Method for producing protein containing non-natural amino acid

Abstract

To provide a non-natural amino acid-containing protein production method that is capable of producing a protein containing a non-natural amino acid at low cost.SOLUTION: A method for producing a protein containing a non-natural amino acid includes a precursor administration step S2 involving the administration of a precursor of a phenylalanine analog to a silkworm that expresses an aminoacyl-tRNA synthetase in the silk gland, the synthetase having activity to bind the phenylalanine analog to phenylalanine-specific tRNA.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to methods for producing proteins that contain unnatural amino acids. [Background technology]

[0002] Conventionally, techniques for producing proteins containing unnatural amino acids are known.

[0003] For example, Patent Document 1 discloses a method for producing a protein containing Nε-acetyllysine, an analog of lysine, using Escherichia coli.

[0004] Furthermore, Non-Patent Document 1 discloses a technique for causing silkworms to produce a protein (fibroin) containing 4-chlorophenylalanine, which is an analogue of phenylalanine.

[0005] The silkworm described in Non-Patent Document 1 has a gene for an aminoacyl-tRNA synthetase with extended amino acid recognition ability incorporated therein, and when fed feed containing 4-chlorophenylalanine, it is capable of producing proteins containing 4-chlorophenylalanine. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2011-500077 [Non-patent literature]

[0007] [Non-Patent Document 1] Teramoto et al. (2015) Physical properties of silkworm silk with unnatural amino acid (4-chlorophenylalanine) introduced. Journal of the Japanese Society of Silk Science, Vol. 23, 27-35. Summary of the Invention [Problem to be solved by the invention]

[0008] However, unnatural amino acids are generally more expensive than natural amino acids, and therefore, when producing a protein containing unnatural amino acids, the production costs may be higher than when producing a protein composed of natural amino acids.

[0009] In view of the above circumstances, an object of the present invention is to provide a method for producing a protein containing an unnatural amino acid, which can produce a protein containing an unnatural amino acid at low cost. [Means for solving the problem]

[0010] The present inventors focused on phenylalanine analogs, which are a type of unnatural amino acid, and as a result of extensive investigation, discovered that some endogenous enzyme having the activity of converting a precursor of a phenylalanine analog into a phenylalanine analog is expressed in the body of silkworms, thereby completing the present invention.

[0011] That is, one aspect of the present invention includes the following aspects.

[0012] [1] A method for producing a protein containing an unnatural amino acid, comprising the step of administering a precursor of a phenylalanine analog to a silkworm expressing an aminoacyl-tRNA synthetase in its silk gland, the aminoacyl-tRNA synthetase having the activity of binding a phenylalanine analog to a phenylalanine-specific tRNA. [2] The method for producing an unnatural amino acid-containing protein according to [1], wherein the precursor has a structure represented by the following formula (1): [ka] [In formula (1), R represents a halogen atom, a cyano group, an azide group, or an ethynyl group. n represents the number of R and is an integer of 1 to 4, and when n is 2 or more, R may be the same or different.] [3] The method for producing an unnatural amino acid-containing protein according to [1] or [2], wherein the precursor has a structure represented by the following formula (1-1): [ka] [In formula (1-1), R represents a halogen atom, a cyano group, an azide group, or an ethynyl group.] [4] The method for producing a non-natural amino acid-containing protein according to [2] or [3], wherein R is a chlorine atom or a bromine atom. [5] The method for producing a protein containing an unnatural amino acid according to [2] or [3], wherein R is an azide group. [6] The method for producing a non-natural amino acid-containing protein according to any one of [1] to [5], wherein the aminoacyl-tRNA synthetase comprises any one of the following amino acid sequences (a) to (c): (a) an amino acid sequence having an amino acid mutation at least in one of the 407th and 432nd amino acids from the N-terminus of the amino acid sequence shown in SEQ ID NO:1; (b) an amino acid sequence in which one or several amino acids have been deleted, inserted, substituted or added at a site other than the 407th and 432nd amino acids from the N-terminus of the amino acid sequence of (a) above; (c) an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 at a site other than the 407th and 432nd positions from the N-terminus of the amino acid sequence of (a) [7] the amino acid mutation at position 407 is T407A or T407G; The method for producing a protein containing an unnatural amino acid according to [6], wherein the amino acid mutation at position 432 is F432A, F432V, F432L, F432M, F432T, F432C, or F432Y. [8] The method for producing a protein containing an unnatural amino acid described in [6] or [7], wherein the silkworm has a gene encoding the aminoacyl-tRNA synthetase and a promoter that causes the gene to be expressed specifically in the silk gland. [9] The method for producing a protein containing an unnatural amino acid according to [8], wherein the promoter is a promoter that causes the gene to be expressed specifically in the posterior silk gland.

[10] The method for producing a protein containing an unnatural amino acid described in [8] or [9], wherein the promoter is a promoter of a gene encoding a fibroin L chain, a gene encoding a fibroin H chain, or a gene encoding P25 / fhx. Effect of the Invention

[0013] INDUSTRIAL APPLICABILITY The present invention provides a method for producing an unnatural amino acid-containing protein, which can inexpensively produce a protein that contains a phenylalanine analog, which is a type of unnatural amino acid. [Brief description of the drawings]

[0014] [Figure 1] 1 is a flow diagram showing a method for producing an unnatural amino acid-containing protein according to a preferred embodiment of the present invention. [Diagram 2] 1 is a graph showing the amount of phenylalanine analogs introduced into proteins when BmN cells are cultured in a medium containing 4-chlorophenylpyruvic acid. [Diagram 3] 1 is a graph showing the amount of phenylalanine analogs introduced into proteins when BmN cells are cultured in a medium containing 4-chlorophenylpyruvic acid or chlorophenylalanine. [Figure 4] 1 is a graph showing the amount of fibroin produced by transgenic silkworms and wild-type silkworms fed an artificial diet containing chlorophenylalanine or 4-chlorophenylpyruvic acid. [Diagram 5] 1 is a graph showing the amount of chlorophenylalanine introduced into the fibroin L chain produced by transgenic silkworms and wild-type silkworms fed chlorophenylalanine or 4-chlorophenylpyruvic acid. [Figure 6]1 is a graph showing the amount of fibroin produced by transgenic silkworms and wild-type silkworms fed an artificial diet containing azidophenylalanine or 4-azidophenylpyruvic acid. [Figure 7] The figures show bright-field images of fibroin obtained from transgenic silkworms administered with azidophenylalanine or 4-azidophenylpyruvic acid and wild-type silkworms, which were subjected to SDS-PAGE and stained with CBB. [Figure 8] Fluorescence images showing the results of SDS-PAGE of fibroin obtained from transgenic silkworms and wild-type silkworms administered azidophenylalanine or 4-azidophenylpyruvic acid, which were labeled with an azide-reactive probe. [Figure 9] 1 is a graph showing the amount of azidophenylalanine introduced into the fibroin L chain produced by transgenic silkworms and wild-type silkworms fed azidophenylalanine or 4-azidophenylpyruvic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, preferred embodiments of the present invention will be described in detail, with reference to the drawings where necessary.

