover an enzyme exhibiting a higher specific activity. An equivalent amount of 5 mL of the crude enzyme solution was loaded onto a 1 mL HiTrap SP HP column (GE Healthcare, Little Chalfont, UK) equilibrated with 20 mM phosphate buffer (pH 7.6). The column was washed with 20 mM phosphate buffer (pH 7.6) 5 times the column volume, and then the protein was eluted with 20 mM phosphate buffer (pH 7.6) containing 0.5 M NaCl. Gel filtration column chromatography was performed with the AKTAprime system (GE Healthcare). The eluate from the cation exchange column was loaded onto a HiLoad 16/600 superdex 75 pg column (1.6 × 60 cm; GE Healthcare) equilibrated with 50 mM phosphate buffer (pH 7.0) containing 0.15 M NaCl. The protein was detected with absorbance at 280 nm, eluting with the same buffer at a flow rate of 0.7 mL / min with a fraction volume of 2 mL.

<Chymotrypsin-like protease assay>
Chymotrypsin protease activity was determined by hydrolysis using SAAPFpNA (Sigma Aldrich, St. Louis, MO) as a substrate. The reaction mixture contained 386 μL of 50 mM Tris-HCl buffer (pH 9.0), 2 μL of 50 mM SAAPFpNA in dimethylformamide, and 10 μL of sample and incubated at 37 ° C. for 10 minutes. The reaction was stopped by adding 20 μL of glacial acetic acid. The absorbance of free p-nitroaniline was measured at 405 nm. One unit of activity corresponds to the amount of enzyme that liberates 1 μmol of p-nitroaniline per minute under standard assay conditions.

<Carboxypeptidase-like protease assay>
Carboxypeptidase activity was determined using Z-Gly-Phe (Sigma Aldrich) as a substrate. The reaction mixture contained 190 μL of 1 mM Z-Gly-Phe dissolved in Tris-HCl buffer (pH 9.0) containing 0.1 M NaCl and 10 μL of sample, and incubated at 37 ° C. for 30 minutes. The reaction was stopped by adding 600 μL of 0.4 M acetate buffer (pH 5.0). The absorbance of free phenylalanine was determined by ninhydrin assay (Non-patent Document 28). 100 μL of sample was collected in a separate tube and 100 μL of ninhydrin reagent (2% solution, Sigma Aldrich) was added. Samples were incubated at 100 ° C. for 15 minutes. After incubation, 1 mL of ice-cold ethanol was added to the sample. The absorbance of free phenylalanine was determined at 570 nm. One unit of activity corresponds to the amount of enzyme that liberates 1 μmol of phenylalanine per minute under standard assay conditions.

<SDS-PAGE>
Proteins in the fraction containing carboxypeptidase activity (Fraction I) and the fraction containing chymotrypsin activity (Fraction II) recovered by column chromatography were separated by SDS-PAGE.

  The result of SDS-PAGE of fraction I is shown in FIG. 1 (A), and the result of SDS-PAGE of fraction II is shown in FIG. 1 (B). Among the proteins contained in fraction I separated by SDS-PAGE, the bands with numbers 1 to 7 in FIG. 1 (A) indicate protein spots excised for ESI-ion trap MS. . Similarly, among the proteins contained in fraction II separated by SDS-PAGE, the bands labeled Nos. 1 to 7 in FIG. 1 (B) are electrospray ionized ion trap mass spectrometry (ESI−). The protein spots excised for ion trap MS) are shown.

<Mass spectrometry and database search>
After SDS-PAGE, the excised protein spots were digested in gel with trypsin (Non-patent Document 29). Samples were separated using a nanoflow multidimensional HPLC system (Paradigm MS4; Michrom BioResources, Auburn, Calif.) And under optimal conditions (ESI-ion trap MS (LCQDECAXP; Thermo Fisher Scientific, Waltham, Mass.)) (Capillary temperature, 200 ° C .; capillary pressure, 3.0 V; and spray pressure, 1.90 kV). Peptide sequences were searched against the protein database (MSDB) using MASCOT software (Matrix Science Ltd., London, UK).

  Each protein obtained from the SDS-PAGE gel was analyzed by LC-MS / MS. Table 1 shows the results of identification of proteases in the carboxypeptidase-like protease fraction and chymotrypsin-like protease fraction. From the bee genome database, No. 1 was approximately 40 kDa in fraction I. 4 band (FIG. 1 (A)) was identified as a carboxypeptidase B-like protease (XP — 623727) and No. 2 was approximately 25 to 28 kDa in fraction II. One band (FIG. 1 (B)) was identified as chymotrypsin-like protease (XP — 394370) (SEQ ID NO: 1). These results were consistent with the results of the SAAPFpNa or Z-Gly-Phe enzyme assay.

<N-terminal amino acid sequence>
After SDS-PAGE, the fraction II protein was transferred to a PVDF membrane by semi-driving, and each band was cut out. The N-terminal amino acid sequence of chymotrypsin-like protein was analyzed from the transcribed PVDF membrane pieces using a peptide sequencer (Model 491; Applied Biosystems, Foster City, Calif.).

