JP2015015927A - Transgenic silkworm and producing method of non-natural amino acid-containing protein therewith - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transgenic silkworm which can produce a non-natural amino acid-containing protein easily and a producing method of the non-natural amino acid-containing protein using the transgenic silkworm.SOLUTION: A transgenic silkworm has DNA coding a protein having activity to attach desired non-natural amino acid to phenylalanine-specific tRNA, and a promoter expressing the DNA specifically to a silk gland.

Description

  The present invention relates to a transgenic silkworm and a method for producing an unnatural amino acid-containing protein using the silkworm.

  Silk, which has high physical strength and is used as a surgical suture, is one of the few biological materials that have been proven safe for the human body. Recent research has made it possible to process silk into various shapes (films, sponges, nanofibers, etc.), and a wide range of applications are being developed as medical materials (Non-Patent Documents 1 to 3). Furthermore, it has been reported that it has unique characteristics as an optical material and an electronic material (Non-Patent Document 4).

Silk itself has many useful properties, but it is expected to be utilized industrially as a more useful material by modifying its properties. Two methods, “chemical methods” and “genetic engineering methods”, have been used so far to modify the properties of silk.
“Chemical method” is a method of modifying the properties of a silk protein by a chemical reaction with respect to a side chain functional group (Non-patent Document 5). Although chemical methods can greatly modify the properties of silk proteins, there are problems such as difficulty in controlling reaction sites and reaction amounts, and concern about toxicity due to by-products and residual reagents involved in the reaction.
On the other hand, the “gene engineering method” is a method of modifying the properties of silk protein using silkworm genetic recombination technology (Non-patent Document 6). The genetic engineering method is an extremely effective method because it can avoid toxicity, which is a serious problem of the chemical method, and further technical development is desired. Among them, the method of introducing non-natural amino acids into proteins attracts attention because non-natural amino acids can be used as constituent elements and proteins having characteristics that are not found in nature can be created.

Two methods are known for introducing unnatural amino acids into proteins. One is “site-specific unnatural amino acid introduction method”. This method requires administration of an unnatural amino acid and modification of the target protein gene sequence, and has a tRNA having an anticodon corresponding to the codon of the modified sequence, and aaRS (aminoacyl) for aminoacylating the tRNA with the unnatural amino acid. -TRNA synthetase). However, in this site-specific unnatural amino acid introduction method, both the target protein gene and the target aaRS gene must be modified, and the process is complicated. In addition, there is a limitation that the sequence of the target protein gene must be known.
On the other hand, the “residue-specific unnatural amino acid introduction method” is known as the second method. In this method, in addition to administration of an unnatural amino acid, the uptake efficiency of an unnatural amino acid is improved by forced expression of a mutant aaRS in which a mutation is introduced into an amino acid binding site. This residue-specific non-natural amino acid introduction method can be performed without modifying the target protein gene sequence, so it is easier to use than the site-specific non-natural amino acid introduction method, and the target protein gene sequence is unknown. However, since it has the advantage of being usable, it is a technology with high utility value.
As examples of residue-specific unnatural amino acid introduction methods so far, there are reports in E. coli (Non-patent Document 7) and silkworm cultured cells (Non-patent Document 8).

Rockwood, DN, et. al., Nat. Protoc. , (2011) Vol. 6, pp. 1612-1631. Yagi, T .; , et. al. , J .; Artif. Organs, (2011) Vol. 14, pp. 89-99. Kawakami, M., et. al., Biomed. Mater. Eng., (2011) Vol. 21, pp. 53-61. Omenetto, F.G. and Kaplan, D.L., Science, (2010) Vol. 329, pp. 528-531. Murphy, A.R. and Kaplan, D.L., J. Mater. Chem., (2009) Vol. 19, pp. 6443-6450. Katsura Kojima, Kei Tamada, BIO INDUSTRY, (2007) Vol. 24, pp. 11-18. Kirshenbaum, K., et.al., ChemBioChem, (2002) Vol. 3, pp. 235-237. Teramoto, H., et.al., ChemBioChem, (2012), Vol. 13, pp. 61-65. Teramoto, H. and Kojima, K. J., Insect Biotechnol. Sericol. , (2010) Vol. 79, pp. 53-65

  However, the purpose of introducing unnatural amino acids into proteins that has been published in the past is mainly to elucidate the structure and functions of proteins, and silk proteins that function as materials such as fibers for industrial use. There is still room for study on methods to dramatically increase the usefulness. In addition, there have been no reports of successful use of the residue-specific unnatural amino acid introduction method in multicellular organisms such as silkworms.

