Signal peptide

Abstract

To suppress the production of poorly cleaved hedgehog protein during hedgehog protein production in a plant expression system.SOLUTION: Provided is use of a signal peptide to produce hedgehog protein in a plant expression system.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to the use of signal peptides in the production of hedgehog proteins in plant expression systems. [Background technology]

[0002] Signal peptides are peptides present in the N-terminal region of secretory protein precursors and membrane-bound protein precursors, and contribute to the translation, transport, and localization of these proteins. Signal peptides are typically cleaved by signal peptidases after transmembrane transport. Rice α-amylase-derived signal peptides (RSPs) are known to be used as signal peptides for protein expression in plants (Non-patent document 1: Kalthoff, D. et al. (2010) Journal Virology 84: 12002-12010). Hedgehog proteins are proteins that contribute to embryonic development, and known hedgehog proteins include sonic hedgehog (SHH), Indian hedgehog (IHH), and desert hedgehog (DHH). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Kalthoff, D. et al. (2010) Journal Virology 84: 12002-12010 [Overview of the project] [Problems that the invention aims to solve]

[0004] In plant expression systems, it is known that signal peptides (RSPs) derived from rice α-amylase are used to express secreted proteins. However, when the inventors used RSP to produce hedgehog protein in plants, they found that the polypeptide containing RSP and hedgehog protein was cleaved at a site different from the intended cleavage site by the signal peptidase, resulting in the production of hedgehog protein with an amino acid sequence different from the intact N-terminal amino acid sequence. It is desirable to suppress the production of such poorly cleaved hedgehog protein as much as possible. The inventors first identified this problem by attempting to use RSP for the production of hedgehog protein. [Means for solving the problem]

[0005] The inventors conducted diligent research to solve the above problems and, as a result, have succeeded in significantly suppressing the production of poorly cleaved hedgehog proteins compared to the conventional RSP technology by using a signal peptide in which the second amino acid from the C-terminus is an acidic amino acid (glutamic acid (Glu) or aspartic acid (Asp)) or phenylalanine (Phe) in the production of hedgehog proteins in a plant expression system, thereby completing the present invention. In other words, the present invention is as follows:

[0006] [1] The use of a signal peptide in the production of hedgehog protein in a plant expression system, wherein the signal peptide is defined by the following formula I: M-(X1)i-X2-X3-X4-X5 Formula I (In the formula, M is Met, X1 is an amino acid sequence consisting of 1 to 22 amino acids, where i is either 0 or 1. X2 is an amino acid sequence consisting of 3 to 19 amino acids, including at least 3 Leu molecules. X3 is either Ala or Val. X4 is Glu, Asp, or Phe. X5 is either Ala or Ser. The above-mentioned use includes an amino acid sequence consisting of 7 to 45 amino acids as shown, and has the activity to be cleaved by a signal peptidase. [2] The signal peptide, (a) Polypeptides containing the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16 (b) A polypeptide having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16, and having the activity to be cleaved by a signal peptidase, or (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16, and which is cleaved by a signal peptidase. The use described in [1] above. [3] (i) Equation I below: M-(X1)i-X2-X3-X4-X5 Formula I (In the formula, M is Met, X1 is an amino acid sequence consisting of 1 to 22 amino acids, where i is either 0 or 1. X2 is an amino acid sequence consisting of 3 to 19 amino acids, including at least 3 Leu molecules. X3 is either Ala or Val. X4 is Glu, Asp, or Phe. X5 is either Ala or Ser. A polynucleotide comprising an amino acid sequence consisting of 7 to 45 amino acids as shown, and encoding a signal peptide having the activity to be cleaved by a signal peptidase, (ii) Polynucleotides encoding the hedgehog protein and A nucleic acid construct comprising the signal peptide, wherein the polynucleotide encoding the signal peptide is ligated to the 5' end of the polynucleotide encoding the hedgehog protein. [4] The signal peptide, (a) A polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 5, 11, 13 or 16, (b) A polypeptide having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 3, 5, 11, 13 or 16 and having the activity of being cleaved by signal peptidase, or (c) A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 3, 5, 11, 13 or 16 and having the activity of being cleaved by signal peptidase is the nucleic acid construct according to [3] above. [5] A recombinant vector comprising the nucleic acid construct according to [3] or [4] above. [6] A transformant comprising the nucleic acid construct according to [3] or [4] above and / or the recombinant vector according to [5] above. [7] The transformant according to [6] above, wherein the transformant is a plant, a part thereof or a plant cell. [8] A method for producing a hedgehog protein, comprising the step of culturing or cultivating the transformant according to [6] or [7] above. [9] A method for producing a hedgehog protein, comprising the following steps (a) and (b): (a) A step of introducing the nucleic acid construct according to [3] or [4] above and / or the recombinant vector according to [5] above into a host to obtain a transformant, and (b) A step of recovering the hedgehog protein produced by the transformant obtained in step (a) comprising the method.

Advantages of the Invention

[0007] According to the present invention, it is possible to suppress the production of a hedgehog protein with poor cleavage in a plant expression system.

Brief Description of the Drawings

[0008] [Figure 1] This figure shows the alignment of various hedgehog proteins. [Figure 2] This figure shows the results of measuring the signal intensity of poorly cleaved hedgehog proteins when various signal peptides were used to generate hedgehog proteins. [Figure 3] This figure shows the alignment analysis results of the amino acid sequences of various signal peptides. [Figure 4] This figure shows the results of LC-MS analysis of hedgehog proteins generated using signal peptides RSP, C11S, and C20S. [Modes for carrying out the invention]

[0009] The present invention will now be described in detail. The following embodiments are illustrative for illustrating the present invention and are not intended to limit the present invention to these embodiments only. The present invention can be implemented in various forms without departing from its spirit.

[0010] 1. overview In plant expression systems, it is known that signal peptides (RSPs) derived from rice α-amylase are used to express secreted proteins. However, when the inventors used RSP to produce hedgehog protein in plants, they found that the polypeptide containing RSP and hedgehog protein was cleaved at a site different from the intended cleavage site by signal peptidase, resulting in the production of hedgehog protein with an amino acid sequence different from the intact N-terminal amino acid sequence. It is desirable to suppress the production of such poorly cleaved hedgehog protein as much as possible. To address these challenges, the inventors have succeeded in significantly suppressing the production of poorly cleaved hedgehog proteins compared to the conventional RSP technique by using a signal peptide in which the second amino acid from the C-terminus is Glu, Asp, or Phe during the generation of hedgehog proteins in a plant expression system. This invention improves the accuracy of signal peptidase cleaving signal peptides and hedgehog proteins at their intended cleavage sites during hedgehog protein production in plant expression systems. As a result, compared to conventional techniques, it is possible to suppress the production of poorly cleaved hedgehog proteins and improve the production rate of hedgehog proteins with intact N-terminal amino acid sequences. Having such effects, this invention is extremely useful industrially because it can provide homogeneous hedgehog proteins.

[0011] 2. Signal peptide The signal peptide of the present invention is not limited as long as the amino acid at the second position from the C-terminus (X4 in formula I below) is an acidic amino acid (specifically, glutamic acid (Glu) or aspartic acid (Asp)) or phenylalanine (Phe).

[0012] More specifically, see equation I below: M-(X1)i-X2-X3-X4-X5 Formula I (In the formula, M is Met, X1 is an amino acid sequence consisting of 1 to 22 amino acids, where i is either 0 or 1. X2 is an amino acid sequence consisting of 3 to 19 amino acids, including at least 3 Leu molecules. X3 is either Ala or Val. X4 is Glu, Asp, or Phe. X5 is either Ala or Ser. Preferably, the amino acid sequence contains the sequence shown and has the activity to be cleaved by a signal peptidase.

[0013] In general, signal peptides are known to lack amino acid sequence homology among different signal peptides, but they are known to share a common structural motif. This structural motif is typically known to consist of an N-terminal region, a hydrophobic region (H region), and a C-terminal region. The N-terminal region contains positively charged amino acids, the hydrophobic region contains at least three residues (e.g., 3-5 residues) of Leu, and the C-terminal region contains uncharged amino acids (Hajar Owji, et al., European Journal of Cell Biology, 97 (2018) 422-441, K. Hatsuzawa, et al., J Biochem. 1997 Feb;121(2):270-7). The hydrophobic region is located between the N-terminal and C-terminal regions. In the above formula I of the present invention, the N-terminal region of the signal peptide corresponds to the amino acid sequence M-X1, the hydrophobic region and part of the C-terminal region correspond to the amino acid sequence X2, and the 3rd to 1st amino acids from the C-terminus correspond to X3-X4-X5.