  That is, two types of proteases that can hydrolyze carboxypeptidase substrate Z-Gly-Phe and chymotrypsin substrate SAAPFpNa, respectively, from fractions isolated from 2-3 days old queen bee larva homogenates. Identified. Mass spectrometry and MASCOT searches suggested that these activities were derived from a protein similar to bee carboxypeptidase B (XP — 623727) or chymotrypsin (XP — 394370) (FIG. 1, Table 1).

[Example 2]
The identified chymotrypsin-like protease was expressed by an E. coli expression system and functional analysis was performed.

<Target DNA amplification and recombinant vector construction>
Total RNA of bee queen larvae was prepared from whole larvae using FastPure RNA kit (Takara, Shiga Prefecture, Japan). Total RNA was stored at −80 ° C. until use. cDNA was prepared from total RNA using oligo (dT) primers (Toyobo, Osaka, Japan) and reverse transcriptase (ReverTraAce, Toyobo). Mix 1 μg of total RNA with 0.5 μL oligo (dT) primer, 4 μL 4 × buffer, 8 μL 2.5 mM dNTP mixture, 1 μL RNase inhibitor, and 1 μL RiverTraace at 37 ° C. The reverse transcription reaction was performed by incubating for 30 minutes. After the reverse transcription reaction, the reaction solution was heated at 99 ° C. for 1 hour to inactivate the enzyme. The bee chymotrypsin-like protease DNA is composed of a sense primer containing a SalI restriction enzyme site (5′-AAA GTC GAC ATT GTT GGC GGC AGT GAT G-3 ′; the underlined portion is a restriction enzyme site) (SEQ ID NO: 2) and EcoRI. Amplified by PCR using an antisense primer containing a restriction enzyme site (5′-AAAAA GAA TTC TAA GAA ATG TAG AAA CCG TCT GCT G-3 ′; the underlined part is a restriction enzyme site) (SEQ ID NO: 3) . PCR conditions are as follows: 1 cycle at 98 ° C. for 30 seconds, 10 cycles at 98 ° C., 15 seconds at 58 ° C., 30 cycles at 72 ° C. for 20 seconds, and 1 cycle at 72 ° C. for 5 minutes . As a result, a PCR product containing a base sequence portion encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 was obtained.

  To construct recombinant E. coli, PCR products and Gateway® pENTR® 1A vector (Life Technologies, Carlsbad, Calif.) Were digested with SalI and EcoRI, and T4 DNA ligase (New England Biolabs, Ipswich, Massachusetts). Ligated entry clones were transformed into competent E. coli DH5α strains and selected on LB media plates containing 50 μg / mL kanamycin. The coding region of the entry clone chymotrypsin-like protein was converted to the pDEST15 vector by LR Klonase (Invitrogen) according to the manufacturer's instructions. Recombinant pDEST15 vector was transformed into competent E. coli DH5α strain and selected on LB media plates containing 100 μg / mL ampicillin. Positive clones were selected by PCR and nucleotide sequence analysis (ABI Genetic Analyzer PRISM 3100, Applied Biosystems).

<Expression of recombinant protein>
The recombinant protein was incorporated into a recombinant vector and then expressed in E. coli BL21 (DE3). Recombinant E. coli was cultured at 20 ° C. in LB medium containing 100 μg / mL ampicillin and 200 μM IPTG (isopropyl β-D-1-thiogalactopyranoside). E. coli was recovered by centrifugation at 5000 × g and 4 ° C. for 10 minutes.

<Purification of recombinant protein>
The expressed protein was refolded using Rapid GST Inclusion Body Solubilization and Regeneration Kit (Cell Biolabs, San Diego, Calif.). E. coli was resuspended in a solution containing 0.2 mg / mL lysozyme and 1 mM dithiothreitol in the STE buffer included in the kit, and then lysed by sonication on ice. Each E. coli lysate was subjected to SDS-PAGE and compared to lysate without IPTG to confirm recombinant protein expression. Furthermore, in order to confirm the structure of inclusion bodies, the E. coli lysate was centrifuged at 12000 × g for 15 minutes at 4 ° C. to separate the soluble protein fraction and the insoluble protein fraction, and the protein composition was analyzed by SDS-PAGE. Examined. Inclusion bodies were solubilized and refolded using the kit according to the manufacturer's instructions. That is, since recombinant GST-chymotrypsin was expressed as an insoluble protein, it was solubilized and refolded using Rapid GST Induction Body Solubilization and Regeneration Kit (Cell Biolabs, San Diego, Calif.).

