JP2006271332A - New trypsin derived from starfish and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new trypsin that has higher reliability than that derived from a mammal and contributes to the effective use of resources and to provide its representative use technology. <P>SOLUTION: The polypeptide has trypsin activity, does not require Ca<SP>2+</SP>and an optimal temperature in the vicinity of 50°C. The polypeptide is found in pyloric caecum of Asteria pectinifera and comprises 264 residue amino acids. The mature type trypsin comprises 237 residue amino acids. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel trypsin and a typical use thereof, and specifically to a novel trypsin derived from the pyloric blindness of a starfish starfish and the use of this novel trypsin.

  Trypsin is synthesized in the pancreatic acinar cells as trypsinogen, is secreted into the duodenum through the pancreatic duct, and becomes active trypsin by limited degradation. Trypsin is positioned as a key enzyme in digestion because it converts other precursor forms of digestive enzymes biosynthesized in the pancreas to activated forms. Therefore, trypsin is an enzyme that has been studied for a long time, and one of the most advanced studies on the molecular level regarding structure and function.

  The trypsin is not only widely used as a reagent for various biochemistry, but also used for medicine and medical use. Specifically, since trypsin has a high pancreatic specificity, it is used to differentiate these diseases when pancreatic diseases such as acute pancreatitis, chronic pancreatitis, pancreatic cancer and the like are suspected. In addition, an enzyme preparation in which trypsin is mixed with bromelain is used as a therapeutic agent for reducing swelling caused by inflammation under the product name Kimotab (registered trademark).

  Examples of trypsin that have been marketed so far include those derived from bovine pancreas, porcine pancreas, and human pancreas. Further, as trypsin derived from sea products, those derived from caperin (see non-patent document 1), anchovy (see non-patent document 2), Atlantic cod (see non-patent document 3), and Atlantic salmon (see 4) are known. ing. All of these are fish that can live in cold regions (cold-adapted fish), and it has been shown that trypsin of these cold-applied fish has higher catalytic activity than mammalian trypsin.

  By the way, starfish have become a problem as an insect pest of cultured scallops and oysters, and about 5000 tons of starfish are exterminated annually in Hokkaido alone. Exterminating starfishes are only partially used as fertilizers, most of which are unused. That is, since starfish are important unused biomass, it is required to cultivate fields of use such as recovery of useful components.

Here, as a result of studying a starfish, a kind of starfish, the present inventors have uniquely found that trypsin derived from the pyloric blindness of the starfish starfish has a unique property (Non-patent Document 5). (See 6.) Specifically, trypsin derived from the pyloric blindness of the starfish starfish is different in nature from vertebrate trypsin because it does not require Ca ions for enzyme activation and stability. The present inventors have also examined the N-terminal amino acid sequence of the above trypsin (see Non-Patent Document 7).
Hjelmeland and Raa, Comp.Biochem.Physiol., 71B, 557-562, 1982 Martinez and Serra, Comp.Biochem.Physiol., 93B, 61-66 1989 Asgeirsson et al., Eur. J. Biochem., 180, 85-94, 1989 Smalas et al., Proteins, 20, 149-166, 1994 H. Kishimura & K. Hayashi; Comp.Biochem.Physiol.132B, 485-490, 2002 Kishimura, Hayashi: Journal of Japanese Fisheries Society, 55 (8), 1415-1420, 1989 H. Kishimura & K. Hayashi; Fisheries Science, 69, 867-869, 2003

  In recent years, trypsin derived from mammals has been difficult to use because components derived from mammalian organs tend to be avoided from the influence of bovine spongiform encephalopathy. Therefore, development of more reliable trypsin that does not originate from mammalian organs and the like is expected.

  On the other hand, the trypsin derived from the low-temperature applied fish is widely used as a marine resource so that the low-temperature applied fish is typified by food, and thus is more reliable than a mammal-derived trypsin. In recent years, however, overfishing of marine fish and a decrease in catch due to this have become a problem in some parts of the world. For example, the above-mentioned anchovy and sardine can be cited as one of the marine fish that have been reported to reduce the resource amount due to overfishing.

  Therefore, if trypsin can be obtained from biomass that is not yet used as a resource, such as the above-mentioned starfish, it is highly preferable because it is highly reliable and resources can be used effectively. it is conceivable that. However, a technique for effectively acquiring trypsin from unused biomass such as starfish has not been known so far.

  The present invention has been made in view of the above problems, and its object is a novel trypsin that is more reliable than a mammal-derived one and can contribute to effective use of resources, and its It is to provide typical utilization technology.

  As a result of intensive studies in view of the above problems, the present inventors have succeeded in finding for the first time a polypeptide having trypsin activity and a polynucleotide encoding the same in the starfish starfish, a kind of starfish, based on this finding. The present invention has been completed by making it possible not only to use the starfish starfish as biomass but also to produce highly reliable trypsin using molecular biological techniques.

That is, the present invention includes the following polypeptides.
(1) A polypeptide having trypsin activity comprising:
(A) the amino acid sequence shown in SEQ ID NO: 1; or (b) the amino acid sequence shown in SEQ ID NO: 1 consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added. A polypeptide characterized by.
(2) A polypeptide having trypsin activity comprising:
(A) a polynucleotide comprising the base sequence shown in SEQ ID NO: 2; or (b) a base in which one or several bases have been deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 2. A polypeptide encoded by a polynucleotide comprising a sequence.
(3) A polypeptide having trypsin activity comprising:
(C) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 2; or
(D) A polypeptide encoded by a polynucleotide comprising a base sequence that is at least 80% identical to a base sequence complementary to the base sequence shown in SEQ ID NO: 2.

  The present invention also includes a polynucleotide encoding any one of the above polypeptides, an expression vector containing the polynucleotide, a transformant obtained by introducing the expression vector, at least a partial base sequence of the polynucleotide or A gene detection instrument using the complementary sequence as a probe, an antibody that specifically binds to the polypeptide, and a pharmaceutical composition containing the polypeptide are also included.

  In the present invention, as described above, a polypeptide having a novel amino acid sequence that has not been known so far and a polynucleotide encoding the polypeptide have been found. In particular, the polypeptide according to the present invention has an amino acid sequence different from that of bovine pancreatic trypsin known so far, and not only has an optimum temperature lower than that of mammalian trypsin but also has trypsin activity. It became clear that Ca ions are not required to exert. Therefore, in the present invention, it is possible to provide a novel and highly reliable trypsin that has not been obtained so far, and it is possible to effectively use starfish that has not been used as biomass.

  An embodiment of the present invention will be described as follows, but the present invention is not limited to this.

[1] Polypeptide As used herein, the term “polypeptide” is used interchangeably with “peptide” or “protein”. In addition, “fragment” of a polypeptide is intended to be a partial fragment of the polypeptide. The polypeptides according to the invention may also be isolated from natural sources or produced recombinantly.

  The term “isolated” polypeptide or protein is intended to be a polypeptide or protein that has been removed from its natural environment. For example, recombinantly produced polypeptides and proteins expressed in a host cell can be isolated in the same manner as natural or recombinant polypeptides and proteins that have been substantially purified by any suitable technique. It is thought that there is.

  Polypeptides according to the present invention include natural purified products and products produced by recombinant techniques from eukaryotic hosts (including, for example, yeast cells, higher plant cells, insect cells, and mammalian cells). Depending on the host used in the recombinant production procedure, the polypeptides according to the invention may be glycosylated. Furthermore, the polypeptides according to the invention may also contain an initiating modified methionine residue in some cases as a result of a host-mediated process.