[0016] FIG. 1 is a flow diagram showing a method for producing an unnatural amino acid-containing protein according to a preferred embodiment of the present invention.

[0017] The method for producing an unnatural amino acid-containing protein according to the embodiment shown in FIG. 1 includes the steps of producing transgenic silkworms expressing an aminoacyl-tRNA synthetase having an activity of binding a phenylalanine analog to a phenylalanine-specific tRNA in their silk glands (transgenic silkworm production step S1), administering a precursor of the phenylalanine analog to the transgenic silkworms (precursor administration step S2), and producing a phenylalanine analog-containing protein by the silkworms administered the precursor of the phenylalanine analog (phenylalanine analog-containing protein production step S3).

[0018] It is not necessary for the method for producing a protein containing an unnatural amino acid to include the transgenic silkworm production step S1. Transgenic silkworms expressing an aminoacyl-tRNA synthetase in the silk gland that has the activity of binding a phenylalanine analog to a phenylalanine-specific tRNA may be prepared in advance and subjected to the precursor administration step S2.

[0019] <Transgenic silkworm production process S1> The transgenic silkworms produced in the transgenic silkworm production step S1 express, in the silk gland, an aminoacyl-tRNA synthetase having an activity of binding a phenylalanine analog to a phenylalanine-specific tRNA.

[0020] Here, the aminoacylation of tRNA is catalyzed by aminoacyl-tRNA synthetases corresponding to each amino acid. Aminoacyl-tRNA synthetases have strict substrate specificity among the 20 types of amino acids that make up proteins. For example, the aminoacyl-tRNA synthetase corresponding to phenylalanine is called phenylalanyl-tRNA synthetase (PheRS).

[0021] A phenylalanyl-tRNA synthetase having an activity of recognizing a phenylalanine analog and binding the phenylalanine analog to a phenylalanine-specific tRNA comprises any one of the following amino acid sequences (a) to (c):

[0022] (a) an amino acid sequence having an amino acid mutation at least in one of the 407th and 432nd amino acids from the N-terminus (the left side in the sequence listing) of the amino acid sequence shown in SEQ ID NO:1; (b) an amino acid sequence in which one or several amino acids have been deleted, inserted, substituted or added at a site other than the 407th and 432nd amino acids from the N-terminus of the amino acid sequence of (a) above; (c) an amino acid sequence having 80% or more sequence identity with SEQ ID NO: 1 at a site other than the 407th and 432nd positions from the N-terminus of the amino acid sequence of (a)

[0023] The mutation at the 407th amino acid from the N-terminus is a mutation from threonine to alanine or glycine (T407A or T407G).

[0024] The amino acid mutation at the 432nd position from the N-terminus is preferably a mutation from phenylalanine to alanine, valine, leucine, methionine, threonine, cysteine, or tyrosine (F432A, F432V, F432L, F432M, F432T, F432C, or F432Y), and more preferably a mutation to alanine, valine, or methionine (F432A, F432V, or F432M).

[0025] In the amino acid sequence of (b), the number of deleted, inserted, substituted or added amino acids is preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, particularly preferably 1 to 5, and most preferably 1 to 2.

[0026] The sequence identity in the amino acid sequence of (c) is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more. In the amino acid sequence of (c), at least one of the amino acids corresponding to the 407th amino acid and the 432nd amino acid from the N-terminus of the amino acid sequence of (a) has the same amino acid mutation as the 407th and 432nd amino acids in the amino acid sequence of (a). That is, in the amino acid sequence of (c), the amino acid corresponding to the 407th amino acid from the N-terminus of the amino acid sequence of (a) may have a mutation of T407A or T407G. In the amino acid sequence of (c), the amino acid corresponding to the 432nd amino acid from the N-terminus of the amino acid sequence of (a) may have a mutation of F432A, F432V, F432L, F432M, F432T, F432C, or F432Y. The amino acid sequence of (c) has any of these mutations at least at one of these two positions.

[0027] In this specification, the sequence identity of a subject amino acid sequence to the reference amino acid sequence shown in SEQ ID NO: 1 can be determined, for example, as follows. First, the reference amino acid sequence and the subject amino acid sequence are aligned. Each amino acid sequence may contain gaps to maximize sequence identity. Next, the number of matching amino acids in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be calculated according to the following formula (A). Sequence identity (%) = number of matching amino acids / total number of amino acids in the target amino acid sequence × 100...(A) The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the α subunit of phenylalanyl-tRNA synthetase in the wild-type silkworm (Bombyx mori).

[0028] The phenylalanyl-tRNA synthetase containing any of the amino acid sequences (a) to (c) may further have any sequence such as a protein tag (for example, a histidine tag, an HA tag, or GFP).

[0029] A phenylalanyl-tRNA synthetase containing any one of the amino acid sequences (a) to (c) has a mutation in an α subunit having amino acid recognition activity, and has an activity of binding a phenylalanine analog to a phenylalanine-specific tRNA.

[0030] By expressing a phenylalanyl-tRNA synthetase having any of the amino acid sequences (a) to (c) in the silk gland, it is possible to highly efficiently introduce an unnatural amino acid that does not normally correspond to a native codon into a protein produced in the silk gland.

[0031] In order to avoid toxicity that may occur when an aminoacyl-tRNA synthetase that has any one of the amino acid sequences (a) to (c) and recognizes (tolerates) a phenylalanine analog is expressed in the entire silkworm, it is preferable to use a promoter that expresses the phenylalanyl-tRNA synthetase specifically in the silk gland of a transgenic silkworm.