  The refolded recombinant GST protein sample was filtered through a 0.2 μm membrane filter (Millipore, Billerica, Mass.). An equivalent amount of 500 μL of glutathione sepharose 4B beads (50% slurry, GE Healthcare) was added to 10 mL of the sample solution, and incubated at room temperature while rotating. The beads were collected by centrifugation at 500 × g for 5 minutes at room temperature, and the supernatant was discarded. This washing step was repeated a total of 3 times. Recombinant protein was cleaved from the GST tag in 200 μL of PBS solution containing 25 U / mL thrombin (GE Healthcare) for 16 hours at room temperature. Recombinant protein was recovered by centrifugation at 500 × g for 5 minutes at room temperature, and 200 μL of PBS was again added to the beads and centrifuged, and the two supernatants were mixed. The protein concentration in the recombinant protein sample was measured by the Bradford method using Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, Calif.) And BSA as a standard.

  FIG. 2 (A) shows the results of SDS-PAGE analysis using CBB staining of recombinant chymotrypsin-like protease extracted from E. coli lysate. In the left panel, each lane represents total E. coli lysate with and without IPTG stimulation. In the right panel, each lane shows the soluble and insoluble fractions of IPTG-stimulated E. coli lysate and the protein purified by glutathione sepharose 4B beads. As shown in FIG. 2A, expression of a protein having a molecular weight of about 53 kDa was induced by IPTG. From the amino acid sequence information, the molecular weight of the identified chymotrypsin-like protease was predicted to be about 26 kDa, and conjugated with a 26 kDa GST tag, so that the molecular weight of the recombinant chymotrypsin-like protease was predicted to be about 52 kDa. That is, the protein having a molecular weight of about 53 kDa, whose expression was induced by IPTG, was the target recombinant chymotrypsin-like protease (recombinant GST-chymotrypsin).

  After refolding, the GST tag was cleaved by thrombin from GST-tagged chymotrypsin (solubilized chymotrypsin) recovered using glutathione sepharose 4B beads. FIG. 2B shows the results of SDS-PAGE analysis using silver staining of the recombinant protein after thrombin treatment. As shown in FIG. 2 (B), the size of the obtained protein coincided with the expected size of about 26 kDa.

<Optimum pH and temperature for the hydrolysis reaction of SAAPFpNa>
The recombinant chymotrypsin-like protease with the GST tag cleaved was examined in the same manner as in Example 1 for the optimum pH and temperature of chymotrypsin-like protease activity using SAAPFpNa as a substrate.
Specifically, the optimum pH for enzyme activity is 50 mM pH 4, 5, and 6 citrate buffer, pH 6, 7, and 8 50 mM phosphate buffer, pH 8 and 9 50 mM Tris-HCl buffer, and Measurements were made using 50 mM glycine-NaOH buffer at pH 9 and 10. Enzyme activity was measured at 37 ° C. using SAAPFpNa as a substrate.
The optimum temperature was measured at 20 ° C., 30 ° C., 37 ° C., 40 ° C., 45 ° C., and 50 ° C. using 50 mM Tris-HCl buffer (pH 9.0) and SAAPFpNA.

  FIG. 3 (A) shows the pH dependence of the protease activity of recombinant chymotrypsin-like protease. In FIG. 3 (A), the results for 50 mM citrate buffer (pH 4, 5, and 6) are indicated by white dots and black lines, and the results for 50 mM phosphate buffer (pH 6, 7, and 8) are indicated by dark gray dots and lines. The results for 50 mM Tris-Hcl buffer (pH 8 and 9) are indicated by light gray dots and lines, and the results for 50 mM glycine-NaOH buffer (pH 9 and 10) are indicated by black dots and dotted lines, respectively. Data were expressed as mean ± SD. Protease activity using the recombinant chymotrypsin-like protease SAAPFpNa as a substrate showed maximum activity at pH 9.0. The activity at pH 7 was about half that at pH 9.0.

  FIG. 3B shows the temperature dependence of the protease activity of the recombinant chymotrypsin-like protease. Data were expressed as mean ± SD. The protease activity using the recombinant chymotrypsin-like protease SAAPFpNa as a substrate was highest at 40 ° C. Recombinant chymotrypsin-like protease showed maximum activity at alkaline pH, and the enzyme was not cold compatible and heat resistant.

  That is, the 26 kDa chymotrypsin-like protease produced by E. coli (FIG. 2) showed chymotrypsin activity. The activity was highest at pH 9 and 40 ° C. (FIG. 3). The optimal temperature of the recombinant chymotrypsin-like protease is such that the temperature of the bee larvae is such that the temperature of the bee colonies is maintained at approximately 35 ° C. for normal brood development (Non-patent Document 30). It seemed to be consistent with the growth environment. Furthermore, the optimum pH of the enzyme was in the alkaline region. Non-Patent Document 9 and Non-Patent Documents 16 to 18 suggest that the optimum pH of the larval digestive enzyme is in the alkaline region, but the pH dependence of the recombinant chymotrypsin-like protease derived from the honey bee larva according to the present invention. Was consistent with these reports.