The polypeptide according to the present invention has trypsin activity, amino acid residues involved in Ca ²⁺ binding existing in known trypsin are substituted or deleted, and the optimum temperature is around 50 ° C. Mention may be made of polypeptides. This polypeptide is derived from the pyloric blindness of the starfish starfish, and preferably has the amino acid sequence shown in SEQ ID NO: 1 (see Examples described later). As used herein, “trypsin activity” intends an activity that specifically degrades the peptide bond at the carboxy terminus of Arg or Lys in a protein.

  In one embodiment, the polypeptide according to the present invention is preferably a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3. As will be described later, this polypeptide is a mature trypsin in which the signal sequence is removed from the amino acid sequence shown in SEQ ID NO: 1 (see Examples).

  In another embodiment, the polypeptide according to the present invention is preferably a polypeptide that is a variant of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and has trypsin activity.

  As used herein, a “polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1” is encoded by a polynucleotide having the base sequence shown in SEQ ID NO: 2, and a signal sequence (shown in SEQ ID NO: 2). The amino acid sequence represented by SEQ ID NO: 1 encoded by positions 1 to 81 of the nucleotide sequence). The mature polypeptide having trypsin activity is a form in which the signal sequence is cleaved (polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3), but as used herein, “SEQ ID NO: The polypeptide comprising the amino acid sequence shown in FIG.

  Such variants include deletions, insertions, inversions, repeats, and type substitutions (eg, replacement of a hydrophilic residue with another residue, but usually strongly hydrophilic residues are strongly hydrophobic) Mutants containing residues that do not substitute). In particular, “neutral” amino acid substitutions in a polypeptide generally have little effect on the activity of the polypeptide.

  It is well known in the art that several amino acids in the amino acid sequence of a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is also well known that there are variants in natural proteins that do not significantly alter the structure or function of the protein, not only artificially modified.

  Preferred variants have conservative or non-conservative amino acid substitutions, deletions, or additions. Silent substitution, addition, and deletion are preferred, and conservative substitution is particularly preferred. These do not alter the trypsin activity of the polypeptides according to the invention.

  Typically conservative substitutions are seen where one amino acid in the aliphatic amino acids Ala, Val, Leu, and Ile is replaced with another amino acid; replacement of hydroxyl residues Ser and Thr, acidic residue Asp And Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.

  As detailed above, further guidance on which amino acid changes are likely to be phenotypically silent (ie likely not to have a significantly detrimental effect on function) can be found in Bowie, JU “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions”, Science 247: 1306-1310 (1990), incorporated herein by reference.

  One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, according to a known point mutation introduction method (mutagenesis method), an arbitrary base of a polynucleotide encoding a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide. Furthermore, by using the method described herein, it can be easily determined whether or not the produced mutant has a desired trypsin activity.

Therefore, the polypeptide according to this embodiment is a polypeptide having trypsin activity,
(A) an amino acid sequence represented by SEQ ID NO: 1; or (b) a polymorph comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1. A peptide is preferred. As described above, such a mutant polypeptide is not limited to a polypeptide having a mutation artificially introduced by a known mutant polypeptide production method, and a naturally occurring polypeptide is isolated and purified. It may be what you did.

In another aspect, the polypeptide according to this embodiment is a polypeptide having trypsin activity,
(A) a polynucleotide comprising the base sequence shown in SEQ ID NO: 2; or (b) a base in which one or several bases have been deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 2. It is preferably encoded by a polynucleotide consisting of a sequence.

In another aspect, the polypeptide according to this embodiment is a polypeptide having trypsin activity,
(C) It is preferably encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 2.

  Hybridization can be performed by a well-known method such as the method described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained. The appropriate hybridization temperature varies depending on the base sequence and the length of the base sequence. For example, when a DNA fragment consisting of 18 bases encoding 6 amino acids is used as a probe, a temperature of 50 ° C. or lower is preferable.

  As used herein, the term “stringent hybridization conditions” refers to hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate. (Containing pH 7.6), 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA) overnight at 42 ° C., then 0.1 × at about 65 ° C. It is intended to wash the filter in SSC. Depending on the polynucleotide that hybridizes to a “portion” of the polynucleotide, at least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably about Polynucleotides (either DNA or RNA) that hybridize to polynucleotides longer than 30 nt are contemplated. Polynucleotides (oligonucleotides) that hybridize to “parts” of such polynucleotides are also useful as detection probes as discussed in more detail herein.

In yet another aspect, the polypeptide according to this embodiment is a polypeptide having trypsin activity,
(D) at least 80% identical to the base sequence complementary to the base sequence shown in SEQ ID NO: 2, more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% It is preferably encoded by a polynucleotide having the same base sequence.

  For example, the target base sequence encodes the polypeptide according to the present invention by “a polynucleotide comprising a base sequence at least 95% identical to the reference (QUERY) base sequence of the polynucleotide encoding the polypeptide according to the present invention”. It is intended to be identical to the reference sequence, except that it may contain up to 5 mismatches per 100 nucleotides (bases) of the polynucleotide reference base sequence. In other words, up to 5% of the bases in the reference sequence can be deleted or replaced with another base to obtain a polynucleotide comprising a base sequence that is at least 95% identical to the reference base sequence, or Many bases up to 5% of the total bases in the reference sequence can be inserted into the reference sequence. These discrepancies in the reference sequence are distributed either individually at the 5 ′ or 3 ′ end position of the reference base sequence or at the base in the reference sequence or in one or more adjacent groups within the reference sequence. Can occur anywhere between the end portions of the. This reference sequence can be a polynucleotide, variant, derivative or analog encoding the amino acid sequence set forth in SEQ ID NO: 1, as described herein.

  Any particular nucleic acid molecule is, for example, at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% relative to the base sequence shown in SEQ ID NO: 3 Whether they are the same or not is known computer program (for example, Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix (registered trademark), Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711)) Can be determined using Bestfit uses Smith and Waterman's local homology algorithm to find the best homology segment between two sequences (Advances in Applied Mathematics 2: 482-489 (1981)). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the percent identity is the reference base. Parameters are set so that gaps in homology up to 5% of the total number of nucleotides in the reference sequence are allowed, calculated over the entire length of the sequence.

  In certain embodiments, the identity (also referred to as global sequence alignment) between a reference (QUERY) sequence (a sequence according to the invention) and a subject sequence is determined by the algorithm of Brutlag et al. (Comp. App. Biosci. 6). : 237-245 (1990)). The preferred parameters used in FASTDB alignment of DNA sequences to calculate% identity are: Matrix = Unitary, k-tuple = 4, Mismatch Penalty = 1, Joining Penalty = 30, Randomization Group Length = 0, Cutoff Score = 1, Gap Penalty = 5, Gap Size Penalty = 0.05, Window Size = 500, or the length of the target base sequence (whichever is shorter). According to this embodiment, if the subject sequence is shorter than the QUERY sequence due to a 5 ′ or 3 ′ deletion (not because of an internal deletion), the FASTDB program calculates the percent identity Considering the fact that 5 ′ and 3 ′ shortening of the target sequence is not taken into account, a manual correction is made to the result. For subject sequences that are truncated at the 5 'end or 3' end relative to the query sequence, the percent identity is the number of bases in the query sequence that are 5 'and 3' of the subject sequence that are not matched / aligned. Is corrected as a percentage of the total base of the QUERY sequence. The determination of whether a nucleotide is matched / aligned is determined by the result of the FASTDB sequence alignment.