[0032] In other words, it is preferable that the transgenic silkworm subjected to the precursor administration step S2 described in detail below has a gene encoding an aminoacyl-tRNA synthetase having any of the amino acid sequences (a) to (c) and having the activity of binding a phenylalanine analog to a phenylalanine-specific tRNA, and a promoter that causes the gene to be expressed specifically in the silk gland.

[0033] The silk gland of the silkworm is roughly divided into an anterior silk gland, a middle silk gland, and a posterior silk gland.

[0034] Examples of promoters that express the genes specifically in the middle silk gland include promoters of DNA (genes) that code for sericin 1 protein or sericin 2 protein. By using promoters that express the genes specifically in the middle silk gland, phenylalanine analogs can be introduced into proteins produced in the middle silk gland. An example of a protein produced in the middle silk gland of silkworms is sericin.

[0035] Furthermore, examples of promoters that cause the gene to be expressed specifically in the posterior silk gland include the promoter of the gene encoding the fibroin L chain (FibL) protein, the promoter of the gene encoding the fibroin H chain (FibH) protein, the promoter of the gene encoding the P25 / fhx protein, etc. Among these, the promoter of the gene encoding the fibroin L chain (FibL) protein is particularly preferred.

[0036] By using a promoter that expresses the gene specifically in the posterior silk gland, a phenylalanine analog can be introduced into the protein produced in the posterior silk gland. Since the protein produced in the posterior silk gland of silkworms is fibroin, which accounts for the majority of silkworm cocoon proteins, by using a promoter that expresses the gene specifically in the posterior silk gland, it becomes easier to control the properties of the silk protein.

[0037] A promoter that causes silk gland-specific expression of phenylalanyl-tRNA synthetase containing any of the amino acid sequences (a) to (c) may be a promoter that can cause expression at any one of the above three sites, or at a site consisting of a combination of two or more of the above three sites.

[0038] <Precursor administration step S2> In the precursor administration step S2, a precursor of a phenylalanine analog represented by the following formula (1) is administered to silkworms.

[0039] [ka] [In formula (1), R represents a halogen atom, a cyano group, an azide group, or an ethynyl group. n represents the number of R and is an integer of 1 to 4, and when n is 2 or more, R may be the same or different.]

[0040] The halogen atom of R may be a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and is preferably a chlorine atom or a bromine atom.

[0041] The precursor of the phenylalanine analogue represented by the above formula 1) is a molecule in which one to four hydrogen atoms bonded to the carbon skeleton of the phenyl group of phenylpyruvic acid are each substituted with a halogen atom, a cyano group, an azide group, or an ethynyl group.

[0042] The method of administering the precursor of the phenylalanine analogue represented by the above formula (1) to the transgenic silkworm may be, for example, oral administration. Oral administration may be, for example, feeding the larvae of the transgenic silkworm with a feed containing the precursor of the phenylalanine analogue. The time of feeding the larvae of the transgenic silkworm is preferably, but not limited to, the 3rd to 6th day of the 5th instar silkworm. The proportion of the precursor of the phenylalanine analogue in the feed is not particularly limited, but may be, for example, 0.05 to 5 wt %, 0.05 to 1.5 wt %, 0.08 to 1.2 wt %, or 0.1 to 1 wt % by dry weight.

[0043] In this embodiment, the precursor of the phenylalanine analog administered to the transgenic silkworm may be administered simultaneously with phenylalanine and / or a phenylalanine analog. The precursor of the phenylalanine analog administered may be one type or two or more types.

[0044] The precursor of the phenylalanine analog represented by the above formula (1) may be a phenylalanine analog having one R at the 4-position of the phenyl group of phenylpyruvic acid. Such a phenylalanine analog has a structure represented by the following formula (1-1).

[0045] [ka] [In formula (1-1), R represents a halogen atom, a cyano group, an azide group, or an ethynyl group.]

[0046] Among the precursors of the phenylalanine analogues represented by the above formula (1-1), for example, a precursor in which R is a chlorine atom, i.e., a precursor in which a chlorine atom is bonded to the 4-position of the phenyl group of phenylpyruvic acid (4-chlorophenylpyruvic acid), has a structure represented by the following formula (1-1A).

[0047] [ka]

[0048] Furthermore, among the precursors of the phenylalanine analogues represented by the above formula (1-1), for example, a precursor in which R is an azide group, i.e., a precursor in which an azide group is bonded to the 4-position of the phenyl group of phenylpyruvic acid (4-azidophenylpyruvic acid), has a structure represented by the following formula (1-1B).

[0049] [ka]

[0050] Surprisingly, as a result of intensive research by the present inventors, it was found that, although phenylalanine is an essential amino acid in silkworms, when a precursor of a phenylalanine analog represented by the above formula (1) is administered to the above transgenic silkworms, the precursor is converted into a phenylalanine analog in the silkworm's body, and further, cocoon filaments containing the phenylalanine analog are produced, as described in detail later in the Examples.

[0051] Therefore, by administering a precursor of a phenylalanine analog, which can generally be produced more cheaply than a phenylalanine analog, to silkworms, a phenylalanine analog-containing protein can be produced more cheaply than in the case of administering a phenylalanine analog to silkworms.

[0052] Furthermore, since it is not necessary to introduce into silkworms that produce proteins containing unnatural amino acids a gene encoding an aminoacyl-tRNA synthetase having the activity of binding a phenylalanine analog to a phenylalanine-specific tRNA as well as a gene encoding an enzyme having the activity of converting a precursor of an unnatural amino acid into the unnatural amino acid, the effort of introducing the genes can be eliminated.

[0053] The method for producing the precursor of the phenylalanine analogue is not particularly limited, but for an example, see the following literature: Junhua T., et al., Development of a Continuous Enzymatic Process for the Preparation of (R)-3-(4-Fluorophenyl)-2-hydroxy Propionic Acid. Organic Process Research & Development,2002, 6, 520-524. Also, as an example of a method for producing 4-azidophenylpyruvic acid, please refer to the following literature. Jae-Eun J. et al., Genetic incorporation of unnatural amino acids biosynthesized from α-keto acids by an aminotransferase. Chemical Science, 2014, 5, 1881-1885.