<Substrate specificity of recombinant chymotrypsin-like protease>
In order to investigate the substrate specificity of recombinant chymotrypsin-like protease, the activity of several serine proteases on substrates was examined. As substrates, SAAPFpNA, SAAPLpNA, SAAApNA, and BAPA were used. Enzyme activity was measured for each substrate at pH 9, 37 ° C.
Specifically, 10 μL of a sample (recombinant chymotrypsin-like protease) solution and 90 μL of 50 mM Tris-HCl buffer (pH 9.0) containing 1 mM of each substrate were added to a 96-well plate and incubated at 37 ° C. With respect to the reaction solution after incubation, a change in absorbance at 405 nm was measured. One unit of protease activity corresponded to the amount of enzyme that liberates 1 μmol of p-nitroaniline per minute.

  The measurement results of protease activity for each substrate are shown in FIG. Data are shown as specific activity (relative value) against SAAPFpNa (100%). Data were expressed as mean ± SD. As a result, the activity against elastase (SAAPLpNa and SAAApNa) and trypsin (BAPA) was lower than the activity against chymotrypsin substrate. The relative values of activity against SAAPLpNa, SAAApNa, and BAPA were 32.7%, 13.5%, and 8.16% of SAAPLpNa, respectively. These results suggested that the recombinant enzyme is a chymotrypsin protease. In addition, it has been clarified that the recombinant enzyme is different from the queen bee-derived protease described in Non-Patent Document 12 that does not have the activity of degrading the chymotrypsin substrate.

<Inhibition assay>
The effect of various protease inhibitors on the activity of the recombinant protein was measured at 37 ° C. in 50 mM Tris-HCl buffer (pH 9.0) containing 1 mM SAAPFpNA. Inhibition assays include inhibitors as PMSF, TLCK, pepsin, EDTA (Nacalai Tesque, Kyoto, Japan), 4- (2-aminoethyl) AEBSF, TPCK (Sigma Aldrich), antipine, leupeptin, and E- 64 (Peptide Institute Ltd., Osaka, Japan) was used. The effect of protease inhibitors on the hydrolysis reaction of the recombinant protease SAAPLpNa was determined relative to the activity of the sample without inhibitor.

  Table 2 shows the effect of protease inhibitors on recombinant chymotrypsin-like protease. As a result, PMSF strongly inhibited the activity. Furthermore, antipain also showed an inhibitory effect. Both of these inhibitors are inhibitors of serine type protease activity. On the other hand, aspartic protease inhibitor pepstatin A and cysteine protease inhibitor E-64 did not inhibit the activity of the recombinant chymotrypsin-like protease. From these results, the recombinant chymotrypsin-like protease was regarded as a serine protease.

  In addition, EDTA, which is a metalloprotease inhibitor, exhibits weak inhibitory activity against the recombinant chymotrypsin-like protease, and is an inhibitor of AEBSF, which is generally used as a substrate for PMSF, and some serine or cysteine type proteases. Certain leupeptins showed weak inhibitory activity. Furthermore, neither TPCK nor TLCK inhibited the activity of the recombinant protease. These results suggested that the recombinant chymotrypsin-like protease has a different active site from mammalian chymotrypsin.

  That is, the recombinant chymotrypsin-like protease was stronger than the trypsin substrate and the elastase substrate, and hydrolyzed SAAPFpNa, which is a chymotrypsin substrate (FIG. 4). PMSF and antipain inhibited the activity, but pepstatin A and E-64 did not (Table 2). These results suggested that this enzyme is a serine type protease. Furthermore, EDTA showed weak inhibitory activity. On the other hand, TLCK and TPCK did not show inhibitory activity against the recombinant protease. From the results of this inhibition assay, it was considered that the chymotrypsin-like protease according to the present invention has a reaction mechanism different from that of mammalian chymotrypsin. Furthermore, in order to clarify the reaction mechanism, structural analysis such as crystallography of the protease is necessary.

  The inventors have named the recombinant protease the bee chymotrypsin-like protease (HCLPASE). In addition, HCLPASE is the first identified honey bee larvae protease.

[Example 3]
HCLPASE is a recombinant protein in which two types of tags, a SUMO tag that can form a more accurate three-dimensional structure without refolding and a His tag for protein purification, are used instead of the GST tag. And expressed its function.

<Recombinant vector construction and expression of recombinant protein>
For the construction of a recombinant vector, a SUMO tag is linked downstream of a 6 × His tag, and a SUMO tag expression has a multi-cloning site for inserting a polynucleotide encoding a target protein to be expressed downstream of the SUMO tag. The vector used was an E. coli expression vector (pE-SUMOpro vector) and an insect cell expression vector (pI-secSUMOstar vector) (both LifeSensors).