[0014] Signal peptides have the activity or function of transporting proteins containing the signal peptide at its N-terminus within cells (e.g., to the endoplasmic reticulum) and then being cleaved by signal peptidases after transport. On the other hand, as mentioned above, generally, signal peptides do not have amino acid sequence homology with each other (K. Hatsuzawa, et al., J Biochem. 1997 Feb;121(2):270-7). In other words, the amino acid sequence of a signal peptide is not limited to a specific amino acid sequence in order to have its activity or function. Furthermore, in the present invention, whether or not the signal peptide has activity or function can be easily confirmed by those skilled in the art by examining whether or not the signal peptide is cleaved in a plant expression system, according to known methods (Raif Musa-Aziz, et al., Proc Natl Acad Sci US A. 2009 Mar 31;106(13):5406-11). Specifically, if it can be confirmed that a hedgehog protein of the appropriate molecular weight (19.5 kDa) is produced in the transformant using known immunological methods, such as Western blotting, then it can be confirmed that the signal peptide attached to the hedgehog protein has the activity to be cleaved by signal peptidase or has exhibited such function.

[0015] As stated above, generally speaking, the amino acid sequence of a signal peptide is not limited to a specific amino acid sequence in order to have its activity or exert its function. In the signal peptide of the present invention, X1 is an amino acid sequence consisting of 1 to 22 amino acids, but the type and number of amino acids or amino acid sequences constituting X1 are not limited. That is, in the signal peptide of the present invention, X1 includes an amino acid or amino acid sequence consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids. Furthermore, the signal peptide of the present invention includes polypeptides that do not have the amino acid sequence of X1 (i.e., i in (X1)i in formula I is 0) (for example, "C20S" described later). Such polypeptides have also been shown to function as signal peptides (e.g., Example 4).

[0016] In the signal peptide of the present invention, the amino acid sequence constituting X2 is an amino acid sequence consisting of 3 to 19 amino acids, including at least 3 leucine (Leu) molecules. Generally, the amino acid sequence constituting the hydrophobic region of a peptide that functions as a signal peptide contains at least three (e.g., 3 to 5) leucine molecules. In the present invention, the amino acid sequence of X2 may contain leucine as a continuous sequence or discontinuously (for example, with another amino acid between leucine molecules). In the present invention, the amino acid sequence of X2 includes, for example, amino acid sequences consisting of 19, 18, 17, 16, and 15 amino acids, but is not limited to these. In the signal peptide of the present invention, X3 is alanine (Ala) or valine (Val), X4 is the negative (acidic) amino acid glutamic acid (Glu), aspartic acid (Asp), or phenylalanine (Phe), and X5 is alanine (Ala) or serine (Ser). In this invention, by using a signal peptide in which X4 is Glu, Asp, or Phe in the generation of hedgehog protein in a plant expression system, the generation of poorly cleaved hedgehog protein can be significantly suppressed compared to the conventional RSP technique.

[0017] In Formula I of the present invention, the sequence of X3-X4-X5 is not limited to, and examples include valine(V)-aspartic acid(D)-serine(S), valine(V)-aspartic acid(D)-alanine(A), valine(V)-glutamic acid(E)-serine(S), VEA, valine(V)-phenylalanine(F)-serine(S), VFA, ADS, ADA, AES, AEA, AFS, and AFA, with VDS, VDA, VEA, and AFA being preferred.

[0018] In the present invention, the length of the amino acid sequence of the signal peptide is 7 to 60, preferably 10 to 45, more preferably 20 to 45, and even more preferably 23 to 45.

[0019] In the present invention, the "activity of being cleaved by signal peptidase" of a signal peptide means that when a hedgehog protein fused with the signal peptide is expressed in a plant expression system, the signal peptide is cleaved by signal peptidase between the C-terminal amino acid of the signal peptide and the N-terminal amino acid of the hedgehog protein. The presence of this activity of the signal peptide can be easily confirmed by a known method (Raif Musa-Aziz, et al., Proc Natl Acad Sci US A. 2009 Mar 31;106(13):5406-11), for example, by examining whether or not the signal peptide is cleaved in a plant expression system. More specifically, whether or not a signal peptide has "activity to be cleaved by a signal peptidase" can be confirmed by introducing a nucleic acid construct containing a polynucleotide encoding the signal peptide and a polynucleotide encoding the hedgehog protein into a host, collecting the protein from the resulting transformant, and subjecting the collected protein to a known immunological method, such as Western blotting, to see if a signal is detected at a position corresponding to the appropriate molecular weight (19.5 kDa) of the hedgehog protein. Thus, whether or not the signal peptide under test has "activity to be cleaved by signal peptidases" can be easily confirmed by those skilled in the art using known immunological methods.

[0020] In this specification, the terms "host" and "transformer" are not limited to any nucleic acid construct that can ultimately produce the hedgehog protein from a polynucleotide encoding a signal peptide and a polynucleotide encoding the hedgehog protein. Examples of such "hosts" and "transformers" are not limited to, but include, a plant or a part thereof (e.g., organs, tissues), plant cells (including plant cultured cells), etc.

[0021] In this invention, "plant expression system" refers to a system in a plant in which a target gene is expressed, a target protein is translated, and the target protein is produced. In this specification, "production" means that the target protein (hedgehog protein in this invention) is produced in the plant. In this specification, "generation of hedgehog protein in a plant expression system" means that in a plant, a protein containing a signal peptide and a hedgehog protein is expressed from a nucleic acid construct containing a polynucleotide encoding a signal peptide and a polynucleotide encoding a hedgehog protein, and the signal peptide is cleaved by a signal peptidase to produce a hedgehog protein. In this specification, “plant” includes, but is not limited to, a plant body or a part thereof (e.g., organs, tissues), or plant cells (including plant cultured cells).

[0022] The signal peptides of the present invention are not limited as long as they function in a plant expression system. Examples of such signal peptides include plant signal peptides. Plant signal peptides can be searched for by in silico analysis using known databases such as The Universal Protein Resource (UniProt). Specifically, for example, by using the search formula taxonomy:viridiplantae annotation:(type:signal evidence:experimental) in the UniProt database (https: / / www.uniprot.org / uniprot / ), it is possible to search for plant signal peptides that have been experimentally demonstrated to function as signal peptides in plants and obtain their amino acid sequence information. Furthermore, it is also possible to obtain CDS sequences from publicly available genome databases or sequencing information such as proprietary RNAseq, and predict and obtain sequences that may function as signal peptides using analysis tools such as SignalP (https: / / services.healthtech.dtu.dk / service.php?SignalP-6.0).

[0023] The table below shows examples of plant signal peptides that have been experimentally demonstrated to function as signal peptides in plants through in silico analysis, but such plant signal peptides are not limited to these.

[0024] [Table 1]

[0025] Examples of signal peptides in the present invention include, but are not limited to, the signal peptides shown in the table below.

[0026] [Table 2]

[0027] Table 3 shows the correspondence between the amino acid sequences of the signal peptides exemplified in Table 2 above and the following formula I of the present invention. M-(X1)i-X2-X3-X4-X5 Formula I (In the formula, M is Met, X1 is an amino acid sequence consisting of 1 to 22 amino acids, where i is either 0 or 1. X2 is an amino acid sequence consisting of 3 to 19 amino acids, including at least 3 Leu molecules. X3 is either Ala or Val. X4 is Glu, Asp, or Phe. X5 is either Ala or Ser.

[0028] [Table 3] When C20S shown in Table 3 above is represented by equation I, i is 0.

[0029] The signal peptide in this invention is one of the following polypeptides (a) to (c): (a) Polypeptides containing the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16 (b) A polypeptide having at least 80% sequence identity with the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16, and having the activity to be cleaved by a signal peptidase, or (c) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16, and which is cleaved by a signal peptidase. It includes.

[0030] In the present invention, "polypeptide containing the amino acid sequence of SEQ ID NO: 3, 5, 11, 13, or 16" includes polypeptides consisting of the amino acid sequence of SEQ ID NO: 3, 5, 11, 13, or 16.

[0031] Furthermore, the signal peptides in the present invention include polypeptides that contain the amino acid sequences of SEQ ID NOs. 3, 5, 11, 13, or 16, as well as amino acid sequences having 80% or more sequence identity with the amino acid sequences of SEQ ID NOs. 3, 5, 11, 13, or 16, and that have the activity to be cleaved by a signal peptidase. Such polypeptides include polypeptides that contain an amino acid sequence with approximately 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16, and that have the activity to be cleaved by a signal peptidase. Sequence identity can be checked using homology searches such as FASTA, BLAST, and PSI-BLAST at homology search sites using the internet, such as the DNA Data Bank of Japan (DDBJ). It can also be checked using BLAST at the National Center for Biotechnology Information (NCBI).