In addition, as described above, a residue-specific unnatural amino acid introduction method has been conventionally used in silkworm cultured cells. However, when silkworm cultured cells are used, the amount of unnatural amino acid-containing protein produced is less than that of unnatural amino acids. There was a problem of a significant decrease compared to the amount of protein not contained.
Moreover, even if it was a non-natural amino acid (for example, p-Br-Phe) whose recognition by the mutant type aaRS was confirmed by an in vitro experiment, there was a problem that the protein was not incorporated into a silkworm cultured cell (non-patent) Reference 9).
In general, when culturing silkworm cultured cells, it is necessary to add serum to the culture medium. Therefore, in the system using silkworm cultured cells, strict mixing of protein material components including unnatural amino acids is required. It was very difficult to control.
Because of these problems, in particular, it is not possible to produce proteins in silkworm cultured cells that contain certain unnatural amino acids (for example, pN ₃ -Phe), such as unnatural amino acids whose structure differs greatly from natural amino acids. It was very difficult.

  The present invention has been made in view of the above circumstances, and provides a transgenic silkworm that can easily produce a non-natural amino acid-containing protein, and a method for producing a non-natural amino acid-containing protein using the transgenic silkworm.

  As a result of diligent research to solve the above problems, the present inventors have found a transgenic silkworm that specifically expresses a silk gland that encodes a protein having an activity of binding a desired unnatural amino acid to tRNA. Was completed.

  That is, the present invention provides a transgenic silkworm having the following characteristics and a method for producing an unnatural amino acid-containing protein using the transgenic silkworm.

(1) (a) the amino acid sequence shown in SEQ ID NO: 1,
(B) the amino acid sequence shown in SEQ ID NO: 2,
(C) an amino acid sequence in which one to several amino acids have been deleted, inserted, substituted or added at a site other than the amino acid number 450 position of the amino acid sequence shown in SEQ ID NO: 1, and desired for phenylalanine-specific tRNA (D) one to several amino acids are deleted, inserted, substituted or added at a site other than amino acid number 407 of the amino acid sequence shown in SEQ ID NO: 2; A DNA encoding a protein having any one amino acid sequence selected from the group consisting of amino acid sequences of proteins having an activity of binding a desired unnatural amino acid to a phenylalanine-specific tRNA, and Has a promoter that expresses the DNA specifically in the silk gland A transgenic silkworm characterized by that.
(2) The transgenic silkworm according to (1), wherein the promoter that specifically expresses the DNA is a silk gland-specific promoter.
(3) The transgenic silkworm according to (1) or (2), wherein the promoter that specifically expresses the DNA is a silkworm-derived promoter.
(4) A method for producing an unnatural amino acid-containing protein, comprising the step of administering an unnatural amino acid to the transgenic silkworm according to any one of (1) to (3).
(5) The method for producing an unnatural amino acid-containing protein according to (4), wherein the unnatural amino acid is a phenylalanine analog.
(6) The method for producing a protein containing an unnatural amino acid according to (4) or (5), wherein the unnatural amino acid is an amino acid represented by the following general formula (1).

[Wherein, R represents a halogen atom, an azide group or an ethynyl group. ]
(7) The method for producing an unnatural amino acid-containing protein according to (6), wherein R is an azide group or an ethynyl group.

Non-natural amino acid-containing proteins can be easily produced by the transgenic silkworm of the present invention.
Moreover, the non-natural amino acid containing protein by which the characteristic of the original protein which does not contain an unnatural amino acid was modified can be manufactured by the manufacturing method of the non-natural amino acid containing protein of this invention using the transgenic silkworm of this invention.