[0054] The precursor of the phenylalanine analog represented by the above formula (1) is converted in the body of the silkworm into a phenylalanine analog having a structure represented by the following formula (2). The phenylalanine analog having the structure represented by formula (2) is a molecule in which one to four hydrogen atoms bonded to the carbon skeleton of the phenyl group of phenylalanine are each substituted with a halogen atom, a cyano group, an azide group, or an ethynyl group. The phenylalanine analog converted in the body of the silkworm is an L-form.

[0055] [ka] [In formula (2), R represents a halogen atom, a cyano group, an azide group, or an ethynyl group. n represents the number of R and is an integer of 1 to 4, and when n is 2 or more, R may be the same or different.]

[0056] In the above formula (2), R is equal to R in the precursor before conversion, and n is equal to n in the precursor before conversion.

[0057] A phenylalanyl-tRNA synthetase having any one of the amino acid sequences (a) to (c) and having the activity of binding a phenylalanine analog to a phenylalanine-specific tRNA can bind the phenylalanine analog represented by the above formula (2) to the phenylalanine-specific tRNA.

[0058] The precursor of the phenylalanine analog having the structure represented by the above formula (1-1) is converted in the body of the silkworm into a phenylalanine analog having the structure represented by the following formula (2-1).

[0059] [ka] [In formula (2-1), R represents a halogen atom, a cyano group, an azide group, or an ethynyl group.]

[0060] In the above formula (2-1), R is equal to R in the precursor before conversion. That is, the phenylalanine analog is a compound in which a halogen atom, a cyano group, an azide group, or an ethynyl group is bonded to the 4-position of the phenyl group of phenylalanine.

[0061] In addition, the precursor of the phenylalanine analog having the structure represented by the above formula (1-1A) is converted into a phenylalanine analog (4-chlorophenylalanine) having the structure represented by the following formula (2-1A) in the body of the silkworm.

[0062] [ka]

[0063] In addition, the precursor of the phenylalanine analog having the structure represented by the above formula (1-1B) is converted into a phenylalanine analog (4-azidophenylalanine) having the structure represented by the following formula (2-1B) in the body of the silkworm.

[0064] [ka] <Phenylalanine analog-containing protein production process S3> In the phenylalanine analog-containing protein production step S3, a phenylalanine analog-containing protein is produced in the silk gland of the transgenic silkworm to which the above-mentioned precursor of the phenylalanine analog has been administered.

[0065] The type of the unnatural amino acid-containing protein produced in the silk gland is not particularly limited, but is preferably a silk protein produced as cocoon thread in the silk gland of a transgenic silkworm. Alternatively, an exogenous DNA encoding an arbitrary protein may be forcibly expressed in the silk gland, so that the arbitrary protein is produced in the silk gland.

[0066] The unnatural amino acid-containing protein produced in the silk gland is then recovered. Since the unnatural amino acid-containing protein produced in the silk gland is secreted outside the silkworm body as cocoon filaments, it can be easily recovered without using a special recovery method, and the unnatural amino acid-containing protein can be easily obtained in large quantities.

[0067] Although the preferred embodiment has been described in detail above, the present invention is not limited to the above embodiment, and various modifications are possible within the scope of the invention described in the claims. EXAMPLES

[0068] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0069] <Experimental Example 1: Experimental administration of precursors of phenylalanine analogues using cultured silkworm cells> In this experiment, the following four types of plasmids were purified, mixed, and transfected into silkworm cultured cells (BmN cells). For transfection, 3 μL of transfection reagent (Fu Gene® 4K, Promega) was used for 1 μg of DNA. 1. A plasmid containing a gene encoding an aminoacyl-tRNA synthetase containing an amino acid sequence having a T407A mutation at the 407th amino acid from the N-terminus of the amino acid sequence shown in SEQ ID NO:1 (see SEQ ID NO:2 for the base sequence). 2. A plasmid containing a gene encoding the wild-type β subunit of phenylalanyl-tRNA synthetase (see SEQ ID NO: 3 for the base sequence). 3. A plasmid containing a gene encoding a GFP protein tagged for purification (see SEQ ID NO: 4 for the base sequence) 4. A plasmid containing a gene encoding phenylpyruvate aminotransferase (an enzyme having the activity of converting a precursor of a phenylalanine analogue into a phenylalanine analogue, GlnAT) (see SEQ ID NO: 5 for the base sequence).

[0070] In addition, a baculovirus-derived SK16 promoter is inserted into each of the plasmids 1 to 4. In addition, the base sequence of the gene encoding phenylpyruvate aminotransferase is codon-optimized based on the base sequence of the gene in Thermus thermophilus (see SEQ ID NO: 6).

[0071] The BmN cells transfected with the above four types of plasmids were cultured for 7 days in a medium containing 0.2 mM (mol / L) or 0.5 mM 4-chlorophenylpyruvic acid (an example of a precursor of phenylalanine analogues, see formula (1-1A) above). The medium was Grace Insect Medium (product number: 11300027) from Thermo Fisher Scientific supplemented with 4-chlorophenylpyruvic acid.

[0072] Next, the BmN cells were disrupted. In detail, first, the BmN cells were collected and washed with PBS (phosphate-buffered saline). Next, the cells were suspended in a buffer containing 1% by volume of Igepal® CA-630 (Sigma-Aldrich, model number: I7771). Then, the cells were disrupted by gentle stirring on ice.

[0073] After disruption of the BmN cells, GFP was purified using a Strep-Spin Protein Miniprep Kit (Zymo Research) according to the protocol of the kit.

[0074] Thereafter, a trypsin digest of the purified GFP was subjected to mass spectrometry to analyze the amount of chlorophenylalanine (see formula (2-1A) above), an example of a phenylalanine analog, introduced into GFP. In addition, a system in which 4-chlorophenylpyruvic acid was not added to the medium and a system in which only the plasmid described in 4. above (plasmid containing the gene encoding GlnAT) was not transfected into BmN cells were also prepared.

[0075] FIG. 2 is a graph showing the amount of phenylalanine analogs introduced into proteins when BmN cells were cultured in a medium containing 4-chlorophenylpyruvic acid.