  This percentage is then subtracted from the percent identity calculated by the above FASTDB program using the specified parameters to arrive at a final percent identity score. This corrected score is used for the purpose of this embodiment. Only bases outside the 5 'and 3' bases of the subject sequence that do not match / align with the QUERY sequence are calculated for the purpose of manually adjusting the percent identity score as shown in the FASTDB alignment.

  For example, a 90 base subject sequence is aligned with a 100 base query sequence to determine percent identity. The deletion occurs at the 5 'end of the subject sequence, so the FASTDB alignment does not show the first 10 bases match / alignment at the 5' end. Ten unpaired bases represent 10% of the sequence (number of bases at unmatched 5 'and 3' ends / total number of bases in the QUERY sequence), so 10% is generated by the FASTDB program. Subtracted from the calculated percent identity score. If the remaining 90 residues are perfectly matched, the final percent identity is 90%. In another example, a 90 residue subject sequence is compared to a 100 base query sequence.

  In this case, the deletion is an internal deletion, so there is no base at the 5 'end or 3' end of the subject sequence that is not aligned / aligned with the QUERY sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only the 5 'and 3' terminal bases of the subject sequence that do not match / align with the QUERY sequence are manually corrected. No other manual correction is made for the purposes of this embodiment.

  In a further embodiment, the polypeptide according to the present invention preferably has a sugar chain added thereto. The polypeptide according to this embodiment is physicochemically stable and has high biological activity due to the addition of a specific sugar chain. Preferable sugar chains include high mannose type N bond, bisecting GlcNAc, sialic acid, or β-linked galactose.

  The polypeptide according to the present invention may be a polypeptide in which amino acids are peptide-bonded, but is not limited thereto, and may be a complex polypeptide including a structure other than the polypeptide. As used herein, examples of the “structure other than the polypeptide” include sugar chains and isoprenoid groups, but are not particularly limited.

  The polypeptide according to the present invention may include an additional polypeptide. Examples of the additional polypeptide include epitope-tagged polypeptides such as His, Myc, and Flag.

  The polypeptide according to the present invention may be in a state in which a polynucleotide encoding the polypeptide according to the present invention is introduced into a host cell and the polypeptide is expressed in the cell, or a cell, tissue, etc. It may be isolated and purified from.

  In other embodiments, the polypeptides of the invention can be recombinantly expressed in a modified form such as a fusion protein. For example, additional amino acids, particularly charged amino acid regions, of the polypeptides according to the invention may be used in host cells to improve stability and persistence during purification or subsequent manipulation and storage. It can be added to the N-terminus or C-terminus of the polypeptide.

  The polypeptide according to this embodiment can be added to the N-terminus or C-terminus, for example, to a tag label (tag sequence or marker sequence) that is a sequence encoding a peptide that facilitates purification of the fused polypeptide. Such sequences can be removed prior to final preparation of the polypeptide. In certain preferred embodiments of this aspect of the invention, the tag amino acid sequence is a hexa-histidine peptide (eg, a tag provided in a pQE vector (Qiagen, Inc.), among many others Are publicly and / or commercially available. For example, as described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989) (incorporated herein by reference), hexahistidine is a convenient fusion protein. Provide purification. The “HA” tag is another peptide useful for purification corresponding to an epitope derived from influenza hemagglutinin (HA) protein, which is described in Wilson et al., Cell 37: 767 (1984) (herein) Which is incorporated by reference). Other such fusion proteins include a polypeptide according to this embodiment or a fragment thereof fused to Fc at the N or C terminus.

  In another embodiment, the polypeptides according to the invention may be produced recombinantly as detailed below.

  Recombinant production can be performed using methods well known in the art, for example using vectors and cells as detailed below.

  Thus, it can be said that the polypeptide according to the present invention is a polypeptide having trypsin activity, and may be at least the variant consisting of the amino acid sequence shown in SEQ ID NO: 1 or retaining the activity. That is, it should be noted that the present invention includes a polypeptide in which the above-described polypeptide and an arbitrary amino acid sequence having a specific function (for example, a tag) are linked. Moreover, the said polypeptide and arbitrary amino acid sequences which have a specific function (for example, tag) may be connected with the appropriate linker peptide so that each function may not be inhibited.

  That is, an object of the present invention is to provide a novel polypeptide having trypsin activity, and does not exist in the method for producing a polypeptide specifically described in the present specification. Therefore, it should be noted that polypeptides having trypsin activity obtained by methods other than the above methods also belong to the technical scope of the present invention.

[2] Polynucleotide or Oligonucleotide In one aspect, the present invention provides a polynucleotide encoding a polypeptide having trypsin activity or a fragment thereof. As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. As used herein, the term “base sequence” is used interchangeably with “nucleic acid sequence” or “nucleotide sequence” and refers to the sequence of deoxyribonucleotides (abbreviated A, G, C, and T). As shown. The “polynucleotide comprising the base sequence shown in SEQ ID NO: 3 or a fragment thereof” refers to a polynucleotide containing the sequence shown by each deoxynucleotide A, G, C and / or T of SEQ ID NO: 3 or a fragment thereof Is intended. In the present specification, for the purpose of clearly distinguishing from abbreviations and the like, formal names may be added as appropriate for the nucleotides A, G, C and T (for example, A (adenine)).

  The polynucleotide according to the present invention may exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand).

  The polynucleotide according to the present invention can be fused to the polynucleotide or oligonucleotide encoding the tag tag (tag sequence or marker sequence) described above on the 5 'side or 3' side.

  As used herein, the term “oligonucleotide” is intended to be a combination of several to several tens (or several hundreds) nucleotides, and is used interchangeably with “polynucleotide”. . Oligonucleotides are called dinucleotides (dimers) and trinucleotides (trimers) as short ones, and are represented by the number of nucleotides polymerized such as 30 mers or 100 mers as long ones. Oligonucleotides can be produced as fragments of longer polynucleotides or synthesized.

  Oligonucleotides according to the present invention are intended to be fragments of a length of at least 12 nt (nucleotide), preferably about 15 nt, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably at least about 40 nt. Is done. By a fragment having a length of at least 20 nt, for example, a fragment comprising 20 or more consecutive bases from the base sequence shown in SEQ ID NO: 3 is contemplated. By referring to the present specification, the base sequence shown in SEQ ID NO: 3 is provided, so that those skilled in the art can easily prepare a DNA fragment based on SEQ ID NO: 3. For example, restriction endonuclease cleavage or ultrasonic shearing can be readily used to create fragments of various sizes. Alternatively, such fragments can be made synthetically. Appropriate fragments (oligonucleotides) are synthesized, such as by Applied Biosystems Incorporated (ABI, 850 Lincoln Center Dr., Foster City, CA 94404) type 392 synthesizer.

  In another aspect, the oligonucleotide according to the present invention preferably hybridizes to a polynucleotide comprising the base sequence represented by SEQ ID NO: 3 or a variant thereof. A “variant” can occur naturally, such as a natural allelic variant. By “allelic variant” is intended one of several interchangeable forms of a gene occupying a given locus on an organism's chromosome. Non-naturally occurring variants can be generated, for example, using mutagenesis techniques well known in the art.