[0032] Furthermore, "amino acid sequences in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequences of SEQ ID NOs: 3, 5, 11, 13, or 16" include, for example, (i) an amino acid sequence in which 1 to 10 amino acids (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) are deleted from the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16, (ii) an amino acid sequence in which 1 to 10 amino acids (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) in the amino acid sequence of SEQ ID NOs. 3, 5, 11, 13, or 16 are replaced by other amino acids. (iii) Amino acid sequences in which 1 to 10 amino acids (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) are inserted into the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16. (iv) an amino acid sequence to which 1 to 10 amino acids (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) have been added to the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16, (v) Amino acid sequences mutated by the combinations of (i) to (iv) above These are some examples.

[0033] In the present invention, the presence or absence of "activity that can be cleaved by signal peptidase" is as follows: Those skilled in the art can easily confirm this by examining whether or not the signal peptide is cleaved in a plant expression system, following known methods (Raif Musa-Aziz, et al., Proc Natl Acad Sci US A. 2009 Mar 31;106(13):5406-11). Specifically, if it can be confirmed that a hedgehog protein of the appropriate molecular weight (19.5 kDa) is produced in the transformant using known immunological methods, such as Western blotting, then it can be confirmed that the signal peptide attached to the hedgehog protein has the activity to be cleaved by a signal peptidase. In this invention, "signal peptidase" refers to an enzyme that cleaves signal peptides, and is not limited to any signal peptidase present in plants. For example, signal peptidases are found in the endoplasmic reticulum. Furthermore, "having activity that can be cleaved by signal peptidase" means having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and preferably 90% or more activity compared to the activity of a polypeptide consisting of the amino acid sequence of SEQ ID NOs. 3, 5, 11, 13, or 16, which is set to 100%.

[0034] To prepare polypeptides having the above mutations, mutations can be introduced into the polynucleotides encoding the polypeptides using site-directed mutagenesis methods such as the Kunkel method or the Gapped duplex method, as well as mutagenesis kits such as QuikChange. TM Site-Directed Mutagenesis Kit (manufactured by Stratagene), GeneTailor TM This can be performed using a Site-Directed Mutagenesis System (Invitrogen), a TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio), or similar systems. Alternatively, methods such as site-directed mutagenesis described in "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)) can be used.

[0035] 3. Hedgehog protein Hedgehog proteins are known to be involved in embryonic development. The hedgehog proteins of the present invention include sonic hedgehog (SHH) protein, Indian hedgehog (IHH) protein, and desert hedgehog (DHH) protein. These hedgehog proteins exhibit high sequence identity with one another. Figure 1 shows the amino acid sequence alignment of human SHH protein, human IHH protein, and human DHH protein. In Figure 1, the cleavage sites by signal peptidase and the cleavage sites by autolysis (auto-processing) are indicated by lines, respectively. In the present invention, the nucleotide sequence of the hedgehog protein is designed such that a stop codon is added to the amino acid immediately N-terminal to the self-processing cleavage site. A polypeptide having an amino acid sequence obtained by removing the amino acid sequence of the signal peptide (MLLLARCLLLVLVSSLLVCSGLA) and the amino acid sequence removed by self-processing (265 amino acid residues from the C-terminus in the case of human hedgehog protein) from the amino acid sequence of the hedgehog protein precursor protein is then produced.

[0036] The hedgehog protein in this invention may be derived from any mammal. Such mammals are not limited to, but include, for example, mice, rats, rabbits, cats, dogs, goats, monkeys, and humans, preferably mice, rats, cats, dogs, and humans, and more preferably humans. As an example of the nucleotide and amino acid sequences of these hedgehog proteins, the nucleotide and amino acid sequences of the precursor protein (preproprotein) of the human sonic hedgehog (SHH) protein are shown in SEQ ID NOs. 18 and 19, respectively. The nucleotide and amino acid sequences of the precursor protein of the human SHH protein are registered in the GenBank database under the following predetermined accession numbers.

[0037] Nucleotide sequence of the DNA encoding the human SHH protein: NM_000193.4 (SEQ ID NO: 18) Amino acid sequence of human SHH protein: NP_000184.1 (SEQ ID NO: 19)

[0038] The hedgehog protein in the present invention includes polypeptides having an amino acid sequence obtained by subtracting the amino acid sequence of the signal peptide (MLLLARCLLLVLVSSLLVCSGLA) and the amino acid sequence removed by self-processing from the amino acid sequence of the precursor protein of the human SHH protein described above. Specifically, the hedgehog protein in the present invention includes polypeptides having the following amino acid sequence. Amino acid sequence of human SHH protein (excluding the amino acid sequence of the signal peptide and amino acid sequences removed by self-processing): CGPGRGFGKRRHPKKLTPLAYKQFIPNVAEKTLGASGRYEGKISRNSERFKELTPNYNPDIIFKDEENTGADRLMTQRCKDKLNALAISVMNQWPGVKLRVTEGWDEDGHHSEESLHYEGRAVDITTSDRDRSKYGMLARLAVEAGFDWVYYESKAHIHCSVKAENSVAAKSGG (Sequence ID 21)

[0039] Furthermore, the hedgehog protein in this invention includes those with modified amino acid sequences. Specifically, the hedgehog protein in this invention is not limited to, but includes, for example, a modified human SHH protein containing the following amino acid sequence. Amino acid sequence of modified human SHH protein: IIGPGRGFGKRRHPKKLTPLAYKQFIPNVAEKTLGASGRYEGKISRNSERFKELTPNYNPDIIFKDEENTGADRLMTQRCKDKLNALAISVMNQWPGVKLRVTEGWDEDGHHSEESLHYEGRAVDITTSDRDRSKYGMLARLAVEAGFDWVYYESKAHIHCSVKAENSVAAKSGG (Sequence ID 23)

[0040] In the amino acid sequence of this modified human SHH protein, the N-terminal amino acid (cysteine ​​(C)) in the amino acid sequence of SEQ ID NO: 21 has been modified to two isoleucine (II) molecules. However, the hedgehog proteins in the present invention are not limited to these. For example, the hedgehog proteins in the present invention include those in which a tag and a protease recognition sequence have been added to the C-terminal amino acid of the amino acid sequence of the above-mentioned human SHH protein or modified human SHH protein, as well as those in which the amino acid sequence containing the tag and protease recognition sequence has been excised with a protease after the protein has been expressed. Examples of the above tags include, but are not limited to, His tags, HA tags, FLAG tags, and Myc tags. Examples of proteases include, but are not limited to, Furin, TEV protease, HRV 3C protease, thrombin, factor Xa, and enterokinase. Examples of protease recognition sequences include, but are not limited to, Furin recognition sequences, TEV protease recognition sequences, HRV 3C protease recognition sequences, thrombin recognition sequences, factor Xa recognition sequences, and enterokinase recognition sequences. In this specification, when we refer to the "N-terminal amino acid" or "N-terminal amino acid sequence" of the hedgehog protein, we mean the N-terminal amino acid or N-terminal amino acid sequence in the amino acid sequence of the polypeptide obtained by removing the signal peptide from the precursor protein of the hedgehog protein (e.g., SEQ ID NO: 21 or 23). Similarly, when we refer to the "C-terminal amino acid" or "C-terminal amino acid sequence" of the hedgehog protein in this specification, we mean the C-terminal amino acid or C-terminal amino acid sequence in the amino acid sequence of the polypeptide obtained by removing the amino acid sequence removed by self-processing from the precursor protein of the hedgehog protein (e.g., SEQ ID NO: 21 or 23).

[0041] The hedgehog protein in this invention includes the following polypeptides (a) to (c): (a) A polypeptide containing the amino acid sequence of SEQ ID NO: 21 or 23 (b) A polypeptide having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 21 or 23, and having hedgehog protein activity, or (c) A polypeptide having hedgehog protein activity, comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 21 or 23. It includes.

[0042] In the present invention, "polypeptide containing the amino acid sequence of SEQ ID NO: 21 or 23" includes polypeptides consisting of the amino acid sequence of SEQ ID NO: 21 or 23.

[0043] Furthermore, the hedgehog protein in the present invention includes a polypeptide that contains the amino acid sequence of SEQ ID NO: 21 or 23, as well as an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 21 or 23, and that has the activity of a hedgehog protein. Such polypeptides include polypeptides that contain an amino acid sequence with approximately 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 21 or 23, and that also possess hedgehog protein activity. Sequence identity can be checked using homology searches such as FASTA, BLAST, and PSI-BLAST at homology search sites using the internet, such as the DNA Data Bank of Japan (DDBJ). It can also be checked using BLAST at the National Center for Biotechnology Information (NCBI).

[0044] Furthermore, "amino acid sequences in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 21 or 23" include, for example, (i) an amino acid sequence in which 1 to 10 amino acids (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) are deleted from the amino acid sequence of SEQ ID NO: 21 or 23, (ii) an amino acid sequence in which 1 to 10 amino acids (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) in the amino acid sequence of SEQ ID NO: 21 or 23 are replaced by other amino acids. (iii) An amino acid sequence in which 1 to 10 amino acids (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) are inserted into the amino acid sequence of SEQ ID NO: 21 or 23. (iv) an amino acid sequence to which 1 to 10 amino acids (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, even more preferably 1) have been added to the amino acid sequence of SEQ ID NO: 21 or 23, (v) Amino acid sequences mutated by the combinations of (i) to (iv) above These are some examples.