[0076] A tryptic digest of GFP contains the following five types of polypeptides having phenylalanine or a phenylalanine analog: A phenylalanine analog can be introduced at the "F" position in the following five polypeptides: Polypeptide 1: FEGDTLVNR (see SEQ ID NO: 7, approximately 1051 Da) Polypeptide 2: SAWSHPQFEK (see SEQ ID NO:8, approximately 1217 Da) Polypeptide 3: TIFFKDDGNYK (see SEQ ID NO: 9, approximately 1348 Da) Polypeptide 4: AEVKFEGDTLVNR (see SEQ ID NO: 10, approximately 1478 Da) Polypeptide 5: GIDFKEDGNILGHK (see SEQ ID NO: 11, approximately 1543 Da)

[0077] When a phenylalanine analog (chlorophenylalanine) is introduced into the "F" position of the above polypeptides, the peak of each polypeptide shifts in the direction of increasing by 34 Da. Therefore, the peak intensity (peak intensity after shift) of the polypeptide into which the phenylalanine analog has been introduced into each of the above five types of polypeptides is divided by the peak intensity of each of the above five types of polypeptides to calculate the peak intensity ratio, thereby making it possible to obtain a numerical value that serves as a guide for the amount of the phenylalanine analog introduced into each of the above five types of polypeptides.

[0078] In Figure 2, "without GlnAT" indicates the amount introduced in a system that was not transfected with the plasmid described in 4. above, and "with GlnAT" indicates the amount introduced in a system that was transfected with four types of plasmids, including the plasmid described in 4. above.

[0079] Furthermore, "none" indicates the amount of chlorophenylalanine introduced in a system where 4-chlorophenylpyruvic acid was not added to the medium, "0.2 mM" indicates the amount of chlorophenylalanine introduced in a system cultured in a medium containing 0.2 mM 4-chlorophenylpyruvic acid, and "0.5 mM" indicates the amount of chlorophenylalanine introduced in a system cultured in a medium containing 0.5 mM 4-chlorophenylpyruvic acid. For example, the value "0.100" on the vertical axis in Figure 2 indicates that the above peak intensity ratio is 10.0%.

[0080] As shown in FIG. 2, the amount of chlorophenylalanine (phenylalanine analogue) introduced in the "GlnAT-free" system was comparable to that in the "GlnAT-present" system. These results demonstrated that 4-chlorophenylpyruvic acid was converted to chlorophenylalanine in the BmN cells of the silkworm by the action of some endogenous enzyme.

[0081] <Experimental Example 2: Comparative experiment between phenylalanine analogues and their precursors using cultured silkworm cells> In this experiment, first, three of the four plasmids mentioned above, excluding plasmid 4 (the plasmid containing the gene encoding GlnAT), were purified and mixed, and transfected into cultured silkworm cells (BmN cells).

[0082] The BmN cells transfected with the three plasmids were then cultured for 7 days in a medium containing 0.2 mM chlorophenylalanine or 0.2 mM 4-chlorophenylpyruvic acid, which was the Grace's insect medium supplemented with chlorophenylalanine or 4-chlorophenylpyruvic acid.

[0083] Then, the BmN cells were disrupted and GFP was purified. The methods for disrupting BmN cells and purifying GFP were the same as those described above.

[0084] Then, a trypsin digest of the purified GFP was subjected to mass spectrometry to analyze the amount of chlorophenylalanine, an example of a phenylalanine analog, introduced into GFP. In addition, a system was also prepared in which wild-type BmN cells not transfected with the above-mentioned plasmid were cultured in a medium containing 0.2 mM chlorophenylalanine or 0.2 mM 4-chlorophenylpyruvic acid.

[0085] FIG. 3 is a graph showing the amount of phenylalanine analogs introduced into proteins when BmN cells were cultured in a medium containing 4-chlorophenylpyruvic acid (4-ClPhePyr) or chlorophenylalanine (ClPhe).

[0086] In Figure 3, "without PheRS-T407A" indicates the amount of chlorophenylalanine introduced in a wild-type BmN cell system not transfected with the above plasmids, and "with PheRS-T407A" indicates the amount of chlorophenylalanine introduced in a system transfected with a total of three types of plasmids, including the plasmid in 1 above (the three types excluding the plasmid in 4). Additionally, "none" indicates the amount of chlorophenylalanine introduced in a system in which neither 4-chlorophenylpyruvic acid nor chlorophenylalanine was added to the medium.

[0087] As shown in Figure 3, first, in the system "without PheRS-T407A," phenylalanyl-tRNA synthetase containing any of the amino acid sequences (a) to (c) was not introduced, and therefore, chlorophenylalanine was not introduced into the protein.

[0088] Furthermore, among the systems with PheRS-T407A, the amount of chlorophenylalanine introduced in the system in which BmN cells were cultured in a medium containing 0.2 mM 4-chlorophenylpyruvic acid was similar to the amount introduced in the system in which BmN cells were cultured in a medium containing 0.2 mM chlorophenylalanine.

[0089] These results demonstrated that 4-chlorophenylpyruvate is converted to chlorophenylalanine with high efficiency by some endogenous enzyme in BmN cells.

[0090] <Experimental Example 3: Comparative experiment 1 between phenylalanine analogues (chlorophenylalanine) and their precursors using transgenic silkworm larvae> In this experiment, first, 5th instar larvae of transgenic silkworms (H06 line) expressing phenylalanyl-tRNA synthetase containing an amino acid sequence having an amino acid mutation of F432V at the 432nd amino acid from the N-terminus side of the amino acid sequence shown in SEQ ID NO: 1 were fed a diet containing 1.0% by weight of 4-chlorophenylpyruvic acid or 1.0% by weight of chlorophenylalanine based on the dry weight on the 3rd to 6th days. A system in which 4-chlorophenylpyruvic acid and chlorophenylalanine were not added to the diet was also prepared.

[0091] In addition, this transgenic silkworm (H06 line) has a promoter for a gene encoding a fibroin L chain protein introduced into it so that phenylalanyl-tRNA synthetase containing an amino acid sequence with the F432V amino acid mutation is expressed specifically in the posterior silk gland.

[0092] Next, the cocoon shells obtained from the transgenic silkworms were collected and weighed.