  In another aspect, the present invention provides a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3, a fragment of a variant thereof, or an oligonucleotide consisting of a complementary sequence thereof.

  Even when the oligonucleotide according to the present invention does not encode a polypeptide having trypsin activity, the person skilled in the art creates the polypeptide according to the present invention as a primer for polymerase chain reaction (PCR). Easy to understand that can be used for. Other uses of the oligonucleotide according to the present invention that does not encode the polypeptide according to the present invention include: (i) isolation of trypsin gene or alleles or splicing variants thereof in a cDNA library; ii) In situ hybridization (eg, “FISH”) to metaphase chromosomal spreads to provide the exact chromosomal location of the trypsin gene (Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York ( (Iii)) Northern blot analysis to detect trypsin gene mRNA expression in specific tissues.

  The polynucleotide or oligonucleotide according to the present invention includes not only double-stranded DNA but also single-stranded DNA or RNA such as a sense strand and an antisense strand constituting the double-stranded DNA. The polynucleotide or oligonucleotide according to the present invention can be used as a tool for gene expression manipulation by an antisense RNA mechanism. With antisense RNA technology, a decrease in the gene product derived from the endogenous gene is observed. The polynucleotide or oligonucleotide according to the present invention may contain a sequence such as an untranslated region (UTR) sequence or a vector sequence (including an expression vector sequence).

  Examples of the method for obtaining the polynucleotide or oligonucleotide according to the present invention include various known methods for isolating a DNA fragment containing the polynucleotide or oligonucleotide according to the present invention. For example, by preparing a probe that specifically hybridizes with a part of the nucleotide sequence of the polynucleotide according to the present invention and screening a genomic DNA library or cDNA library, the polynucleotide or oligonucleotide according to the present invention can be obtained. Can be acquired. Such a probe may be a polynucleotide (oligonucleotide) that specifically hybridizes to at least a part of the base sequence of the polynucleotide according to the present invention or its complementary sequence. Polynucleotides selected by such hybridization include, but are not limited to, natural polynucleotides.

  Another method for obtaining the polynucleotide according to the present invention is a method using PCR. In this PCR amplification method, for example, a step of preparing a primer using the 5 ′ and / or 3 ′ sequence (or its complementary sequence) of the cDNA of the polynucleotide according to the present invention, using these primers It includes a step of PCR amplification using genomic DNA (or cDNA) or the like as a template. If this method is used, a large amount of DNA fragments containing the polynucleotide according to the present invention can be obtained.

  The source for obtaining the polynucleotide according to the present invention is not particularly limited, and examples of the organism include starfishes, particularly starfish starfish (scientific name: Asterina pectinifera), more specifically, starfish. In the case of a kind, it is preferably a biological material such as pyloric blind. As used herein, the term “biological material” intends a biological sample (a tissue sample or cell sample obtained from an organism).

  By using the polynucleotide or oligonucleotide according to the present invention, an organism expressing a polypeptide having trypsin activity can be easily detected by detecting the hybridizing polynucleotide.

  The polynucleotide or oligonucleotide according to the present invention is used as a hybridization probe for detecting a polynucleotide encoding a polypeptide having trypsin activity or as a primer for amplifying a polynucleotide encoding a polypeptide having trypsin activity. By this, an organism or tissue expressing a polypeptide having trypsin activity can be easily detected. Furthermore, expression of a polypeptide having trypsin activity in the organism or its tissue or cell can be suppressed by using the oligonucleotide as an antisense oligonucleotide.

  That is, an object of the present invention is to provide a polynucleotide that encodes a polypeptide having trypsin activity, and an oligonucleotide that hybridizes with the polynucleotide, and is specifically described in the present specification. It does not exist in the production method of polynucleotides and oligonucleotides. Therefore, it should be noted that polynucleotides encoding polypeptides having trypsin activity obtained by methods other than the above methods also belong to the technical scope of the present invention.

[3] Use of polypeptide or polynucleotide according to the present invention (A) Vector The present invention provides a vector used for producing a polypeptide having trypsin activity. The vector according to the present invention is not particularly limited as long as it contains a polynucleotide encoding the above-described polypeptide according to the present invention, and a known expression vector or the like can be used. Moreover, for example, a recombinant expression vector into which cDNA of a polynucleotide encoding a polypeptide having trypsin activity (which may or may not include a signal sequence) is inserted. A method for producing a recombinant expression vector includes, but is not limited to, a method using a plasmid, phage, cosmid or the like.

  The specific type of vector is not particularly limited, and a vector that can be expressed in a host cell can be appropriately selected. That is, according to the type of the host cell, a promoter sequence is appropriately selected in order to reliably express the polynucleotide according to the present invention, and a vector in which this and the polynucleotide according to the present invention are incorporated into various plasmids is used as an expression vector. Use it.

  The expression vector according to the present invention contains an expression control region (for example, promoter, terminator, and / or origin of replication) depending on the type of host to be introduced. Examples of the promoter for yeast include glyceraldehyde 3-phosphate dehydrogenase promoter and PH05 promoter, and examples of the promoter for filamentous fungi include amylase and trpC. Examples of animal cell host promoters include viral promoters (eg, SV40 early promoter, SV40 late promoter, etc.). The expression vector can be prepared according to a conventional method using a restriction enzyme and / or ligase. Transformation of a host with an expression vector can also be performed according to conventional techniques.

  After the host transformed with the above expression vector is cultured, cultivated or raised, conventional techniques (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography) are used from the culture. Etc.), the target protein can be recovered and purified.

  The expression vector preferably contains at least one selectable marker. Such markers include eukaryotic cell culture, dihydrofolate reductase, or drug resistance genes such as neomycin, Zeocin, geneticin, blasticidin S, hygromycin B, and culture in E. coli and other bacteria. May include drug resistance genes such as kanamycin, Zeocin, actinomycin D, cefotaxime, streptomycin, carbenicillin, puromycin, tetracycline or ampicillin.

  By using the above selectable marker, it can be confirmed whether or not the polynucleotide according to the present invention has been introduced into the host cell, and whether or not it is reliably expressed in the host cell. Alternatively, the polypeptide according to the present invention may be expressed as a fusion polypeptide. For example, the green fluorescence polypeptide GFP (Green Fluorescent Protein) derived from Aequorea jellyfish is used as a marker, and the polypeptide according to the present invention is used as a GFP fusion polypeptide. It may be expressed as

  The host cell is not particularly limited, and various conventionally known cells can be suitably used. Specifically, for example, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), nematode (Caenorhabditis elegans), Xenopus laevis oocytes, animal cells, CHO cells, COS cells, and Bowes black Tumor cells).

  A method for introducing the expression vector into a host cell, that is, a transformation method is not particularly limited, and a conventionally known method such as an electroporation method, a calcium phosphate method, a liposome method, or a DEAE dextran method can be suitably used. . In addition, for example, when the polypeptide according to the present invention is expressed in insects, an expression system using baculovirus may be used.

  When the above-described polynucleotide is introduced into an organism or cell using the vector according to the present invention, a polypeptide having trypsin activity can be expressed in the organism or cell.

  It is to be noted that an object of the present invention is to provide an expression vector containing a polynucleotide encoding the polypeptide according to the present invention, and the individual vector species and cells specifically described in the present specification. It does not exist in the species, and the vector production method and the cell introduction method. Therefore, it should be noted that expression vectors obtained using vector species and vector production methods other than those described above also belong to the technical scope of the present invention.