In the same manner as in Example 2, the total RNA bee queen larvae obtained by performing a reverse transcription reaction using as a template cDNA as a template, a sense primer containing a BsaI restriction site (5'- AA G GTC TC A AGG TAT TGT TGG CGG CAG-3 ′; underlined restriction enzyme site) (SEQ ID NO: 4) and HindIII restriction enzyme antisense primer (5′-AAA AAG CTT TTA AGA AAT GTA GAA ACC GTC TGC TG-3 ′; the underlined portion is a restriction enzyme site.) Honey bee chymotrypsin-like protease DNA was amplified by PCR using (SEQ ID NO: 5). PCR conditions were the same as in Example 2.

  The obtained PCR product and the pE-SUMOpro vector were digested with BsaI and HindIII and ligated using T4 DNA ligase to obtain a SUMO tag expression vector for recombinant Escherichia coli. Similarly, the PCR product and the pI-sec SUMOstar vector were ligated to obtain a SUMO tag expression vector for recombinant insect cells.

<Expression of recombinant protein in E. coli and preparation of lysate>
The resulting SUMO tag expression vector for recombinant E. coli was introduced into competent cells of E. coli BL21 (DE3) to obtain recombinant E. coli. Recombinant E. coli was cultured at 20 ° C. in LB medium containing 100 μg / mL ampicillin and 100 μM IPTG to express the recombinant protein. E. coli was recovered by centrifugation at 5000 × g and 4 ° C. for 10 minutes.

  The recovered recombinant E. coli was resuspended in 50 mM phosphate buffer (pH 7.0) containing 0.2 M NaCl, and then sonicated on ice to prepare a recombinant E. coli lysate. Recombinant E. coli lysate was centrifuged at 12000 × g and 4 ° C. for 15 minutes to separate a soluble protein fraction and an insoluble protein fraction.

<Expression of recombinant protein in insect cells>
The obtained SUMO tag expression vector for recombinant insect cells was introduced into E. coli DH10Bac1 competent cells by the heat shock method, and LB medium plate (50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 100 μg / mL X -Gal, containing 40 μg / mL IPTG). White colonies were selected from the obtained colonies and cultured to obtain a recombinant bacmid. The obtained bacmid was introduced into SF9 cells derived from sugarcane ovary using the transfection reagent Cellfectin II reagent (Invitrogen). After the introduction treatment, SF9 cells were cultured in SF900IIISFM medium (Life Technologies) at 27 ° C., and then the culture solution was collected by centrifugation at 500 × g for 5 minutes at room temperature to obtain a recombinant baculovirus stock. To SF9 cells seeded at 2 × 10 ⁶ cells / well in a 6-well plate, add 100 μL of baculovirus stock and incubate at 27 ° C. If virus growth is observed, the culture solution is removed by centrifugation. It was collected. This operation was repeated twice to obtain a high-titer recombinant baculovirus stock. The baculovirus stock was added to and infected with SF9 cells seeded in a 75 cm ² flask at 1.5 × 10 ⁷ cells / flask to express the recombinant protein. The expressed recombinant protein is secreted into the culture medium. Therefore, the culture solution (recombinant baculovirus-infected insect cell culture solution) containing the expressed recombinant protein was recovered by centrifuging the culture solution at 500 × g and room temperature for 5 minutes.

<Purification of recombinant protein>
Only the His-tagged protein was purified from the soluble fraction of the recombinant E. coli lysate and the recombinant baculovirus-infected insect cell culture medium by metal affinity chromatography using TALON resin (Clontech).

  The purified His tag protein was confirmed by SDS-PAGE. The result of SDS-PAGE is shown in FIG. As a result, a band was confirmed at a little less than 45 kDa in both recombinant E. coli and recombinant insect cells. The reason why the recombinant protein expressed in the recombinant insect cells is slightly larger is that the secretory signal protein gp67 (about 5 kDa) is added.

  The purified recombinant protein was activated by cleaving the tag with a SUMO tag cleavage protease (SUMO protease 1 and SUMOstar protease 1) at a concentration of 100 μg / mL. The SUMO protease, the cleaved 6 × His-SUMO tag, and the uncleaved protein were removed by purification with TALON resin again. As a result, 40 to 50 μg / mL purified recombinant protein was obtained.

  The purified recombinant protein was confirmed by SDS-PAGE. The result of SDS-PAGE is shown in FIG. As a result, the size of HCLPase was predicted to be about 26 kDa from the amino acid sequence, but a band of the expected size could be confirmed (black arrow in the figure). Since the recombinant protein expressed in both recombinant E. coli and recombinant insect cells had the same size, it was speculated that HCLPase had no added sugar chain.

  However, in both recombinant E. coli and recombinant insect cells, another band was seen in the vicinity of 40 kDa in addition to the target band (white arrow in the figure). The band did not occur when a serine protease inhibitor (PMSF or SBTI) was added (not shown), and thus was presumed to be due to autolysis by HCLPase.

<Measurement of chymotrypsin activity>
The chymotrypsin activity of the purified recombinant HCLPase was measured in the same manner as in Example 2 under the conditions of pH 9 and 37 ° C. using SAAPFpNa as a substrate. The measurement results of the amount of recovered HCLPase expressed in E. coli and the recombinant HCLPase expressed in insect cells (protein concentration, μg / mL) and chymotrypsin specific activity (U / mg) were obtained by cleaving the GST tag of Example 2. It shows in Table 3 with the result of recombinant HCLPase.