[0045] In the present invention, the presence or absence of "hedgehog protein activity" can be evaluated based on known methods. For example, as described in Example 7 of this specification, the presence or absence of hedgehog protein activity for the test polypeptide can be determined by treating mouse embryo-derived C3H10T1 / 2 cells with the test polypeptide, culturing them, and measuring alkaline phosphatase (ALP) activity. Furthermore, "hedgehog protein activity" is ED 50 It can be evaluated by measuring the value. The strength of hedgehog protein activity is measured by ED. 50 It can be evaluated based on the absolute value of the value. Also, ED 50 The fact that a value was measured means that the polypeptide being measured possesses hedgehog protein activity.

[0046] The method for introducing mutations to prepare polypeptides having the above mutations is the same as for the "signal peptides" described above.

[0047] In this specification, the "intact" N-terminus of a hedgehog protein means the N-terminal amino acid or N-terminal amino acid sequence of the hedgehog protein that is produced when it is cleaved at its original cleavage site by a signal peptidase. Here, the "original cleavage site" refers to the area between the C-terminal amino acid of the signal peptide and the N-terminal amino acid of the hedgehog protein. For example, if the signal peptide is C2S in Table 2 above, and the hedgehog protein is the modified human SHH protein described above, the original cleavage site by the signal peptidase is between the C-terminal amino acid Ala of the signal peptide and the N-terminal amino acid Ile of the modified human SHH protein (Figure 3). Furthermore, "when cleaved" refers to the time when the signal peptide and the hedgehog protein are cleaved. The intact N-terminus is the N-terminal amino acid or N-terminal amino acid sequence that, compared to the N-terminus of the original amino acid sequence, does not have any added or deleted amino acids or amino acid sequences. Here, "original amino acid sequence" refers to the amino acid sequence of the Hedgehog protein before the signal peptide is fused. More specifically, the "intact N-terminus" refers to the N-terminal amino acid sequence containing "CGP" in the case of the human SHH protein, and to the N-terminal amino acid sequence containing "IIGP" in the case of the modified human SHH protein described above. The same applies to other Hedgehog proteins.

[0048] On the other hand, in this specification, an N-terminus in which a signal peptide and a hedgehog protein are cleaved at a site different from the original cleavage site and has the addition or deletion of amino acids or amino acid sequences is referred to as a "malcleaved" N-terminus, and a protein having such a malcleaved N-terminus is referred to as a "malcleaved" protein. Examples of malcleaved N-termins are not limited to those specified, and include N-terminal amino acid sequences in which the amino acid sequence VAHA is added to the N-terminus, and N-terminal amino acid sequences in which the amino acid sequence IIGPGRGFGK or IIGPGRGFG is missing.

[0049] 4. Polynucleotides, nucleic acid constructs The present invention provides a nucleic acid construct comprising a polynucleotide encoding a signal peptide and a polynucleotide encoding a hedgehog protein. In the nucleic acid construct of the present invention, the 3' end of the polynucleotide encoding the signal peptide is ligated to the 5' end of the polynucleotide encoding the hedgehog protein. In the present invention, "polynucleotide" includes DNA and RNA. The nucleic acid constructs of the present invention are specifically as follows: (i) Equation I below: M-(X1)i-X2-X3-X4-X5 Formula I (In the formula, M is Met, X1 is an amino acid sequence consisting of 1 to 22 amino acids, where i is either 0 or 1. X2 is an amino acid sequence consisting of 3 to 19 amino acids, including at least 3 Leu molecules. X3 is either Ala or Val. X4 is Glu, Asp, or Phe. X5 is either Ala or Ser. A polynucleotide comprising an amino acid sequence consisting of 7 to 45 amino acids as shown, and encoding a signal peptide having the activity to be cleaved by a signal peptidase, (ii) Polynucleotides encoding the hedgehog protein and A nucleic acid construct that includes, A nucleic acid construct in which the polynucleotide encoding the signal peptide is ligated to the 5' end of the polynucleotide encoding the hedgehog protein.

[0050] The polynucleotide encoding the signal peptide of the present invention is not limited in its nucleotide sequence, as long as it encodes the signal peptide described in "2. Signal Peptides" above. The polynucleotide encoding the signal peptide of the present invention can be prepared by obtaining information on the nucleotide sequence encoding the plant signal peptide by in silico analysis as described in "2. Signal Peptides" above. Specifically, this information can be obtained, for example, by searching for the accession number in a known gene database, such as the UniProt database. Alternatively, it can be obtained by purchasing commercially available polynucleotides.

[0051] The nucleotide sequences of the polynucleotides encoding the signal peptides of the present invention, as exemplified in Table 2 above, are shown in the table below.

[0052] [Table 4]

[0053] The polynucleotides encoding the signal peptide of the present invention are not limited to those exemplified in Table 4 above, and their nucleotide sequences can be optimized for the host type by optimizing their codons.

[0054] As the polynucleotide encoding the signal peptide of the present invention, in addition to a polynucleotide containing or consisting of the nucleotide sequence of SEQ ID NOs: 2, 4, 10, 12, or 15, a polynucleotide encoding a signal peptide that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NOs: 2, 4, 10, 12, or 15 and has the activity to be cleaved by a signal peptidase can be used. In the present invention, "stringent conditions" may be any of low stringent conditions, medium stringent conditions, or high stringent conditions. "Low stringent conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 32°C. "Medium stringent conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 42°C. "Highly stringent conditions" include, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 50°C. For detailed procedures of the hybridization method, refer to "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)), etc. Furthermore, as the polynucleotide of the present invention, it is possible to use a polynucleotide that has 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the nucleotide sequence of SEQ ID NOs. 2, 4, 10, 12, or 15, and that encodes a signal peptide that has the activity to be cleaved by a signal peptidase.

[0055] The polynucleotide encoding the hedgehog protein of the present invention is not limited to those encoding the hedgehog protein described in "3. Hedgehog Protein" above. That is, examples of polynucleotides encoding the hedgehog protein of the present invention include the polynucleotides encoding the sonic hedgehog (SHH) protein, the Indian hedgehog (IHH) protein, and the desert hedgehog (DHH) protein, respectively, as described in "3. Hedgehog Protein," preferably the polynucleotide encoding the human SHH protein. In the preparation of nucleic acid constructs, a polynucleotide containing the nucleotide sequence encoding the Hedgehog protein can be used. This polynucleotide is obtained by excluding the nucleotide sequence encoding the Hedgehog protein precursor from the nucleotide sequence encoding the signal peptide and the portion removed by self-processing. Alternatively, a polynucleotide containing a nucleotide sequence with optimized codons depending on the host type can be used. Examples of such polynucleotides include those encoding human SHH protein or modified human SHH protein containing the amino acid sequence of SEQ ID NO: 21 or 23. The nucleotide sequences of these polynucleotides encoding human SHH protein and modified human SHH protein are shown as SEQ ID NOs: 20 and 22, respectively. The nucleotide sequences of SEQ ID NOs: 20 and 22 have been optimized to match the codon frequency of Nicotiana benthamiana.

[0056] The polynucleotides encoding the hedgehog protein of the present invention are not limited to those exemplified above.

[0057] As the polynucleotide encoding the hedgehog protein of the present invention, in addition to a polynucleotide containing or consisting of the nucleotide sequence of SEQ ID NO: 20 or 22, a polynucleotide encoding a polypeptide having hedgehog protein activity can be used, which is hybridized under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 20 or 22. In the present invention, the stringent conditions and the detailed procedure of the hybridization method are as described above. Furthermore, as the polynucleotide encoding the hedgehog protein of the present invention, a polynucleotide encoding a polypeptide having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology to the nucleotide sequence of SEQ ID NO: 20 or 22, and having the activity of the hedgehog protein, can be used. The presence or absence of "hedgehog protein activity" can be evaluated based on known methods. For example, as described in Example 7 of this specification, the presence or absence of hedgehog protein activity for the test polypeptide can be determined by treating mouse embryo-derived C3H10T1 / 2 cells with the test polypeptide, culturing them, and measuring alkaline phosphatase (ALP) activity.

[0058] The method for introducing mutations into polynucleotides is the same as that described above for "signal peptides."

[0059] Nucleic acid constructs in which the 3' end of a polynucleotide encoding a signal peptide is ligated to the 5' end of a polynucleotide encoding a hedgehog protein can be prepared, for example, by artificial synthesis of the ligated gene, overlap extension PCR, in-fusion cloning, or golden gate cloning.