(B) Transformant The present invention provides a transformant into which a polynucleotide encoding the above-described polypeptide having trypsin activity has been introduced. As used herein, the term “transformant” is intended to include not only cells, tissues or organs, but also individual organisms. In addition, the organism to be transformed is not particularly limited, and examples thereof include various microorganisms, plants and animals exemplified in the host cell.

  In the transformant according to the present invention, the above-mentioned polypeptide having trypsin activity is expressed. The transformant according to the present invention preferably stably expresses a polypeptide having trypsin activity.

(C) Polypeptide Production Method The present invention provides a method for producing a polypeptide according to the present invention. If the method for producing a polypeptide according to the present invention is used, it is possible to provide a polypeptide having trypsin activity at low cost and under a low environmental load. Moreover, if the method for producing a polypeptide according to the present invention is used, a polypeptide having trypsin activity can be easily produced.

  In one embodiment, the method for producing a polypeptide according to the present invention can include a method using the vector according to the present invention.

  In one aspect of the present embodiment, the polypeptide production method according to the present embodiment preferably uses a recombinant expression system using a eukaryotic host. When using a recombinant expression system, the polynucleotide according to the present invention is incorporated into a recombinant expression vector, introduced into a host so that it can be expressed by a known method, and the polypeptide obtained by translation in the host is purified. Such a method can be adopted. The recombinant expression vector may or may not be a plasmid, as long as the target polynucleotide can be introduced into the host. Preferably, the method for producing a polypeptide according to this embodiment includes a step of introducing the vector into a host.

  Thus, when introducing a foreign polynucleotide into a host, the expression vector preferably incorporates a promoter that functions in the host so as to express the foreign polynucleotide. The method for purifying a recombinantly produced polypeptide differs depending on the host used and the nature of the polypeptide, but the target polypeptide can be purified relatively easily by using a tag or the like.

  In another embodiment, the method for producing a polypeptide according to the present invention preferably purifies the polypeptide from cells or tissues that naturally or recombinantly express the polypeptide according to the present invention. Since the polypeptide according to the present invention is secreted from cells in a mature form, it can be recovered from the culture supernatant of cells that naturally or recombinantly express the polypeptide according to the present invention. Moreover, the method for producing a polypeptide according to this embodiment may further include a step of purifying the above-described polypeptide.

  The method for producing a polypeptide according to the present invention preferably further includes a step of purifying the polypeptide from an extract of cells or tissues containing the polypeptide according to the present invention. The step of purifying a polypeptide is to prepare a cell extract from cells or tissues by a well-known method (for example, a method in which a cell or tissue is disrupted and then centrifuged to collect a soluble fraction). Known methods (eg ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography) The step of purifying by a) is preferred, but not limited thereto. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification.

  That is, an object of the present invention is to provide a method for producing a polypeptide having trypsin activity using a eukaryotic host, and a production method including steps other than the various steps described above is also provided. It should be noted that it belongs to the technical scope of the invention.

(D) Antibody The present invention provides an antibody that specifically binds to the polypeptide according to the present invention. More specifically, the antibody according to the present invention binds to the polypeptide having the amino acid sequence shown in SEQ ID NO: 1, but does not bind to trypsin derived from humans or other mammals. In one embodiment, an antibody according to the present invention can inhibit trypsin activity. Preferably, the antibody according to this embodiment is a monoclonal antibody. In addition, the production method of the antibody concerning this invention is not specifically limited, A well-known method can be used suitably.

(E) Specific Field of Use The present invention provides a pharmaceutical composition containing the polypeptide according to the present invention as an active ingredient. Specifically, a pharmaceutical composition that contains a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 (preferably SEQ ID NO: 3) and can be used as various reagents and pharmaceuticals is provided. As described above, the polypeptide according to the present invention has higher reliability than mammalian trypsin, and can use starfishes which are unused biomass as raw materials. In addition, since the optimum temperature is around 50 ° C. and it does not require Ca ²⁺ , it can be used in fields that could not be used with conventional trypsin.

  In addition, all of the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  The present invention will be described more specifically with reference to the following examples and FIGS. 1 to 5, but the present invention is not limited thereto. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

[I. Preparation of total RNA from Itomaki starfish pyloric blindness]
Total RNA was prepared from the pyloric blindness of Itomas starfish using the following steps using Invitrogen's trade name: TRIZOL.

  First, 1.5 ml of TRIZOL reagent was added to 141 mg of pyloric blind, and after homogenization, 0.3 ml of chloroform was added, shaken vigorously by hand, and allowed to stand at room temperature for 3 minutes. Next, the mixture was centrifuged at 12,000 × g for 15 minutes at 4 ° C. Next, the upper layer was taken in another tube, 0.75 ml of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 minutes. Next, it was centrifuged (12,000 × g, 10 minutes, 4 ° C.), and the supernatant was removed. To the resulting precipitate (total RNA), 1.5 ml of cooled 75% ethanol was added and stirred gently. Next, the mixture was centrifuged at 10,000 × g for 10 minutes at 4 ° C., and the supernatant was removed. To the resulting precipitate (total RNA), 1 ml of cooled 75% ethanol was added and gently stirred. Next, it was centrifuged at 10,000 × g for 10 minutes at 4 ° C., and the supernatant was removed. The precipitate was lightly dried and then dissolved with 125 μl of distilled water. A part of the dissolved total RNA was subjected to electrophoresis and UV absorption measurement, and the properties of the total RNA were confirmed and the concentration was measured.

  As a result of the above, the result of electrophoresis of RNA was as shown in FIG. 4, and 28S and 18S were the main components. Yield of total RNA of pyloric blindness was 0.242 mg per 100 mg of pyloric blindness.

[II. Preparation of single-stranded cDNA from total RNA of Itomaki starfish pyloric cecum
Single-stranded cDNA was prepared from the total pyloric blindness RNA of Itomaki starfish using Invitrogen's trade name: SUPERSCRIPT II in the following steps.

  A mixed solution of total RNA and primer was prepared by adding 1 μl of reverse transcription primer RTRACE (10 μM) to 2 μg of total pyloric blind RNA obtained in I above, and making the total volume 11 μl with distilled water. The base sequence of the reverse transcription primer RTRACE is shown below.

RTRACE: GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TT (SEQ ID NO: 4)
In order to destroy the higher-order structure of RNA, it was heated at 70 ° C. for 10 minutes, then rapidly cooled and held for 5 minutes. 5 × 1st. 4 μl of standard buffer, 2 μl of 0.1 M DTT, 1 μl of 10 mM dNTP and 1 μl of RNase inhibitor were added, and preincubation was performed at 42 ° C. for 5 minutes. 1 μl of reverse transcriptase SUPERSCRIPT II was added to make a total volume of 20 μl, and a reverse transcription reaction was performed at 42 ° C. for 50 minutes. The reverse transcription reaction was stopped by heating at 70 ° C. for 15 minutes. Thereby, single-stranded cDNA was prepared.

[III. Partial amplification of the trypsin gene
Based on the alignment of amino acid sequences of various animal trypsins registered in the database, a highly homologous sequence necessary for partial amplification of the trypsin gene of Itomas starfish pyloric cecum was found, and degenerate sense primer (TR2) and anti-sense primer A sense primer (TR4) was synthesized. The base sequences of these primers are shown below.