[0093] The cocoon layers of the transgenic silkworms were then dissolved in an 8M LiBr aqueous solution by heating at 35°C for 40 minutes to a concentration of approximately 50mg / mL. The dissolved solution was then diluted with an 8M urea aqueous solution to a concentration of approximately 4.5mg / mL, and further mixed with a sample buffer containing a reducing agent to adjust the concentration to approximately 3mg / mL, and then allowed to stand at room temperature for 30 minutes or more for reduction. The reduced sample solution was separated by SDS-PAGE, and the band of fibroin L chain protein was excised, decolorized, washed, dried, and then digested in-gel with trypsin at 37°C overnight.

[0094] Next, the amount of chlorophenylalanine introduced into the trypsin-digested fibroin L chain protein-derived polypeptide was analyzed by mass spectrometry.

[0095] The tryptic digest of the fibroin L chain protein contains the following polypeptide 6 as a polypeptide having phenylalanine or a phenylalanine analogue. Polypeptide 6: SGNFAGFR (see SEQ ID NO: 12, approximately 855 Da)

[0096] When a phenylalanine analog (chlorophenylalanine) is introduced into any of the "F" positions of the above polypeptide 6, the peak of polypeptide 6 shifts in the direction of increasing by 34 Da (approximately 889 Da). Therefore, the peak intensity ratio before and after the shift was calculated as a guideline for the amount of phenylalanine analog introduced. Note that, since it is statistically rare for a phenylalanine analog (chlorophenylalanine) to be introduced into both of the two "F" positions of polypeptide 6, the peptide of approximately 923 Da was excluded from the calculation of the amount introduced.

[0097] Table 1 below shows information on the cocoon layers obtained from transgenic silkworms in each line, the amount of fibroin L chain protein obtained from the cocoon layers, the intensities of each peak in the trypsin digest, and the peak intensity ratios. [Table 1]

[0098] As shown in Table 1, it was confirmed that chlorophenylalanine was introduced into the fibroin L chain protein in both cases where 4-chlorophenylpyruvic acid and chlorophenylalanine were administered to the transgenic silkworms. The amount of chlorophenylalanine introduced was equivalent when 4-chlorophenylpyruvic acid was administered and when chlorophenylalanine was administered.

[0099] These results demonstrated that in the H06 transgenic silkworms, 4-chlorophenylpyruvic acid is converted to chlorophenylalanine by some endogenous enzyme in the body, and the converted chlorophenylalanine is incorporated into proteins.

[0100] <Experimental Example 4: Administration of phenylalanine analogues and their precursors to wild-type silkworm larvae> In this experiment, wild-type silkworm (white / CS) 5th instar larvae were first fed a diet containing 1.0% 4-chlorophenylpyruvic acid or 1.0% chlorophenylalanine by weight based on dry weight on days 3 to 6. A system was also prepared in which neither 4-chlorophenylpyruvic acid nor chlorophenylalanine was added to the diet.

[0101] Next, the cocoon shells obtained from the wild-type silkworms were collected and weighed.

[0102] Then, the cocoon shell of the wild-type silkworm was dissolved in 8M LiBr aqueous solution by heating at 35℃ for 40 minutes to a concentration of about 50 mg / mL. This solution was diluted with 8M urea aqueous solution to a concentration of about 4.5 mg / mL, and further mixed with a sample buffer containing a reducing agent to adjust the concentration to about 3 mg / mL, and then left at room temperature for more than 30 minutes for reduction treatment. The reduced sample solution was separated by SDS-PAGE, and the band of fibroin L chain protein was excised, decolorized, washed, dried, and then digested in gel with trypsin overnight at 37℃.

[0103] Next, the amount of chlorophenylalanine introduced into the trypsin-digested fibroin L chain protein-derived polypeptide was analyzed by mass spectrometry.

[0104] Table 2 below shows information on the cocoon layers obtained from wild-type silkworms in each line, the amount of fibroin L chain protein obtained from the cocoon layers, the intensities of each peak in the trypsin digest, and the peak intensity ratios. [Table 2]

[0105] As shown in Table 2, in wild-type silkworms, introduction of chlorophenylalanine into the fibroin L chain protein was not confirmed in any of the systems. Note that the peak intensity value of 1140 at 889 Da in the system in which 4-chlorophenylpyruvic acid and chlorophenylalanine were not added to the feed is the background signal (noise).

[0106] <Experimental Example 5: Comparative experiment 2 between phenylalanine analogues (chlorophenylalanine) and their precursors using transgenic silkworm larvae> In this experiment, 5th instar silkworms of the above-mentioned H06 strain transgenic silkworms, which have the amino acid mutation F432V in phenylalanyl-tRNA synthetase, were fed an artificial diet of normal composition until the second day, and from the third day onwards, they were fed an artificial diet (Silkmate original species 1-3 instar M, Nosan Kogyo Co., Ltd.) supplemented with 1.0% by weight (dry diet equivalent) of chlorophenylalanine or its precursor, 4-chlorophenylpyruvic acid.

[0107] In addition, a system was prepared using wild-type 5th instar silkworms instead of transgenic silkworms. In addition, a system was prepared in which chlorophenylalanine and 4-chlorophenylpyruvic acid were not added to the artificial diet. In each system, n=3.

[0108] Figure 4 is a graph showing the amount of fibroin produced by transgenic and wild-type silkworms fed an artificial diet containing chlorophenylalanine or 4-chlorophenylpyruvic acid. The average value of three independent experiments for each line is plotted in Figure 4. The error bars show the standard deviation.

[0109] As shown in Figure 4, in both transgenic and wild-type silkworms, the amount of fibroin produced was reduced in the line in which chlorophenylalanine or 4-chlorophenylpyruvic acid was added to the artificial diet, compared to the line in which chlorophenylalanine and 4-chlorophenylpyruvic acid were not added to the artificial diet (labeled "non-administered" in the figure). The amount of reduction in fibroin production was greater in the transgenic silkworms than in the wild-type silkworms.

[0110] After feeding the artificial diet, as in Experimental Example 3 described above, the cocoon layers of the transgenic silkworms and wild-type silkworms were dissolved in an aqueous LiBr solution, diluted in an aqueous urea solution, mixed with a sample buffer containing a reducing agent for reduction treatment, and then separated by SDS-PAGE.

[0111] Next, the fibroin L chain protein band was excised, destained, washed, dried, and digested in-gel with trypsin overnight at 37°C.