[0060] 5. Recombinant vectors The present invention provides a recombinant vector comprising the nucleic acid construct of the present invention. In the present invention, the recombinant vector is not limited to those containing the nucleic acid construct of the present invention as described in 4. above, but for example, plasmid vectors, viral vectors, etc., can be used. Examples of plasmid vectors when using plants as hosts include, but are not limited to, pRI vectors (pRI101, pRI909, pRI910, pRI201, etc.). Examples of viral vectors when using plants as hosts include, for example, plant virus vectors, specifically, TMV vectors, PVX vectors, CPMV vectors, CMV vectors, PPV vectors, AIMV vectors, ZYMV vectors, etc. The vector used in the present invention may be either a transient expression vector or a constitutive expression vector. Those skilled in the art can appropriately select the vector to be used depending on the type of host and purpose to which the vector is introduced. The recombinant vector of the present invention may optionally include, in addition to the nucleic acid construct of the present invention, cis elements such as enhancers, splicing signals, poly(A) addition signals, ribosome-binding sequences (SD sequences), selection marker genes, reporter genes, etc., and these may also be modified. For example, the 5' untranslated region (5'-UTR) (translational enhancer region) in the recombinant vector can be replaced with another. More specifically, for example, the Arabidopsis thaliana ADH (AtADH) or rice ADH-derived 5'-UTR in the pRI201 vector can be replaced with an AtCOR47-derived 5'-UTR (Yamasaki, S. et al. (2018) Journal of Bioscience and Bioengineering 125: 124-130). Examples of selectable marker genes include dihydrofolate reductase genes, ampicillin resistance genes, neomycin resistance genes, and kanamycin resistance genes. Examples of reporter genes include green fluorescent protein (GFP) or its variants (fluorescent proteins such as EGFP, BFP, and YFP), luciferase, alkaline phosphatase, and LacZ genes.

[0061] 6. Transformed The present invention provides a transformant comprising the nucleic acid construct and / or recombinant vector of the present invention. The transformant of the present invention can be obtained by introducing the nucleic acid construct and / or recombinant vector described in "4." above into a host. The nucleic acid construct of the present invention in the nucleic acid construct and / or recombinant vector introduced into the host may or may not be incorporated into the host genome.

[0062] In this specification, "host" means an organism into which the nucleic acid construct and / or recombinant vector of the present invention is introduced, and which expresses and produces the hedgehog protein of the present invention. In this specification, "host" and "transformer" are not limited to any organism capable of producing hedgehog protein from the nucleic acid construct or recombinant vector of the present invention. Such "hosts" or "transformers" are not limited to, but include, for example, plants, such as plant bodies or parts thereof (e.g., organs, tissues), and plant cells (including plant cultured cells). Examples of plants are not limited to, but include, solanaceous plants (tobacco, tomato, potato, etc.), grasses (rice, wheat, barley, maize, etc.), cruciferous plants (Arabidopsis thaliana, Brassica napus, etc.), daisy family plants (lettuce, etc.), and mosses (Marchantia polymorpha, Physcomitrella patens, etc.), with plants of the genus Nicotiana being preferred. Examples of plants of the genus Nicotiana include, but are not limited to, Nicotiana benthamiana, tobacco (N. tabacum), and Nicotiana excelsior. If the plant is a member of the genus Nicotiana, then the plant body, part thereof, or plant cells include Nicotiana, part thereof, or cells derived from Nicotiana (including cultured cells). The same applies to other plants.

[0063] The introduction of nucleic acid constructs and / or recombinant vectors into a host can be carried out using known methods. Known gene introduction methods include, for example, methods using viral vectors, agro-infection, calcium phosphate method, microinjection method, particle gun method, DEAE-dextran method, electroporation, cationic lipid method, and methods combining these. However, when the host is a plant, methods using plant viral vectors and agro-infection are preferred. Recombinant vectors that can be used in these methods are as described in "5." above.

[0064] In this invention, when a plant is used as a host, the plant is cultured or grown before transformation. If the plant is a part of a plant body or plant cells (including plant cultured cells), these are cultured; if the plant is a plant body, it is grown. Methods for culturing plant cells are well known (Plant Biotechnol J. 2019 Aug; 17(8): 1560-1566 or Front. Plant Sci. 2018 Jan: Volume 9 Article 45). Furthermore, methods for cultivating plants are not limited to those described below. First, sow the seeds in a seedling tray containing fertilizer, and then grow the plants for several days in an artificial climate chamber with adjusted light cycles. If using liquid fertilizer, you can soak a hydroponic urethane mat in the liquid fertilizer and place it in the seedling tray. Next, the seedlings are transplanted into cultivation (early stage) panels, and the transplanted panels are set in an artificial climate chamber and cultivated for several days using, for example, the deep flow technique (DFT method). Subsequently, the plants are removed from the early cultivation panels and transplanted into the late cultivation panels. The transplanted late cultivation panels are then placed in an artificial climate chamber and cultivated for several days using the DFT method to obtain the plants.

[0065] In the method described above, liquid fertilizer can be used, but is not limited to it. When using liquid fertilizer, the liquid fertilizer can be soaked into a hydroponic urethane mat and placed in a seedling tray. In this invention, commercially available liquid fertilizers can be used in appropriate combinations and are not limited to those mentioned above. The liquid fertilizer can be dissolved in dechlorinated water. Furthermore, the liquid fertilizer can be used after adjusting its electrical conductivity and pH, which can be done using methods known to those skilled in the art.

[0066] In the present invention, environmental conditions can be set as follows: temperature of 10-40°C (e.g., 28°C), relative humidity of 60-80% in the early stages of cultivation and 40-60% in the later stages of cultivation, CO2 concentration of 300-5000 ppm (e.g., 400 ppm, 500 ppm), and cultivation period of 0-35 days in the early stages of cultivation (e.g., 6-9 days) and 0-35 days in the later stages of cultivation (e.g., 6-9 days). However, the present invention is not limited to these conditions, and those skilled in the art can appropriately adjust these conditions according to the growth status of the plants, etc. In this invention, the hydroponic cultivation method can mainly consist of the deep flow technique (DFT method) and the nutrient film technique (NFT method).

[0067] As described above, when the host is a plant, methods using plant virus vectors or agro-infection can be used. These methods are well known to those skilled in the art, but a brief explanation of agro-infection as an example is as follows. First, the vector of the present invention described in "5." above is introduced into Agrobacterium by electroporation or the like to transform the Agrobacterium. The Agrobacterium that can be used in the present invention are not limited to, but examples include strain AGL1, strain GV3101, strain LBA4404, strain EHA101, strain EHA105, etc.

[0068] Next, the transformed Agrobacterium are used to infect plant leaves and other parts of the plant. When Agrobacterium infects a plant, polynucleotides containing the nucleic acid construct of the present invention are introduced into the plant. Methods for infecting plants with Agrobacterium include, for example, vacuum infiltration, syringe infiltration, leaf disc method, and foliar spraying. When using the vacuum infiltration method, for example, first, the cultivated plant is turned upside down and immersed in a beaker of Agrobacterium bacterial solution so that all leaves are completely submerged in the solution. Then, the beaker is placed in a vacuum desiccator and left to stand for several minutes (e.g., 1 minute) to reduce the pressure. After that, the valve is opened all at once to restore pressure. After the restoration is complete, the plant is returned to an upright position and placed in an artificial climate chamber. After infection, the plant is cultivated in the artificial climate chamber for 1 to 14 days (e.g., 6 to 9 days) using, for example, the DFT method. The environmental conditions are the same as described above, and those skilled in the art can adjust these conditions as appropriate according to the plant growth conditions, etc.

[0069] This allows for the acquisition of plant transformants. When infecting plants with Agrobacterium, multiple species of Agrobacterium, each containing a different vector, may be used to infect the plants simultaneously. In this case, Agrobacterium species containing vectors other than the vector of the present invention may also be used in combination.

[0070] 7. Use of signal peptides This invention provides the use of signal peptides in the production of hedgehog proteins in plant expression systems. In the generation of hedgehog proteins in plant expression systems, using the signal peptide of the present invention significantly suppresses the generation of poorly cleaved hedgehog proteins compared to the conventional RSP technique, while also improving the generation rate of hedgehog proteins with intact N-terminal amino acid sequences.

[0071] As explained in "2. Signal Peptides" above, in the use of the signal peptides of the present invention, "plant expression system" refers to a system in a plant in which the target gene is expressed, the target protein is translated, and the target protein is produced. In this specification, "production" means that the target protein is produced. As stated above, in this specification, "generation of hedgehog protein in a plant expression system" means that in a plant, a protein containing a signal peptide and a hedgehog protein is expressed from a nucleic acid construct containing a polynucleotide encoding a signal peptide and a polynucleotide encoding a hedgehog protein, and the signal peptide and hedgehog protein are cleaved by a signal peptidase to produce a hedgehog protein. Furthermore, as described in section 6 above, in this specification, "plant" includes, but is not limited to, a plant body or a part thereof (e.g., organs, tissues) or plant cells (including cultured plant cells). The explanation of "Hedgehog protein" is as described in "3. Hedgehog protein" above.