TR2: CAC TTC TGY GGW GSN TCY HT (SEQ ID NO: 5)
TR4: GARTCH CCC TGR CAN GAR TC (SEQ ID NO: 6)
PCR using the above TR2 and TR4 was performed using the single-stranded cDNA prepared in II above as a template. The PCR reaction composition was 2 μl of x10 buffer, 2 μl of 25 mM MgCl ₂ , 2 μl of 2 mM dNTP, 2 μl of 10 μM TR2, 2 μl of 10 μM TR4, 0.3 μl of the above single-stranded cDNA, The DNA polymerase (5 unit / μl) was adjusted to 0.14 μl, and the total volume was adjusted to 20 μl with distilled water. As the DNA polymerase, trade name: AmpliTaq Gold DNA Polymerase manufactured by Applied Biosystems was used.

  As PCR reaction conditions, the product name: GeneAmp PCR System 2400, manufactured by Applied Biosystems, was used. First, the sample was heated at 95 ° C. for 9 minutes, and then cycled at 94 ° C. for 15 seconds, 54 ° C. for 30 seconds, and 72 ° C. for 1 minute. The cycle was repeated 45 times, and finally, the reaction was terminated by heating at 72 ° C. for 7 minutes and cooling to 10 ° C.

  The sample after the completion of the PCR was subjected to 1.2% agarose gel electrophoresis (containing Tris / acetic acid / EDTA buffer, ethidium bromide at a final concentration of 50 μg / 100 ml). As a result, as shown in FIG. 5, the generation of a trypsin gene product having a size of 490 bp was confirmed.

[IV. Determination of the base sequence of the trypsin gene
The base sequence of the trypsin gene product obtained in III above was determined by the sequence reaction in the next step.

  First, the PCR reaction product obtained in III above was subjected to low melting point agarose gel electrophoresis with a gel concentration of 2%, and the target band was cut out with a scalpel. Product name: Wizard SV Gel and PCR Clean-Up System manufactured by Promega Corporation was used to elute cDNA from the cut gel piece. The concentration of the eluted cDNA was determined by subjecting it to 1.2% agarose gel electrophoresis together with a DNA marker of known concentration.

  The base sequence of the eluted cDNA was determined by the dideoxy method. The reaction composition is 9 ng of cDNA template, 2 μl of terminator, 3 μl of × 5 buffer, 0.32 μl of sense primer TR2 (10 μM) to read the base sequence of the sense strand, and to read the base sequence of the antisense strand Was added with 0.32 μl of antisense primer TR4 (10 μM), and the total volume was adjusted to 20 μl with distilled water. The PCR reaction conditions were the product name: GeneAmp PCR System 2400 manufactured by Applied Biosystems, and this cycle was performed 25 times with 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 4 minutes. The reaction was terminated by cooling to ° C.

  After completion of PCR, 2 μl of 3M sodium acetate (pH 5.2) and 50 μl of 100% ethanol were added to the reaction solution, and the reaction product was recovered as a precipitate by centrifugation (conditions: 14,000 rpm, 20 minutes, room temperature). Thereafter, the precipitate was gently washed with 250 μl of 70% ethanol, and then centrifuged (conditions: 14,000 rpm, 10 minutes, room temperature) to remove the ethanol in the supernatant and lightly dried. Thereafter, 20 μl of formamide was added to dissolve the precipitate. After heating at 95 ° C. for 3 minutes, the precipitate was rapidly cooled and analyzed by Perkin Elmer, trade name: ABI PRISM (registered trademark) 3100 Genetic Analyzer.

  The obtained base sequence information was subjected to BLAST search, and the base sequence of the trypsin gene of Itomaki starfish pyloric cecum was determined.

[V. Itomaki starfish pyloric blindness trypsin gene 3'RACE]
3′RACE was performed using the single-stranded cDNA prepared in II above as a template.

  A sense primer TR6 for 3'RACE was synthesized from the nucleotide sequence of the trypsin gene of Itomas starfish pyloric cecum obtained in IV above. The base sequence of the sense primer TR6 is shown below.

TR6: CCT ACC CTT ACG AAC TCA GAC AG (SEQ ID NO: 7)
PCR was performed using the sense primer TR6 and RACE using the single-stranded cDNA prepared in II above as a template. The base sequence of RACE is shown below.

RACE: GGC CAC GCG TCG ACT AGT AC (SEQ ID NO: 8)
The PCR reaction composition is 2 μl of × 10 buffer, 2 μl of 25 mM MgCl ₂ , 2 μl of 2 mM dNTP, 0.8 μl of 10 μM TR6, 0.8 μl of 10 μM RACE, and 0.3 μl of single-stranded cDNA. DNA polymerase (5 unit / μl) was adjusted to 0.14 μl, and the total volume was adjusted to 20 μl with distilled water. As the DNA polymerase, trade name: AmpliTaq Gold DNA Polymerase manufactured by Applied Biosystems was used. As PCR reaction conditions, a product name: GeneAmp PCR System 2400 manufactured by Applied Biosystems was used. First, 95 ° C was heated for 9 minutes, and then 94 ° C for 15 seconds, 58 ° C for 30 seconds, and 72 ° C for 1 minute for one cycle. The cycle was repeated 45 times, and finally, the reaction was terminated by heating at 72 ° C. for 7 minutes and cooling to 10 ° C.

  The obtained PCR reaction product was subjected to low-melting point agarose gel electrophoresis with a gel concentration of 2%, a target band of 850 bp was cut with a scalpel, and the product name: Wizard SV Gel and PCR Clean-Up System manufactured by Promega was used. CDNA was eluted from the cut gel piece. It was subjected to 1.2% agarose gel electrophoresis together with a DNA marker having a known concentration to determine the concentration of the eluted cDNA. The cDNA concentration was 7.6 ng / μl.

  The base sequence of the eluted cDNA was determined by the dideoxy method. As the PCR reaction composition, 2 μl of terminator, 3 μl of × 5 buffer and 0.16 μl of 10 μM TR6 were added to 11 ng of cDNA template, and the total volume was adjusted to 20 μl with distilled water. As PCR reaction conditions, a product name: GeneAmp PCR System 2400 manufactured by Applied Biosystems was used. First, 95 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 4 minutes, and this cycle was performed 25 times. The reaction was terminated by cooling to 10 ° C.

  After the PCR reaction was completed, 2 μl of 3M sodium acetate (pH 5.2) and 50 μl of 100% ethanol were added to the reaction solution, and the reaction product was collected as a precipitate by centrifugation (14,000 rpm, 20 minutes, room temperature). The precipitate was lightly washed with 250 μl of 70% ethanol and then centrifuged again (14,000 rpm, 10 minutes, room temperature). After removing ethanol from the supernatant and lightly drying, 20 μl of formamide was added to dissolve the precipitate. Subsequently, after heating at 95 ° C. for 3 minutes, the sample was rapidly cooled and analyzed by Perkin Elmer, trade name: ABI PRISM (registered trademark) 3100 Genetic Analyzer.

  The obtained base sequence information was subjected to BLAST search, and the base sequence of the trypsin gene of Itomaki starfish pyloric cecum was determined. The 3'RACE product of Itomas starfish pyloric cecum trypsin gene amplified by sense primer TR6 and RACE primer was 850 bp. In addition, in order to determine the entire base sequence of the 3'RACE product, a new sequence primer TR9 was synthesized based on the base sequence obtained by the sequence primer TR6, and a sequence reaction was performed. The base sequence of primer TR9 is shown below.