  As a result, when expressed using the SUMO tag, the specific activity of the enzyme was 3-4 times higher in insect cell expression than in E. coli expression. The cause of the difference in specific activity is not known, but in the case of E. coli expression, it has been confirmed that there are a large amount of insoluble protein in inclusion bodies, so it contains an enzyme that is solubilized but not well folded There was a possibility that it was.

  The specific activity of chymotrypsin is clearly higher in the amount of purified protein recovered and the specific activity of the enzyme in recombinant HCLPase cleaved after expression with SUMO tag than in recombinant HCLPase cleaved after expression with GST tag. It was. This was presumably because, unlike the GST tag that required refolding, it was a more native fold when expressed with the SUMO tag, and a more stable protein was obtained. .

<Optimum pH for the hydrolysis reaction of SAAPFpNa>
The effect of pH on the chymotrypsin activity using the SAAPFpNa substrate of recombinant HCLPase cleaved from the SUMO tag was examined in the same manner as in Example 2.
Specifically, the optimum pH for enzyme activity is 50 mM pH 4 and 5 citrate buffer, pH 6 and 7 50 mM phosphate buffer, pH 8 and 9 50 mM Tris-HCl buffer, pH 10, 11, and 12. Measurements were made using 50 mM glycine-NaOH buffer and pH 13 KCl-NaOH. Enzyme activity was measured by performing an enzyme reaction at 37 ° C. for 30 minutes at 1 mM SAAPFpNa, 2 μg / mL enzyme protein.

  FIG. 6 (A) shows the measurement results of the specific activity of recombinant HCLPase expressed in E. coli, and FIG. 6 (B) shows the measurement results of the specific activity of recombinant HCLPase expressed in insect cells. As a result, in both E. coli expression and insect cell expression, the effect of pH on the obtained recombinant HCLPase was almost the same, the activity was high on the alkali side, and the activity was decreased as the pH decreased. When GST tag was expressed, the activity was highest at pH 9 and decreased at pH 10 (FIG. 3 (A)), but when SUMO tag was expressed, the activity was highest at pH 12, and pH 13 The activity was reduced. Since the concentration of the purified protein was low in the GST-tagged expression sample, there was a possibility that inactivation occurred due to autolysis when the activity increased.

<Optimum temperature for the hydrolysis reaction of SAAPFpNa>
The effect of temperature on chymotrypsin activity using SAAPFpNa as a substrate of recombinant HCLPase cleaved from the SUMO tag was examined in the same manner as in Example 2.
Specifically, the optimal temperature for enzyme activity is 1 mM SAAPFpNA, 2 μg / mL enzyme protein, 50 mM Tris-HCl buffer (pH 9.0), and the enzyme reaction at 20 to 70 ° C. for 30 minutes. Performed and measured.

  FIG. 7A shows the measurement result of specific activity (U / mg) at each temperature, and FIG. 7B shows the relative value of specific activity at each temperature when the specific activity at 37 ° C. is 100%. (%) Is shown respectively. As a result, the specific activity of the recombinant HCLPase expressed by insect cells was higher than that of the recombinant HCLPase expressed by E. coli, but the ratio changes with temperature were almost the same. Further, when the GST tag was expressed, the activity was highest at 40 ° C. (FIG. 3B), but when the SUMO tag was expressed, the activity was highest at 60 ° C. However, the enzyme activity did not change significantly at 50-60 ° C.

<Substrate specificity of recombinant HCLPase>
The substrate specificity of recombinant HCLPase was measured in the same manner as in Example 2. As substrates, SAAPFpNA, SAAPLpNA, SAAApNA, and BAPA were used.
Specifically, the enzyme activity was measured by performing an enzyme reaction at 37 ° C. for 30 minutes using 1 mM substrate, 2 μg / mL enzyme protein, and 50 mM Tris-HCl buffer (pH 9.0).

  The measurement result of the specific activity of the recombinant HCLPase expressed in E. coli is shown in FIG. 8 (A), and the measurement result of the specific activity of the recombinant HCLPase expressed in insect cells is shown in FIG. 8 (B). As a result, both recombinant HCLPase expressed in Escherichia coli and recombinant HCLPase expressed in insect cells showed high activity for SAAPFpNa, which is a substrate for chymotrypsin, but low activity for the other three substrates. Of the three, only the activity against SAAPLpNa was slightly higher. These results were the same as when GST tag was expressed (FIG. 4). From this result, it can be said that HCLPase is a chymotrypsin-like enzyme.