[0072] In one embodiment, the present invention relates to the use of a signal peptide in the production of hedgehog protein in a plant expression system, comprising the following steps (a) and (b): (a) A step of introducing a nucleic acid construct comprising a polynucleotide encoding a signal peptide and a polynucleotide encoding a hedgehog protein and / or a recombinant vector comprising the nucleic acid construct into a host to obtain a transformant, and (b) A step of recovering the hedgehog protein produced from the transformant obtained in step (a), It can include... Details of each step are described in "8. Method for Producing Hedgehog Protein" below.

[0073] 8. Method for producing hedgehog protein This invention provides a method for producing hedgehog protein. The manufacturing method of the present invention can suppress the production rate of poorly cleaved hedgehog proteins compared to the prior art. In other words, the manufacturing method of the present invention can improve the production rate of hedgehog proteins with intact N-terminuses compared to the prior art. An explanation of "intact" N-terminuses is given in "3. Hedgehog Protein" above.

[0074] In one embodiment, the manufacturing method of the present invention includes a step of culturing or growing the transformant of the present invention. By culturing or growing the transformant of the present invention, hedgehog protein is produced in the transformant. Specifically, for example, if the transformant is a plant, hedgehog protein is produced in the transformant by culturing the transformant for 1 to 14 days (for example, 6 to 9 days) using an artificial climate chamber, for example, in a DFT (Deep Fever Treatment) method. Hedgehog protein can be obtained by recovering the produced hedgehog protein. The method for recovering hedgehog protein will be described later. A detailed description of the transformant of the present invention and the method of culturing or growing the same is as described in "7." above.

[0075] In one embodiment, the present invention relates to the following steps (a) and (b): (a) A step of introducing the nucleic acid construct and / or the recombinant vector of the present invention into a host to obtain a transformant, and (b) A step to recover the hedgehog protein produced from the transformant obtained in step (a). This is a method for producing hedgehog protein, which includes [the specified ingredient].

[0076] Step (a) above is a step of introducing a nucleic acid construct (the nucleic acid construct of the present invention) and / or a recombinant vector (the recombinant vector of the present invention) containing the nucleic acid construct into a host to obtain a transformant. When the transformant is cultured or grown, a polypeptide containing the signal peptide and the hedgehog protein is expressed, and the polypeptide is cleaved into the signal peptide and the hedgehog protein by a signal peptidase to produce the hedgehog protein.

[0077] Specifically, step (a) above includes, for example, the following steps (a1) and (a2): (a1) A step of introducing the nucleic acid construct of the present invention and / or the recombinant vector of the present invention into a host to obtain a transformant, and (a2) A process of culturing or growing transformed organisms. Includes.

[0078] The "host," "transformer," and method for obtaining the transformer related to step (a1) are as described in "6. Transformer" above. Specifically, methods for introducing the nucleic acid construct of the present invention into a host include, but are not limited to, a method of infecting the host with an Agrobacterium transformer into which a recombinant vector containing the nucleic acid construct of the present invention has been introduced, and a method of introducing polynucleotides containing the nucleic acid construct of the present invention into the host by a particle gun or electroporation. Furthermore, methods for introducing a recombinant vector containing the nucleic acid construct of the present invention into a host include, but are not limited to, a method of infecting the host with a viral vector containing the nucleic acid construct of the present invention, and a method of introducing a plasmid vector containing the nucleic acid construct of the present invention into the host by a particle gun or electroporation. Moreover, in the present invention, both the nucleic acid construct of the present invention and a recombinant vector containing it can be introduced into the host. As described above, the host and transformer in the present invention include, for example, plants, for example, plant bodies or parts thereof (e.g., organs, tissues), plant cells (including plant cultured cells), etc. Furthermore, if the plant is a member of the genus Nicotiana, the plant body, a part thereof, or plant cells include the Nicotiana genus, a part thereof, or cells derived from the Nicotiana genus (including cultured cells). The same applies to other plants. Furthermore, step (a2) may include, for example, if the transformant is a plant, cultivating the transformant in an artificial climate chamber for 1 to 14 days (for example, 6 to 9 days) using the DFT method. By culturing or growing the transformant, hedgehog protein is produced in the transformant.

[0079] Step (b) described above is a step of recovering the hedgehog protein produced in the transformant in step (a). While there are no limitations on the method for recovering hedgehog protein, if the transformant is a plant, hedgehog protein can be recovered, for example, as follows. First, leaves are collected from transformed plants cultivated 1 to 14 days (e.g., 6 to 9 days) after Agrobacterium infection, and hedgehog protein is extracted using an extraction buffer. The amount of leaves collected varies depending on the type of plant. The leaves can be frozen and stored at -80°C until extraction. Examples of extraction buffers include, but are not limited to, phosphate buffer, Tris buffer, and acetate buffer. The pH is usually adjusted between pH 2 and 11, including the range in which the buffer works appropriately. Next, the hedgehog protein contained in the extract is purified. Purification can be carried out by conventional methods, such as aqueous two-phase partitioning, ammonium sulfate fractionation, affinity chromatography, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, reverse-phase chromatography, etc., either alone or in appropriate combinations. Confirmation that the obtained purified substance is the target protein, i.e., Hedgehog protein, can be performed by conventional methods such as SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis, Western blotting, enzyme immunosorbent assay (ELISA), and mass spectrometry. This allows us to obtain purified hedgehog protein.

[0080] The activity of the obtained hedgehog protein can be measured, for example, by treating mouse embryo-derived C3H10T1 / 2 cells with the hedgehog protein of interest, culturing them, and measuring the alkaline phosphatase (ALP) activity, as described in Example 7 of this specification.

[0081] 9. Composition containing hedgehog protein The present invention can provide a composition containing the hedgehog protein of the present invention, for example, a hedgehog protein obtained by the method for producing the hedgehog protein of the present invention. In addition to the hedgehog protein, the composition of the present invention may contain known additives such as physiological saline, buffer, and excipients. Compared to hedgehog protein produced using conventional signal peptides, the composition containing the hedgehog protein of the present invention has a lower content of poorly cleaved hedgehog protein and a higher content of hedgehog protein having an intact N-terminal amino acid sequence, making it useful for the industrial use of hedgehog protein.

[0082] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0083] In silico analysis of signal peptides In this example, in silico analysis of plant signal peptides was performed using a sequence database to obtain sequence information for several signal peptides that have been experimentally proven to function in plants. The amino acid sequences of 11 of the obtained signal peptides (C1S, C2S, C3S, C4S, C5S, C6S, C8S, C9S, C11S, C16S, C20S) and the amino acid sequence of the rice α-amylase-derived signal peptide (RSP) are shown in the table below.

[0084] [Table 5] [Examples]

[0085] Construction of a signal peptide-modified human sonic hedgehog (SHH) expression vector 1. Preparation of nucleic acid constructs In this example, based on the information of the plant-derived signal peptide obtained in Example 1, a nucleic acid construct was constructed in which the polynucleotide encoding the signal peptide was ligated to the 5' end of the polynucleotide encoding human SHH. Specifically, polynucleotides were artificially synthesized using GeneArt gene synthesis (Thermo Fisher Scientific) by ligating polynucleotides encoding the signal peptides shown in Table 1 (C1S, C2S, C4S, C5S, C6S, C8S, C9S, C11S, C16S, C20S) to the 5' end of a polynucleotide encoding human SHH. The nucleotide sequence of these polynucleotides was optimized using GeneArt GeneOptimizer software (Thermo Fisher Scientific) to match the codon frequency of *N. benthamiana*. This allowed us to create nucleic acid constructs containing the polynucleotide encoding the signal peptide and the polynucleotide encoding the human SHH protein.

[0086] 2. Preparation of expression vectors The nucleic acid constructs prepared in step 1 above were cloned into the Nde I-Sal I site of the pRI201-AN vector (Takara Bio Inc.). In addition, the AtADH-derived 5'-UTR contained in the pRI201-AN vector was replaced with the AtCOR47-derived 5'-UTR (Yamasaki, S. et al. (2018) Journal of Bioscience and Bioengineering 125: 124-130). This resulted in an expression vector containing a nucleic acid construct that includes a polynucleotide encoding a signal peptide and a polynucleotide encoding a hedgehog protein. [Examples]

[0087] Preparation of transformants 1. Cultivation of host plants In this example, the host plant used was Nicotiana benthamiana, a species of the Nicotiana genus. (1) Seeding Liquid fertilizer for sowing (Otsuka House S1 (Otsuka Agri Techno Co., Ltd.) 0.78 g / L, Otsuka House No. 2 (Otsuka Agri Techno Co., Ltd.) 0.25 g / L, pH 5.0) was soaked into a hydroponic urethane mat (Ematsu Kasei W587.5 mm × D282 mm × H28 mm: 12 × 2 grids, hole diameter φ9 mm), placed in a seedling tray (W600 mm × D300 mm × H300 mm), and seeds of Nepenthes benthamiana were sown. (2) Seedling raising After sowing, the plants were grown for 12 days in an artificial climate chamber (NC-410HC) (Nippon Ika Kikai Seisakusho) at a room temperature of 28°C, relative humidity of 60-80%, and a light cycle of 16 hours of day and 8 hours of night.