[0112] Thereafter, the peptide fragments obtained by digestion were extracted, dried, and then mass analyzed by MALDI-TOF / MS. For the fragment of the above-mentioned polypeptide 6 (SGNFAGFR, see SEQ ID NO: 12) among the peptide fragments generated by trypsin digestion, the peak intensity ratio before and after the peak shift was calculated as the approximate amount of chlorophenylalanine introduced, as in Experimental Example 3.

[0113] Figure 5 is a graph showing the amount of chlorophenylalanine introduced into the fibroin L chain produced by transgenic silkworms and wild-type silkworms fed with chlorophenylalanine or 4-chlorophenylpyruvic acid. The average value of three independent experiments for each line is plotted in Figure 5. Standard deviation is used as the error bar.

[0114] As shown in Figure 5, in the fibroin L chain produced by wild-type silkworms, no incorporation of chlorophenylalanine was observed in either the system to which 4-chlorophenylpyruvic acid was added or the system to which chlorophenylalanine was added.

[0115] In contrast, in the fibroin L chain produced by transgenic silkworms, introduction of chlorophenylalanine was confirmed in both the system to which 4-chlorophenylpyruvic acid was added and the system to which chlorophenylalanine was added.

[0116] There was almost no difference in the amount of chlorophenylalanine introduced between the systems to which 4-chlorophenylpyruvic acid had been added and the systems to which chlorophenylalanine had been added.

[0117] These results confirmed that 4-chlorophenylpyruvic acid was converted to chlorophenylalanine with high efficiency by some endogenous enzyme in the H06 transgenic silkworms.

[0118] <Experimental Example 6: Comparative experiment between phenylalanine analogue (azidophenylalanine) and its precursor using transgenic silkworm larvae> In this experiment, 5th instar silkworms of the above-mentioned H06 strain transgenic silkworms, which have the amino acid mutation F432V in phenylalanyl-tRNA synthetase, were fed an artificial diet of normal composition until the second day, and from the third day onwards, they were fed an artificial diet (Silkmate original species 1-3 instar M, Nosan Kogyo Co., Ltd.) supplemented with 0.1% by weight (dry diet equivalent) of azidophenylalanine or its precursor, 4-azidophenylpyruvic acid.

[0119] In addition, a system was prepared using wild-type 5th instar silkworms instead of transgenic silkworms. A system was also prepared in which azidophenylalanine and 4-azidophenylpyruvic acid were not added to the artificial diet. In this experiment, n=3 was used for each system.

[0120] Figure 6 is a graph showing the amount of fibroin produced by transgenic and wild-type silkworms fed an artificial diet containing azidophenylalanine or 4-azidophenylpyruvic acid. The average value of three independent experiments for each line is plotted in Figure 6. The error bars show the standard deviation.

[0121] As shown in Figure 6, in both transgenic and wild-type silkworms, when 0.1% 4-azidophenylpyruvic acid (4-AzPhePyr) was added to the artificial diet, no significant decrease in fibroin production was observed compared to the system in which azidophenylalanine (AzPhe) and 4-azidophenylpyruvic acid were not added to the artificial diet ("non-administered" in the figure).

[0122] In contrast, in a system in which 0.1% azidophenylalanine was added to the artificial diet and fed to the H06 transgenic silkworms, the amount of fibroin produced was significantly lower than in a system in which wild-type silkworms were fed the artificial diet and in a non-administered system in which azidophenylalanine and 4-azidophenylpyruvic acid were not added.

[0123] These results suggest that administering a precursor of a phenylalanine analog containing an azide group to transgenic silkworms may significantly increase fibroin production compared to administering azidophenylalanine to transgenic silkworms.

[0124] After obtaining cocoon shells from the above-mentioned transgenic silkworms and wild-type silkworms, the cocoon shells were purified with urea to obtain fibroin, which was then dissolved in an 8 M LiBr aqueous solution by heating at 35° C. for 2 hours to a concentration of 25 mg / mL.

[0125] 3.2 μL of the thus obtained lysate was mixed with 16.8 μL of reaction solution (a mixture of 15.4 μL of 8 M urea aqueous solution, 1 μL of 1 M Tris-HCl buffer (pH 8), and 0.4 μL of 5 mM carboxyrhodamine 110 DBCO (azide-reactive probe, Click Chemistry Tools)) and incubated at room temperature overnight.

[0126] Next, 7.5 μL of the reaction solution was mixed with 12.5 μL of an 8 M urea aqueous solution and 10 μL of a 6x sample buffer to adjust the concentration to 1 mg / mL, and reduced by heating at 50° C. for 15 minutes. The reduced sample solution was then separated by SDS-PAGE.

[0127] The SDS-PAGE gel used was a Mini-PROTEAN TGX precast gel (Bio-Rad) with a concentration of 4-15% (w / v), and the electrophoresis voltage was 100 V. After washing the gel with pure water, the fluorescent signal was photographed with a Fusion FX7 imaging system (Vilber). The proteins in the gel were then stained with CBB staining solution (QC Colloidal Coomassie Stain, Bio-Rad) and photographed with the same imaging system.

[0128] Figure 7 is a bright-field image showing the results of SDS-PAGE and CBB staining of fibroin obtained from transgenic silkworms administered with azidophenylalanine or 4-azidophenylpyruvic acid and wild-type silkworms, respectively. Figure 8 is a fluorescent image showing the results of SDS-PAGE of fibroin obtained from transgenic silkworms administered with azidophenylalanine or 4-azidophenylpyruvic acid and wild-type silkworms, respectively, labeled with an azide-reactive probe.

[0129] 7 and 8, the samples in lanes 1 to 6 are as follows: M is a molecular weight marker. 1: Wild-type silkworm, not administered azidophenylalanine or 4-azidophenylpyruvic acid 2: Wild-type silkworms, administered 0.1% 4-azidophenylpyruvic acid 3: Wild-type silkworms, administered 0.1% azidophenylalanine 4: Transgenic silkworms, not administered azidophenylalanine or 4-azidophenylpyruvic acid 5: Transgenic silkworms, administered 0.1% 4-azidophenylpyruvic acid 6: Transgenic silkworms, administered 0.1% azidophenylalanine

[0130] As shown in Figure 7, bands indicating the presence of fibroin H chains and fibroin L chains were detected in all samples, whereas in the fluorescence image shown in Figure 8, bands indicating fibroin H chains and fibroin L chains were confirmed only in the fibroin obtained by administering 0.1% 4-azidophenylpyruvic acid or azidophenylalanine to the transgenic silkworms. The bands in the fluorescence image indicate that an azide-containing molecule (specifically, azidophenylalanine) has been introduced into the fibroin H chains and fibroin L chains.