<Inhibition assay>
The effect of various protease inhibitors on the activity of recombinant HCLPase was measured in the same manner as in Example 2. The same protease inhibitor as in Example 2 was used.
Specifically, the enzyme activity was determined by using 1 mM SAAPFpNA, 2 μg / mL enzyme protein, 50 mM Tris-HCl buffer (pH 9.0) in the presence of each protease inhibitor at 37 ° C. for 30 minutes. Reaction was performed and measured. The concentration of each protease inhibitor was 1 mM for PMSF, 1 mM for AEBSF, 10 mM for EDTA, 100 μM for TLCK, 100 μM for TPCK, 10 μM for leupeptin, 10 μM for antipain, 10 μM for pepstatin A, and 10 μM for E-64. . The effect of protease inhibitors on the hydrolysis reaction of recombinant HCLPase with SAAPLpNa was determined relative to the activity of samples without inhibitors.

  The effect of protease inhibitors on recombinant HCLPase is shown in FIG. As a result, recombinant HCLPase expressed in Escherichia coli and recombinant HCLPase expressed in insect cells showed high inhibitory activity with the serine protease inhibitor PMSF and chymotrypsin inhibitor TPCK, but AEBSF generally used as an alternative to PMSF. Showed no inhibitory activity. Moreover, when GST tag was expressed, it was not inhibited by TPCK (Table 2), but it was considered that TPCK did not function well due to the effect of folding.

<Confirmation of HCLPase>
Whether or not the obtained recombinant HCLPase was definitely HCLPase (XP — 394370) and whether there was no error in the amino acid sequence was examined by MALDI-TOF-MS and PMF methods of digestion of the recombinant HCLPase in gel with trypsin.

  Specifically, after purifying the recombinant recombinant HCLPase by SDS-PAGE, the separated band was excised from the gel and digested in gel with trypsin at 37 ° C. overnight. Subsequently, the peptide fragment was extracted and subjected to mass spectrometry using MALDI-TOF-MS. MS-digest (http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msdigest) was used to calculate the expected mass of the peptide fragment produced during trypsin digestion of HCLPase, and TOF-MS From the obtained peaks, those having the same mass were picked up. Analysis by MALDI-TOF-MS is performed 5 times, and the peaks corresponding to the calculation by MS-digest are collected 5 times, and the protein is identified by PMF method at MASCOT (http://www.matrixscience.com/search_form_select.html) Went.

  As a result, both the recombinant HCLPase expressed by E. coli and the recombinant HCLPase expressed by insect cells were identified as HCLPase (XP — 394370, gi | 48095159) named by the present inventors. Both expression samples covered almost the same position, and the coverage was 89%. In addition, the 41st histidine residue (His-41), the 87th asparagine residue (Asn-87), and the 178th serine in the amino acid sequence represented by SEQ ID NO: 1 presumed to be the active site The amino acid sequence of the region containing the residue (Ser-178) was consistent with the amino acid sequence (XP — 394370) on the database, and it was considered that there was no amino acid mistranslation near the active center. However, neither the expression of E. coli nor the expression of insect cells detected the VVAHR and LASGSK sequences and the C-terminal GEAPADGFYIS sequence in the amino acid sequence of SEQ ID NO: 1.

[Example 4]
RJ was degraded using the recombinant HCLPase expressed in Escherichia coli purified in Example 3 and the recombinant HCLPase expressed in insect cells.
Specifically, recombinant HCLPase was added to an RJ water-soluble protein solution (1 mg protein / mL) having a pH of 4, 7, 9, or 12 at 1 μg protein / mL and incubated at 37 ° C. Sampling was performed at 1, 3, 6 and 24 hours after the start of incubation, and degradation of RJ protein was examined by SDS-PAGE. An RJ water-soluble protein solution with no enzyme added was used as a control.

  The result of SDS-PAGE is shown in FIG. As a result, the degradation pattern of RJ protein showed no significant difference between E. coli expression and insect cell expression, and as the alkalinity became stronger, degradation was faster overall. At pH 4, several distinct bands that appeared to be degradation products were formed in a short time after incubation. In addition, at the time when 24 hours passed from the start of the incubation, the band around 25 kDa was darker as the pH was lower, and was not visible at pH 12. The possibility that the substrate protein was influenced by pH was considered that the degradation pattern was different depending on pH even though it was degraded by a single enzyme.

[Example 5]
RJ was degraded using the recombinant HCLPase expressed in insect cells purified in Example 3 and the crude enzyme (LC) of honey bee queen larvae. For the LC, the crude enzyme solution prepared in Example 1 was used.
Specifically, the enzyme to be added is 1 μg of purified recombinant HCLPase, 2.5 mU of LC (LC-1), or LC (LC-2) that is 1/10 of the amount of protein relative to RJ. RJ was decomposed in the same manner as in Example 4 except that. In addition, since 1 μg of purified recombinant HCLPase expressed in insect cells was about 2.5 mU, LC-1 has the same specific activity of the enzyme as purified recombinant HCLPase. Table 4 shows the protein concentration (μg / mL) and the substrate: enzyme ratio for the enzyme added to each sample.