[0088] (3) Cultivation (first half) The urethane mats used for seedling cultivation were separated into individual cells and transplanted into cultivation (early stage) panels (W600 mm x D300 mm, 30 holes). After transplanting, the cultivation (early stage) panels were set in an artificial climate chamber (LH-410SP) (Nippon Ika Kikai Seisakusho) and cultivated for 9 days using the deep flow technique (DFT method). Environmental conditions and liquid fertilizer conditions were controlled as follows. <Environmental conditions> Temperature: 28℃ Relative humidity: 60-80% Illumination: Average photosynthetic photon flux density (PPFD): 140 μmol / m 2 • seconds, 24-hour continuous illumination, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation) <Liquid fertilizer conditions> Liquid fertilizers were prepared by dissolving fertilizer solution A (150 g / L of Otsuka House S1 and 2.5 g / L of Otsuka House No. 5 (Otsuka Agri Techno Co., Ltd.)) and fertilizer solution B (100 g / L of Otsuka House No. 2) in dechlorinated water and mixing them in equal amounts. pH adjustment was performed using pH adjuster Down (Otsuka Agri Techno Co., Ltd.) and a 4% KOH aqueous solution. The electrical conductivity (EC) and pH of the liquid fertilizer were adjusted to EC: 2.3 mS / cm and pH 6.0 using the "Rakuraku Fertilizer Management Machine 3" (Sem Corporation).

[0089] (4) Cultivation (late stage) Plants were removed from the early cultivation panels and transplanted into late cultivation panels (W600 mm x D300 mm, 6 holes). The transplanted late cultivation panels were set in an artificial climate chamber (LH-410SP) (Nippon Ika Kikai Seisakusho) and cultivated for 7 days (28 days after sowing) using the DFT method. Environmental conditions were controlled as follows. <Environmental conditions> Temperature: 28℃ Relative humidity: 40-60% Illumination: Average photosynthetic photon flux density (PPFD): 140 μmol / m 2 • seconds, 24-hour continuous illumination, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation)

[0090] 2. Preparation of transformed organisms (1) Infection by Vacuum Infiltration The vectors constructed in Example 2 were introduced into Agrobacterium AGL1 strains by electroporation, and these AGL1 strains, along with those containing the Tomato bushy stunt virus P19 expression vector (pRIANP19), were used to infect tobacco plants (N. benthamiana) using the agro-infiltration method. Specifically, the tobacco plants obtained in "1." above, 28 days after sowing, were inverted and submerged in a beaker of Agrobacterium bacterial solution so that all leaves were completely immersed in the liquid. Subsequently, the beaker was placed in a vacuum desiccator (FV-3P) (Tokyo Glass Instruments Co., Ltd.) and left to stand at -0.09 MPa for 1 minute to reduce the pressure. After that, the valve was opened all at once to restore the pressure. After the pressure restoration was complete, the plants were returned to an upright position and placed in an artificial climate chamber (LH-410SP) (Nippon Ika Kikai Seisakusho). The above-mentioned pRIANP19 was prepared by inserting the nucleotide sequence (SEQ ID NO: 24) encoding the RNA silencing repressor P19 derived from TBSV (Tomato bushy stunt virus) into the multi-cloning site MCS1 of pRI 201-AN (Takara Bio Inc.) using a conventional method. The amino acid sequence of P19 is shown in SEQ ID NO: 25.

[0091] (2) Cultivation of infected leaves (expression process) Post-infection cultivation was carried out using an artificial climate chamber (LH-410SP) (Nippon Ika Kikai Seisakusho). Cultivation was performed for 6 days using the DFT method. Environmental conditions were controlled as follows: <Environmental conditions> Temperature: 20℃ Relative humidity: 60-80% Illumination: Average photosynthetic photon flux density (PPFD): 140 μmol / m 2 • seconds, 24-hour continuous illumination, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation) [Examples]

[0092] 1. Evaluation of signal peptide cleavage by Western blotting Proteins were extracted from transformed tobacco leaves (N. benthamiana) in Example 3, and the extracted proteins were subjected to Western blotting. In Western blotting, proteins that appeared at a higher molecular weight position than the predetermined position of the human SHH protein indicate proteins that were not accurately cleaved at the intended cleavage site (poorly cleaved proteins). The signal intensity of proteins appearing at this position was measured. Specifically, extracted proteins obtained by SDS-PAGE on a 4-20% TGX gel (Bio-Rad) were transferred to a PVDF membrane using the Transblot Turbo Transfer System (Bio-Rad) and reacted with goat anti-human / mouse SHH polyclonal antibody (R&D Systems) and HRP-labeled rabbit anti-goat IgG antibody (R&D Systems). Clarity Western ECL Substrate (Bio-Rad) was used as the luminescence reagent, and the signal was detected using ImageQuant LAS 500 (Cytiva). Volume analysis was performed using the ImageQuant TL version 8.1 (Cytiva) program. A commercially available E. coli-expressed human SHH protein (R&D Systems, 1845-SH-025 / CF) was used as a control. The test was conducted twice with different sample types (TEST-1, TEST-2), but the experimental conditions and methods were the same for both tests.

[0093] The evaluation results are shown in Figures 2A and 2B. Figure 2A shows the results of TEST-1, and Figure 2B shows the results of TEST-2. The vertical axis shows the relative signal intensity of poorly cleaved human SHH protein when using rice α-amylase-derived signal peptide (RSP) as the signal peptide, with the signal intensity of poorly cleaved human SHH protein set to 100, for each signal peptide used. As shown in Figures 2A and 2B, when signal peptides C2S, C3S, C9S, C11S, and C20S were used, the intensity of the signal appearing at a higher molecular weight position than the predetermined position on the human SHH protein was significantly lower, approximately 50% or less, compared to when rice α-amylase-derived signal peptide (RSP) was used. These results indicate that when using the five signal peptides C2S, C3S, C9S, C11S, and C20S, human SHH protein is cleaved more effectively at its intended cleavage site compared to when using RSP. In other words, it has been shown that using the signal peptide of the present invention to generate human SHH protein in a plant expression system can significantly suppress the generation of poorly cleaved human SHH protein compared to using RSP.

[0094] 2. Alignment analysis Alignment analysis of signal peptides was performed using the MUSCLE program (Edgar RC. (2004) Nucleic Acids Res 32: 1792-1797). As a result, C2S, C3S, C9S, C11S, and C20S, which were shown to significantly suppress the production of poorly cleaved human SHH protein as described in 1. above, had aspartic acid (Asp, D), glutamic acid (Glu, D), or phenylalanine (Phe, F) as the second (-2) amino acid from the C-terminus (the amino acid X4 in formula I of this specification) (Figure 3). In contrast, C1S, C4S, C5S, C6S, and C8S, which had a similar or greater likelihood of producing poorly cleaved human SHH protein compared to RSP, all had an amino acid other than Asp, Glu, or Phe as the second (-2) amino acid from the C-terminus. These results demonstrate that, surprisingly, the signal peptide of the present invention can significantly suppress the production of poorly cleaved human SHH protein in a plant expression system if the amino acid at the second (-2) position from the C-terminus (the amino acid X4 in formula I herein) is Asp, Glu, or Phe.

[0095] Furthermore, it was shown that C2S, C3S, C9S, C11S, and C20S have an amino acid at the first position (-1) from the C-terminus (the amino acid X5 in Formula I of this specification) that is characteristic of signal peptides, and that the amino acid at the third position (-3) from the C-terminus (the amino acid X3 in Formula I of this specification) is alanine or valine (Figure 3). [Examples]

[0096] Production of human SHH protein 1. Extraction Tobacco leaves were harvested from transformed Neisseria benthamiana (N. benthamiana) six days after infection. They were stored frozen at -80°C until extraction. Extraction was performed using a Polytron homogenizer PT2500E (KINEMATICA) with a buffer solution containing 100 mM potassium phosphate, 500 mM arginine hydrochloride, and 0.4 mg / mL sodium pyrosulfite, pH 8.0.

[0097] 2. Purification (1) Clarification The extract collected using the method described above was adjusted to pH 5.5 by adding 1M hydrochloric acid dropwise while stirring. Tobacco-derived proteins were precipitated by centrifugation at 15,000 × g for 15 minutes. The resulting supernatant was filtered through a 0.22 μm Nalgene bottle-top filter 291-4520 (Thermo Fisher Scientific) pre-coated with Celpure P300 (Sigma Aldrich Fine Chemicals) to obtain a clarified solution.