It is a piggyBac vector map for producing the transgenic silkworm which expresses the alpha subunit gene of silkworm PheRS in the posterior silk gland. An asterisk (*) attached to BmFRS1 indicates a mutant gene. It is the result of RT-PCR amplification of the BmFRS1 ^* (A450G) fragment in the white H01 line. It is a weight gain curve of the silkworm larvae at the time of p-Cl-Phe administration. Fig. 5 shows the total amount of intake (per head) and average cocoon weight of the fifth instar of silkworm larvae administered with p-Cl-Phe. It is a MALDI-TOF-MS spectrum of the fibL digested fragment derived from the lining obtained by p-Cl-Phe administration assay. It is a weight gain curve of the silkworm larvae at the time of p-Br-Phe administration. Fig. 5 shows the total intake (per head) and average cocoon weight of the fifth instar of silkworm larvae administered with p-Br-Phe. It is a MALDI-TOF-MS spectrum of a fibrL digested fragment derived from the capsular layer obtained by p-Br-Phe administration assay. weight gain curves of the silkworm larvae when _p-N 3 -Phe administration. It is the total feeding amount (per head) and average cocoon weight of the fifth instar of the silkworm larvae to which pN ₃ -Phe was administered. It is a MALDI-TOF-MS spectrum of the fibL digested fragment derived from the capsular layer obtained by pN ₃ -Phe administration assay. It is a schematic diagram of a click reaction with p-N ₃ -Phe-containing silk protein and biotin reagent. with biotin antibodies to FibL and FibH post-click response to _p-N 3 -Phe is the result of Western blotting. It is a weight gain curve of the silkworm larva at the time of p-Eth-Phe administration. 5 shows the total amount of intake (per head) and average cocoon weight of the fifth instar of silkworm larvae administered with p-Eth-Phe. It is a MALDI-TOF-MS spectrum of the fibL digested fragment derived from the lining layer obtained by p-Eth-Phe administration assay. It is the result of the western blotting by the biotin antibody with respect to FibL after the click reaction to p-Eth-Phe.

《Transgenic Silkworm》
The transgenic silkworm of the present invention is
(A) the amino acid sequence shown in SEQ ID NO: 1,
(B) the amino acid sequence shown in SEQ ID NO: 2,
(C) an amino acid sequence in which one to several amino acids have been deleted, inserted, substituted or added at a site other than the amino acid number 450 position of the amino acid sequence shown in SEQ ID NO: 1, and desired for phenylalanine-specific tRNA Amino acid sequence of a protein having an activity of binding an unnatural amino acid, and (d) deletion, insertion, substitution or addition of one to several amino acids at a site other than amino acid number 407 of the amino acid sequence shown in SEQ ID NO: 2 A DNA encoding a protein having any one amino acid sequence selected from the group consisting of amino acid sequences of proteins having an activity of binding a desired unnatural amino acid to a phenylalanine-specific tRNA, and Has a promoter that expresses the DNA specifically in the silk gland A transgenic silkworm characterized and.

The aminoacylation of tRNA is catalyzed by aminoacyl-tRNA synthetase (aaRS) corresponding to each amino acid. aaRS has strict substrate specificity among the 20 amino acids constituting the protein. For example, aaRS corresponding to phenylalanine is called phenylalanyl-tRNA synthetase (PheRS). The DNA encoding the protein having the amino acid sequence of (a) to (d) is a DNA having a mutation in the α subunit of silkworm PheRS, and has an activity of binding a desired unnatural amino acid to a phenylalanine-specific tRNA. It is DNA which codes protein which has. The protein having the activity of binding a desired unnatural amino acid to this phenylalanine-specific tRNA only needs to have this activity, and in addition to any of the sequences (a) to (d), It may further have an arbitrary sequence such as a protein tag (for example, histidine tag, HA tag, GFP, etc.).
By expressing a DNA encoding a protein having any one of the sequences (a) to (d) in the silk gland, an amino acid (non-naturally occurring) that does not normally correspond to the original codon for the protein produced in the silk gland Amino acids) can be introduced.

  The unnatural amino acid-containing protein produced in the silk gland is not particularly limited, and silk protein produced by silkworm in the silk gland is preferable. Alternatively, exogenous DNA encoding an arbitrary protein may be forcibly expressed in the silk gland, and the arbitrary protein may be produced in the silk gland.

  Since the protein produced in the silk gland is secreted outside the silkworm body as silk thread, it can be easily recovered without using a special recovery method, and a large amount of protein can be easily obtained.