  The result of SDS-PAGE is shown in FIG. As a result, the degradation pattern of RJ protein by LC-1 and LC-2 was greatly different depending on pH. The degradation pattern under alkaline conditions appeared to be relatively similar between recombinant HCLPase and LC-1 and LC-2, but the degradation pattern under neutral conditions was different. At pH 7, MRJP1 was rapidly digested by LC, and instead a band was generated around 45 kDa. This was presumed to be due to the presence of neutral and strong enzymes such as protease and glycosidase other than HCLPase in LC.

[Example 6]
The influence of SDS on the RJ degradation reaction by the recombinant HCLPase expressed in the insect cells purified in Example 3 was examined.
Specifically, SDS is added to a reaction solution consisting of an RJ water-soluble protein solution (1 mg protein / mL), purified recombinant HCLPase (1 μg / mL), and 50 mM Tris-HCl buffer (pH 9.0) at a final concentration of 0, It added so that it might become 0.01, 0.05, 0.1, 0.5, or 1%, and it incubated at 37 degreeC. Sampling was performed at 1, 3, and 6 hours after the start of incubation, and degradation of RJ protein was examined by SDS-PAGE. An RJ water-soluble protein solution with no enzyme added was used as a control. The result of SDS-PAGE is shown in FIG.

  In addition, SDS was added to the reaction solution so that the final concentration was 0, 0.01, 0.05, 0.1, 0.5, or 1%, and SAAPFpNa was used as a substrate and Example 3 was used as an enzyme. Chymotrypsin activity was examined in the same manner as in Example 2 except that recombinant HCLPase expressed in purified insect cells was used and the pH was adjusted to 9 and 37 ° C. FIG. 13 shows the relative value (%) when the specific activity of chymotrypsin activity when no SDS is added (SDS concentration is 0%) is 100%.

  As shown in FIG. 12, when the SDS concentration was 0.01 to 0.1%, the disappearance of the RJ protein band was accelerated compared to the case where SDS was not added (0%). Moreover, it has confirmed that the appearance pattern of the band considered to be a product in the middle of decomposition was changing depending on the SDS concentration. This was presumed that degradation was promoted because SDS acts on the RJ protein to expose the cleavage site of the protein.

  On the other hand, as shown in FIG. 13, the chymotrypsin activity of HCLPase was increased to about 120% by adding a low concentration of SDS. However, at SDS concentration of 0.1%, chymotrypsin activity was reduced to about 70%. That is, the rate of degradation of the RJ protein was not consistent with the increase or decrease in chymotrypsin activity of HCLPase.

[Example 7]
The expression level of HCLPase in bee queen larvae was examined by measuring the amount of mRNA by real-time RT-PCR. The queen bee larva was obtained from the beekeeping department of Akitaya Honten Co., Ltd. The results of measuring the larval body weight over time are shown in FIG.

  Specifically, the mRNA amount of HCLPase was specifically determined by pulverizing 5 to 10 larvae of each day in liquid nitrogen, extracting total RNA, and reverse transcription using the obtained total RNA as a template. Reaction was performed and cDNA was synthesized. Using the obtained cDNA as a template, PCR was performed using two types of primers {HCLPase-F (5′-AAATGCAAGCAAGCTCACTG, SEQ ID NO: 6) and HCLPase-R (5′-ACAACGAGAGGACCACCAGA, SEQ ID NO: 7)}. The amount of mRNA was measured. Ribosomal protein L32 (RpL32) is used as an internal standard gene, and two types of primers {RpL32-F (5′-CGTCATATGTTGCCAACTGGT, SEQ ID NO: 8) and RpL32-R (5′-TTGAGCACGTTCAACAATGG, SEQ ID NO: 9)} are used. In the same manner, the amount of RpL32 mRNA was measured.

  FIG. 15 shows time-dependent changes in the amount of HCLPase mRNA in queen larvae at 3-5 days of age. Significant differences between each age were tested by Scheffe's test. As a result, the mRNA level of HCLPase was not significantly changed on the 3rd to 4th days in the queen bee larva, but was significantly increased on the 5th day. From this result, it was found that HCLPase was actually expressed in the queen bee larvae, and the expression level increased as the larvae grew.

Claims (4)

(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) a polypeptide having an amino acid sequence in which one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 are deleted, substituted or added, and having chymotrypsin activity, or (C) SEQ ID NO: 1 A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented, and having chymotrypsin activity,
A chymotrypsin-like protease having a chymotrypsin catalytic domain consisting of   A chymotrypsin-like protease according to claim 1 having chymotrypsin activity which is inhibited by phenylmethylsulfonyl fluoride and tosyl-L-phenylalanine chloromethyl ketone but not by 4- (2-aminoethyl) benzenesulfonyl fluoride. .   A method for producing a protein degradation product, comprising degrading a protein at pH 9 to 12 using the chymotrypsin-like protease according to claim 1 or 2 as a catalyst.   The method for producing a protein degradation product according to claim 3, wherein the protein is royal jelly.