TR9: AAG GCT TAT CAG ACA CTC GG (SEQ ID NO: 9)
[VI. Subcloning of 3'RACE product of Itomaki starfish pyloric blind trypsin gene]
When polymorphism (overlapping waveform) is observed in the direct sequence, the sequence must be determined by subcloning. TA cloning is the easiest method for subcloning (see: Biological Experiment Illustrated, 3+ PCR, pages 67-68 and pages 73-84).

  As a result of direct sequencing of the 3'RACE product (TR6 / RACE, 850 bp) of the above-mentioned Itomas starfish pyloric blind trypsin gene, polymorphism was observed. Thus, subcloning was performed using Promega's product name: pGEM-T Easy Vector System. In general, since the 3 ′ end of a PCR product often has a structure in which A (adenine) protrudes by one base, a vector in which T (thymine) protrudes by one base on the 3 ′ side (in this case, pGEM-T The PCR product was incorporated into the vector by ligation using Easy Vector.

  First, 27 μg of trypsin gene 3′RACE product (TR6 / RACE, 850 bp), 5 μl of × 2 rapid ligation buffer, 25 ng of pGEM-T Easy vector, 3 units of T4 DNA ligase were added, and the total volume was 10 μl with distilled water. Incubated overnight.

Next, 2.5 μl of the ligation product was added to 50 μl of competent cells (E. coli JM109), mixed gently and incubated on ice for 30 minutes. Thereafter, heat shock was applied at 42 ° C. for 45 seconds, followed by incubation on ice for 2 minutes. Next, 0.45 ml of MgCl ₂ · MgSO ₄ · glucose-containing SOC medium was added and cultured with shaking at 37 ° C for 1 hour.

  Next, 70 μl of the culture solution was spread on a whole agar plate containing X-gal and IPTG with a spreader and incubated overnight at 37 ° C. Among the grown colonies, colonies showing a white color were isolated.

  From the white colony, a plasmid was prepared using Promega's product name: Wizard Plus SV Minipreps DNA Purification System. First, the isolated white colony was cultured overnight at 37 ° C. in 4 ml of ampicillin-containing LB medium. 2.5 ml of the obtained culture broth was centrifuged at 11,000 rpm for 5 minutes. The plasmid was recovered from the cells recovered as a precipitate by the alkali-SDS method.

  Next, the base sequence of the plasmid was determined by the dideoxy method. First, in 300 ng of plasmid, 2 μl of terminator, 3 μl of × 5 buffer, 0.16 μl of 10 μM primer T7 for reading the base sequence of the sense strand, and 10 μM primer SP6 for reading the base sequence of the antisense strand 0.16 μl was added, and the total volume was adjusted to 20 μl with distilled water. The PCR reaction conditions and the sequence analysis method were the same as in the direct sequence. In addition, the base sequences of primers T7 and SP6 are shown below.

T7: TAA TAC GAC TCA CTA TAG GG (SEQ ID NO: 10)
SP6: TAT TTA GGT GAC ACT ATA G (SEQ ID NO: 11)
[VII. 5'RACE of all genes expressed in Itomaki starfish pyloric blindness]
All genes expressed in Itomaki starfish pyloric blind were subjected to 5'RACE using Clontech's product name: BD SMART RACE cDNA Amplification Kit.

  First, 1 μl of 10 μM 5′RACE cDNA synthesis primer and 1 μl of 10 μM trade name: BD SMART II A Oligo were added to 1 μg of total RNA of pyloric blindness, and the total amount was adjusted to 5 μl with distilled water. The nucleotide sequences of 5'RACE cDNA synthesis primer and BD SMART II A Oligo are shown below.

CDNA synthesis primer for 5 'RACE: 5'-(T) 25N-1V-3 '(N = A, C, G, or T, V = A, G, or C) (SEQ ID NO: 12)
BD SMART II A Oligo: 5'-AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG-3 '(SEQ ID NO: 13)
Next, in order to break the higher-order structure of RNA, it was heated at 70 ° C. for 2 minutes and then rapidly cooled for 2 minutes. Next, reverse transcription reaction was performed using reverse transcriptase BD PowerScript Reverse Transcriptase. The reaction composition was 2 μl of 5 × 1st standard buffer, 1 μl of 20 mM DTT, 1 μl of 10 mM dNTP, 1 μl of reverse transcriptase BD PowerScript Reverse Transcriptase, and the total volume was 10 μl with distilled water. The reaction conditions were set at 42 ° C. for 90 minutes, 100 μl of 10 mM Tricine-EDTA buffer was added, and then heated at 72 ° C. for 7 minutes to stop the reverse transcription reaction. The base sequence of BD SMART II A Oligo used for the reverse transcription reaction is shown below.

  Here, the principle of the SMART method used in the 5'RACE will be described. Oligo T, a cDNA synthesis primer for 5 'RACE, anneals to poly A sequences of various mRNAs in total RNA, and single-stranded cDNA is synthesized by reverse transcriptase. This reaction proceeds to the 5 'end of each mRNA. After synthesis of single-stranded cDNA up to the 5 'end of each mRNA, 3 to 5 C (cytosine) are added to the 3' end of each single-stranded cDNA. Next, as shown in FIG. 1, 3 to 5 GGGs on the 3 ′ side of BD SMART II A Oligo (SEQ ID NO: 13) are added to the 3 ′ end of each single-stranded cDNA (SEQ ID NO: 14). Annealed with C (cytosine). By the reverse transcription reaction, a complementary strand of BD SMART II A Oligo is synthesized on the CCC at the 3 'end of each single-stranded cDNA.

  The important point is that the SMART method synthesizes a template for 5'RACE of all mRNAs in theory.

[VIII. Itomaki starfish pyloric blindness trypsin gene 5'RACE]
An antisense primer TR8 for 5′RACE was synthesized from the base sequence of the tryptic gene of Itomas starfish pyloric cecum obtained in IV above. The base sequence of the antisense primer TR8 is shown below.

TR8: CGG AGC CTG ACG ATG CAA TGC GGA TGG T (SEQ ID NO: 15)
Next, the above VII. PCR was carried out using the above-mentioned antisense primer and NUP, using the single-stranded cDNA prepared in step 1 as a template. The base sequence of NUP is shown below.

NUP: AAG CAG TGG TAA CAA CGC AGA GT (SEQ ID NO: 16)
The PCR reaction composition was 5 μl of x10 buffer, 5 μl of 2 mM dNTP, 1 μl of 10 μM NUP, 1 μl of 10 μM TR8, 2.5 μl of single-stranded cDNA, 1 μl of x50 DNA polymerase, and distilled water. The total volume was 50 μl. As the DNA polymerase, a product name: Advantage 2 Polymerase mix manufactured by Clontech was used. As PCR reaction conditions, the product name: GeneAmp PCR System 2400, manufactured by Applied Biosystems, was used. After heating at 94 ° C. for 1 minute, 94 ° C. for 5 seconds, 68 ° C. for 10 seconds, and 72 ° C. for 2 minutes for one cycle. The cycle was repeated 38 times, and finally, the reaction was terminated by heating at 72 ° C. for 7 minutes and cooling to 10 ° C.