[0098] (2) Purification by cation chromatography Clarification solutions were prepared from tobacco leaves (100 g) expressing human SHH protein using RSP, C11S, and C20S, respectively, following the procedure described above. The clarification solutions were passed through a HiTrap SP HP 5 mL column (Cytiva) pre-equilibrated with column buffer (20 mM potassium phosphate, 150 mM NaCl, 10% (w / v) glycerol, pH 5.5) at a residence time of 1 minute to adsorb the proteins. Subsequently, the column was washed with 25 mL of column buffer, and finally, the NaCl concentration of the column buffer was gradually increased up to 1000 mM while passing the column. The fraction with the absorbance peak at 280 nm was collected to obtain fractions containing each of the human SHH proteins produced using RSP, C11S, and C20S. Each obtained human SHH protein was concentrated using an Amicon Ultra-15 centrifugal filter unit 10K (Merck Millipore) and then solvent-replaced with high-salt PBS (2.7 mM KCl, 587 mM NaCl, 10 mM phosphate buffer, pH 7.4). Further sterilization was performed by filtration using a Millex-GV SLGV004SL filter (Merck Millipore) in a clean bench. To measure the concentration, SDS-PAGE was performed on 4-20% TGX gels (Bio-Rad) with serially diluted Pierce BSA standard solutions (Thermo Fisher Scientific), and colorimetric quantification was performed by CBB staining. ImageQuant LAS 500 (Cytiva) and ImageQuant TL version 8.1 (Cytiva) programs were used for colorimetric quantification. As a result, we were able to produce purified human SHH proteins. [Examples]

[0099] 1.LC-MS analysis The purified human SHH protein obtained in Example 5 was replaced with a 10% acetonitrile solution containing 50 mM ammonium bicarbonate using Amicon Ultra-0.5 3K (Merck UFC500396). The resulting sample was subjected to intact mass spectrometry using a Q Exactive Orbitrap mass spectrometer (ThermoFisher Scientific). A Bio Resolve RP mAb Polyphenyl Column, 450 Å, 2.7 μm, 3 mm × 150 mm (Waters 186008950) was used, and the operation was performed at 60°C. 0.1% formic acid in water was used as mobile phase A, and 0.1% formic acid in acetonitrile was used as mobile phase B. The flow rate was set to 0.2 mL / min. Separation was performed by first holding at 8% B for 5 minutes, then increasing to 100% B over 20 minutes, and holding for 20 minutes. The column was then reduced to 8% B over 0.1 minutes and held for 10 minutes to equilibrate for the next analysis. The capillary voltage was set to 4.0kV with a sheath gas flow rate of 50 arbitrary units and an auxiliary gas flow rate of 15 arbitrary units. The capillary temperature was set to 300°C, and the probe heater temperature was set to 450°C. Mass spectra were acquired in the mass range of m / z 1000 to 3500. Mass spectra were deconvolved using BioPharma Finder 4.0 (ThermoFisher Scientific). Considering variable modifications of +15.995 (oxidation, hydroxylation), -0.984 (amidation), and -17.027 (deamination), proteins were identified from the precise mass of each peak. Finally, the variation ratio of the N-terminus was calculated from the intensity of each peak. For minor peaks with a relative amount of 2.1% or less, those that could not be identified were labeled "Unknown".

[0100] Compared to the case using RSP, the use of C11S and C20S resulted in a higher proportion (production rate) of human SHH protein with an intact N-terminus, and improved cleavage accuracy at the original cleavage site was observed (Figure 4).

[0101] In Figure 4, "Intact" refers to human SHH protein that has been cleaved at its original cleavage site (between the C-terminal amino acid of the signal peptide and the N-terminal amino acid of the human SHH protein), i.e., human SHH protein with an intact N-terminus. In the RSP pie chart in Figure 4, "+VAHA" refers to human SHH proteins with the amino acid sequence VAHA added to the N-terminus, while "-IIGPGRGFGK" and "-IIGPGRGFG" refer to human SHH proteins lacking the amino acid sequences IIGPGRGFGK and IIGPGRGFG, respectively, from the N-terminus. In other words, these hedgehog proteins do not have an intact N-terminus. In Figure 4, "Unknown" refers to substances that cannot be identified, as described above.

[0102] From the above results, it was shown that by using the signal peptide of the present invention, a human hedgehog protein having an intact N-terminal amino acid sequence can be produced at an extremely high production rate as compared with the case of using RSP.

Example

[0103] Activity evaluation The biological activity of the human SHH protein produced in Example 5 was evaluated by the following procedure. Mouse embryo-derived C3H10T1 / 2 cells were seeded in a growth medium containing 10% serum at a range of 10,000 - 25,000 cells / well and cultured for 1 day at 37°C in a 5% CO2 environment. Each human SHH protein produced using RSP, C11S, and C20S was serially diluted in the growth medium and used to treat the cells by medium replacement, followed by continued culture. Thereafter, every 1 and 2 days, the cells were treated twice with the medium containing each human SHH protein prepared in the same manner and the culture was continued. Two days after culture from the final treatment, the cells were collected, and the alkaline phosphatase (ALP) activity of each obtained cell lysate was measured using an ALP activity measurement kit (FUJIFILM Wako Pure Chemical Corporation). Also, the protein content of the above-mentioned cell lysate was measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific), and the ALP activity per 1 μg was calculated. Thereby, the dose-dependent activity depending on the treatment concentration of the human SHH protein was confirmed, and it was fitted to a four-parameter curve using GraphPad Prism 8 (GraphPad Software) to calculate the 50% effective concentration (ED 50 ) The results are shown in the following table

Table 6

[0104] As shown in Table 6 above, the human SHH proteins produced using C11S and C20S showed lower ED 50 values as compared with those produced using RSP. These results demonstrate that the human SHH protein produced using the signal peptide of the present invention has higher activity compared to the one produced using RSP. [Industrial applicability]

[0105] By using the signal peptide of the present invention, human SHH protein having an intact N-terminal amino acid sequence can be produced with a high production rate. [Sequence Listing Free Text]

[0106] Sequence IDs 20-23: Synthetic DNA or synthetic peptides

Claims

1. The use of a signal peptide in the production of a hedgehog protein in a plant expression system, wherein the signal peptide is (a) Polypeptides containing the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16 (b) A polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16, wherein the second amino acid from the C-terminus of the amino acid sequence is aspartic acid (Asp), glutamic acid (Glu), or phenylalanine (Phe), and which has the activity to be cleaved by a signal peptidase, or (c) A polypeptide comprising an amino acid sequence in which 1 to 4 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 3, 1 to 3 amino acids are deleted in the amino acid sequence of SEQ ID NO: 5 or 11, or 1 or 2 amino acids are deleted in the amino acid sequence of SEQ ID NO: 13 or 16, wherein the second amino acid from the C-terminus of the amino acid sequence is aspartic acid (Asp), glutamic acid (Glu), or phenylalanine (Phe), and which has the activity to be cleaved by a signal peptidase. The aforementioned use.

2. (i) a polynucleotide encoding a signal peptide, (ii) Polynucleotides encoding the hedgehog protein and Includes, A nucleic acid construct in which the polynucleotide encoding the signal peptide is ligated to the 5' end of the polynucleotide encoding the hedgehog protein, The signal peptide, (a) Polypeptides containing the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16 (b) A polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NOs: 3, 5, 11, 13, or 16, wherein the second amino acid from the C-terminus of the amino acid sequence is aspartic acid (Asp), glutamic acid (Glu), or phenylalanine (Phe), and which has the activity to be cleaved by a signal peptidase, or (c) A polypeptide comprising an amino acid sequence in which 1 to 4 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 3, 1 to 3 amino acids are deleted in the amino acid sequence of SEQ ID NO: 5 or 11, or 1 or 2 amino acids are deleted in the amino acid sequence of SEQ ID NO: 13 or 16, wherein the second amino acid from the C-terminus of the amino acid sequence is aspartic acid (Asp), glutamic acid (Glu), or phenylalanine (Phe), and which has the activity to be cleaved by a signal peptidase. The nucleic acid construct, as described above.

3. A recombinant vector comprising the nucleic acid construct described in claim 2.

4. A transformant comprising the nucleic acid construct described in claim 2 and / or the recombinant vector described in claim 3.

5. The transformant according to claim 4, wherein the transformant is a plant body or a part thereof or a plant cell.

6. A method for producing hedgehog protein, comprising the step of culturing or growing the transformant described in claim 4 or 5.

7. A method for producing hedgehog protein, comprising the following steps (a) and (b): (a) A step of introducing the nucleic acid construct described in claim 2 and / or the recombinant vector described in claim 3 into a host to obtain a transformant, and (b) A step to recover the hedgehog protein produced from the transformant obtained in step (a). Methods that include...

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