[0131] These results demonstrated that 4-azidophenylpyruvic acid was converted to azidophenylalanine in the transgenic silkworms administered 4-azidophenylpyruvic acid and was then introduced into the fibroin. There was no significant difference in the intensity of the bands in the fluorescent images between the system administered 0.1% 4-azidophenylpyruvic acid and the system administered 0.1% azidophenylalanine.

[0132] Meanwhile, the cocoon layers of each fibroin shown in Figure 6 were dissolved in an aqueous LiBr solution, diluted in an aqueous urea solution, mixed with a sample buffer containing a reducing agent for reduction treatment, and then separated by SDS-PAGE, as in Experimental Example 4 described above.

[0133] The fibroin L chain protein band was then excised, destained, washed, and dried, and then in-gel digested with trypsin overnight at 37°C. The peptide fragments obtained by digestion were then extracted, dried, and solidified, and then analyzed by mass spectrometry using MALDI-TOF-MS.

[0134] Here, when azidophenylalanine is introduced into either of the two "F" positions in the fragment of the above-mentioned polypeptide 6 (SGNFAGFR, see SEQ ID NO: 12) among the peptide fragments obtained by digestion, the peak shifts from about 855 Da to about 870 Da. Therefore, in this experiment, the ratio of the peak intensity at 870 Da to the peak intensity at 855 Da was calculated as the approximate amount of azidophenylalanine introduced into the fibroin L chain.

[0135] 9 is a graph showing the amount of azidophenylalanine introduced into the fibroin L chain produced by H06 transgenic silkworms and wild-type silkworms fed azidophenylalanine or 4-azidophenylpyruvic acid, n=3, and standard deviation was used as the error bar.

[0136] As shown in Figure 9, in both systems where azidophenylalanine or 4-azidophenylpyruvic acid was added to the artificial diet, the peak intensity ratio (directly, the 870 Da peak) of the fibroin L chain of wild-type silkworms was at the background signal (noise) level, and introduction of azidophenylalanine was not observed.

[0137] In contrast, in the H06 transgenic silkworms, introduction of azidophenylalanine into the fibroin L chain was observed in both the systems in which azidophenylalanine and 4-azidophenylpyruvic acid were added to the artificial diet, confirming the replacement of 4-azidophenylpyruvic acid with azidophenylalanine in the bodies of the transgenic silkworms.

[0138] On the other hand, in the system where 4-azidophenylpyruvic acid was added and fed to the transgenic silkworms, the amount of azidophenylalanine introduced into the fibroin L chain was smaller than that in the system where azidophenylalanine was added and fed to the transgenic silkworms. This result suggests that although 4-azidophenylpyruvic acid is converted to azidophenylalanine in the silkworm body, the conversion efficiency is lower than that of 4-chlorophenylpyruvic acid to chlorophenylalanine. [Industrial Applicability]

[0139] According to the present invention, a method for producing a protein containing an unnatural amino acid can be provided, which enables the inexpensive production of a protein containing a phenylalanine analog such as chlorophenylalanine or azidophenylalanine. [Explanation of symbols]

[0140] S1: Transgenic silkworm production process, S2: Precursor administration process, S3: Phenylalanine analog-containing protein production process

Claims

1. A method for producing a protein containing an unnatural amino acid, comprising the step of administering a precursor of a phenylalanine analog to a silkworm expressing, in its silk gland, an aminoacyl-tRNA synthetase having an activity of binding a phenylalanine analog to a phenylalanine-specific tRNA.

2. The method for producing an unnatural amino acid-containing protein according to claim 1 , wherein the precursor has a structure represented by the following formula (1): 【Chemistry 1】 [In formula (1), R represents a halogen atom, a cyano group, an azide group, or an ethynyl group. n represents the number of R and is an integer of 1 to 4. When n is 2 or more, R may be the same or different.]

3. The method for producing an unnatural amino acid-containing protein according to claim 1 , wherein the precursor has a structure represented by the following formula (1-1): 【Chemistry 2】 [In formula (1-1), R represents a halogen atom, a cyano group, an azide group, or an ethynyl group.]

4. The method for producing an unnatural amino acid-containing protein according to claim 3 , wherein R is a chlorine atom or a bromine atom.

5. The method for producing an unnatural amino acid-containing protein according to claim 3 , wherein R is an azide group.

6. The method for producing an unnatural amino acid-containing protein according to any one of claims 1 to 5, wherein the aminoacyl-tRNA synthetase comprises any one of the following amino acid sequences (a) to (c): (a) an amino acid sequence having an amino acid mutation in at least one of the 407th and 432nd amino acids from the N-terminus of the amino acid sequence shown in SEQ ID NO:1; (b) an amino acid sequence in which one or several amino acids have been deleted, inserted, substituted or added at a site other than the 407th and 432nd amino acids from the N-terminus of the amino acid sequence of (a) (c) an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 at a site other than the 407th and 432nd positions from the N-terminus of the amino acid sequence of (a)

7. the 407th amino acid mutation is T407A or T407G; The method for producing an unnatural amino acid-containing protein according to claim 6, wherein the 432nd amino acid mutation is F432A, F432V, F432L, F432M, F432T, F432C or F432Y.

8. 7. The method for producing an unnatural amino acid-containing protein according to claim 6, wherein the silkworm has a gene encoding the aminoacyl-tRNA synthetase and a promoter that causes silk gland-specific expression of the gene.

9. The method for producing an unnatural amino acid-containing protein according to claim 8 , wherein the promoter is a promoter that causes the gene to be expressed specifically in the posterior silk gland.

10. The method for producing an unnatural amino acid-containing protein according to claim 9 , wherein the promoter is a promoter of a gene encoding a fibroin light chain, a gene encoding a fibroin heavy chain, or a gene encoding P25 / fhx.

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