  The obtained PCR reaction product was subjected to low-melting point agarose gel electrophoresis with a gel concentration of 2%, and a 500 bp target band was cut with a scalpel, using a product name: Wizard SV Gel and PCR Clean-Up System manufactured by Promega, CDNA was eluted from the cut gel piece. It was subjected to 1.2% agarose gel electrophoresis together with a DNA marker having a known concentration to determine the concentration of the eluted cDNA. The cDNA concentration was 1.7 ng / μl.

[IX. Subcloning of 5'RACE product of Itomaki starfish pyloric blind trypsin gene]
VI. In the same manner as described above, the 5 ′ RACE product of the trypsin gene was subcloned using the product name: pGEM-T Easy Vector System manufactured by Promega.

  First, 5 μl of × 2 rapid ligation buffer, 25 ng of pGEM-T Easy vector, 3 units of T4 DNA ligase were added to 10 ng of the trypsin gene 5′RACE product (NUP / TR8, 500 bp), and the total amount was 10 μl with distilled water. Incubated overnight.

Next, 2.5 μl of the ligation product was added to 50 μl of competent cells (E. coli JM109), mixed gently and incubated on ice for 30 minutes. Thereafter, heat shock was applied at 42 ° C. for 45 seconds, followed by incubation on ice for 2 minutes. Next, 0.45 ml of MgCl ₂ · MgSO ₄ · glucose-containing SOC medium was added and cultured with shaking at 37 ° C for 1 hour.

  Next, 70 μl of the culture solution was spread on a whole agar plate containing X-gal and IPTG with a spreader and incubated overnight at 37 ° C. Among the grown colonies, colonies showing a white color were isolated.

  From the white colony, a plasmid was prepared using Promega's product name: Wizard Plus SV Minipreps DNA Purification System. First, the isolated white colony was cultured overnight at 37 ° C. in 4 ml of ampicillin-containing LB medium. 2.5 ml of the obtained culture broth was centrifuged at 11,000 rpm for 5 minutes. The plasmid was recovered from the cells recovered as a precipitate by the alkali-SDS method.

  Next, the base sequence of the plasmid was determined by the dideoxy method. First, in 300 ng of plasmid, 2 μl of terminator, 3 μl of × 5 buffer, 0.16 μl of 10 μM primer T7 for reading the base sequence of the sense strand, and 10 μM primer SP6 for reading the base sequence of the antisense strand 0.16 μl was added, and the total volume was adjusted to 20 μl with distilled water. The PCR reaction conditions and the sequence analysis method were the same as in the direct sequence.

[X. result〕
The partial cDNA sequence of the obtained Itomaki starfish pyloric blind trypsin is shown in SEQ ID NO: 3. The size of the cDNA was 1258 bp including a poly A signal (AATAAA). In addition, the amino acid sequence of trypsin of Itomas starfish pyloric cecum coded in the cDNA is shown in SEQ ID NO: 1. The cDNA encoded an amino acid of 264 residues.

  FIG. 2 shows the results of comparing the signal and precursor amino acid sequences of the obtained Itomas starfish pyloric blind trypsin and various vertebrate trypsins. Each vertebrate trypsin has a signal peptide rich in hydrophobic amino acids and a precursor peptide rich in basic amino acids from the start codon Met, and the precursor peptide ends with a Lys residue. On the other hand, it was not possible to confirm the initiation codon Met with Itomas starfish pyloric blind trypsin, but a long signal peptide-like sequence rich in hydrophobic amino acids was confirmed. However, a precursor peptide rich in basic amino acids and ending with a Lys residue was not confirmed. It is considered that the precursor peptide may not be present because trypsin of pylorid pyloric blindness is prepared by preparing crude enzyme from living starfish starfish and measuring the activity.

  Thereafter, the entire amino acid sequence of mature trypsin of Itomas starfish was determined. The amino acid sequence is shown in SEQ ID NO: 2 and FIG. In FIG. 3, the amino acid sequence is compared with that of bovine pancreatic trypsin. The mature starfish trypsin consists of 237 amino acids and has active residues His57, Asp102, Ser195 and Asp189 residues that determine substrate specificity. In addition, Asp194 that forms an ion pair with the N-terminal Ile was also conserved.

In bovine pancreatic trypsin, Glu70, Val75, Glu77, and Glu80, which are involved in Ca ²⁺ binding, were substituted or deleted in Itomas detrypsin. From this, it is not necessary to use Ca ²⁺ like mammalian trypsin in the use of Itomas detrypsin.

  Trypsin of cold-adapted fish such as caperin (Non-patent document 1), anchovy (Non-patent document 2), Atlantic cod (Non-patent document 3), Atlantic salmon (Non-patent document 4) has higher catalytic activity than mammalian trypsin. It is shown. In addition, mammalian trypsin has an optimum working temperature of 60-70 ° C. and is stable to heat. However, the trypsin of Itoshima starfish inhabiting at low temperature has an optimum temperature of around 50 ° C., and is also stable against heat. Lower than that of animals. Therefore, it is considered that trypsin of Itokimaki starfish is particularly suitable for use in food processing performed at low temperatures.

  The present invention is not limited to the configurations described above, and various modifications are possible within the scope shown in the claims, and are described in the best mode for carrying out the invention, examples, and the like. Embodiments and examples obtained by appropriately combining different technical means are also included in the technical scope of the present invention.

  As described above, in the present invention, trypsin derived from the starfish starfish having a novel amino acid sequence that has not been known so far has been found. Therefore, according to the present invention, the reliability is higher than that derived from mammals, and it is possible to contribute to the effective use of starfishes that have been unused biomass. Therefore, the present invention can be applied to a wide range of fields related to reagents and pharmaceuticals using biomass.

It is a figure explaining the principle of the SMART method used by 5'RACE in the Example. It is a figure which shows the result of having compared the sequence of the signal and precursor amino acid in Itomaki starfish pyloric blind trypsin and various vertebrate trypsin. It is a figure which shows the result of having compared the amino acid sequence of the mature type trypsin of Itoma starfish and bovine pancreatic trypsin. It is a figure of electrophoresis which shows the result of having prepared the total RNA from a starfish pyloric blind. It is a figure of electrophoresis which shows the result of having partially amplified the trypsin gene of Itomaki starfish pyloric blind.

Claims (9)

A polypeptide having trypsin activity comprising:
(A) the amino acid sequence shown in SEQ ID NO: 1; or (b) the amino acid sequence shown in SEQ ID NO: 1 consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added. A polypeptide characterized by. A polypeptide having trypsin activity comprising:
(A) a polynucleotide comprising the base sequence shown in SEQ ID NO: 2; or (b) a base in which one or several bases have been deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 2. A polypeptide encoded by a polynucleotide comprising a sequence. A polypeptide having trypsin activity comprising:
(C) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 2; or
(D) A polypeptide encoded by a polynucleotide comprising a base sequence that is at least 80% identical to a base sequence complementary to the base sequence shown in SEQ ID NO: 2.   A polynucleotide encoding the polypeptide according to any one of claims 1 to 3.   An expression vector comprising the polynucleotide according to claim 4.   A transformant into which the expression vector according to claim 5 has been introduced.   A gene detection instrument using, as a probe, at least a part of the base sequence or its complementary sequence in the polynucleotide according to claim 4.   An antibody that specifically binds to the polypeptide according to any one of claims 1 to 3. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 3.