In addition, from the viewpoint of avoiding the toxicity caused by expressing the DNA throughout the transgenic silkworm, a promoter for expressing the DNA in the silk gland is used in the present invention. The silk gland is roughly classified into an anterior silk gland, a middle silk gland, and a posterior silk gland. Here, the promoter for specifically expressing the DNA of the sequences (a) to (d) is not particularly limited as long as it is a promoter capable of expressing the DNA specifically for the silk gland. It may be a promoter that can be expressed at any one of the two sites or a site comprising a combination of two or more.
Examples of the promoter specifically expressed in the middle silk gland include a promoter of DNA encoding sericin 1 protein or sericin 2 protein. By using a promoter that is expressed specifically in the middle silk gland, an unnatural amino acid can be introduced into the protein produced in the middle silk gland. An example of a protein produced in the middle silk gland of silkworm is sericin.
In addition, examples of the promoter specifically expressed in the posterior silk gland include a promoter of DNA encoding a fibroin L chain (FibL) protein. By using a promoter that is expressed specifically in the posterior silk gland, an unnatural amino acid can be introduced into the protein produced in the posterior silk gland. The protein produced in the silkworm's posterior silk gland is fibroin, and fibroin accounts for the majority of silkworm silk silk protein. Becomes easier.

  When the promoter is used, other sequences for controlling the expression of the DNA, the subsequent translation into the protein, etc. may be used at the same time. Examples of such sequences include 3′UTR (untranslated region). ) 5′UTR sequence and the like. The sequence is not particularly limited as long as it can express the DNAs of the sequences (a) to (d) specifically in the silk gland.

  The host silkworm used for the production of the transgenic silkworm of the present invention is not particularly limited as long as it is a silkworm capable of producing a protein containing an unnatural amino acid in the method for producing an unnatural amino acid-containing protein of the present invention described later. In addition, w1-pnd or the like widely used for gene recombination preparation may be used, but from the viewpoint of increasing the amount of unnatural amino acids administered as feed to silkworm individuals, artificial feed feeding properties It is preferable to use silkworms excellent in the above. For example, varieties such as No. 01, No. 603, No. 604, No. 604, and No. 605 are preferable, and white / CS (strain name: MCS601) is more preferred.

  In addition, as described above, the silkworm used for producing the transgenic silkworm of the present invention is not particularly limited, and either a silkworm having the property of producing non-dormant eggs or a silkworm having the property of producing dormant eggs should be used. Is possible. In the case of using silkworms that lay dormant eggs, non-dormant eggs can be laid using the “low-temperature dark blue” method. That is, the embryo is protected at about 15 ° C. after the limb development period, particularly after the bristle development stage. This low temperature period needs 10 days or more in terms of days. Keep dark from embryo reversal to head coloring. Silkworm pupae obtained by rearing hatched larvae then lay non-dormant eggs. (Introduction to Species Author: Takeo Takami Publication: National Species Association (May 31, 1969)).

  As a method other than the low temperature dark blue treatment method, a method (see Japanese Patent Application Laid-Open No. 2003-88274) in which non-dormant eggs are laid by controlling light conditions in the larval stage may be adopted.

  When using a silkworm in which a non-dormant egg cannot be obtained even by using a method such as a low-temperature dark-air bluening method, the egg may be broken diapause by subjecting the silkworm egg to an acid treatment. As a specific method of the soaking treatment, it is possible to immerse silkworm eggs 3 to 4 hours after egg collection in hydrochloric acid at room temperature for about 30 minutes, wash away hydrochloric acid by washing with water, and then air dry the eggs. The hydrochloric acid having a specific gravity of 1.11 can be used, for example (see Ai-Chun Zhao et.al., Insect Science (2012) Vol. 19, pp. 172-182).

  Examples of a method for producing a transgenic silkworm include a method for introducing DNA into a silkworm egg. For example, DNA is introduced into silkworm eggs by, for example, a method of injecting transposon into a silkworm early embryo as a vector (Tamura, T., et. Al., Nature Biotechnology, (2000) Vol. 18, pp. 81- 84)).

  For example, the inverted terminal repeat of a transposon (Handler, AM, et al., Proc. Natl. Acad. Sci. USA, (1998) Vol. 95, pp. 7520-7525. And a vector having a DNA encoding a transposon transferase (helper vector) introduced into a silkworm egg together with a vector in which the DNA is inserted. Examples of the helper vector include, but are not limited to, pHA3PIG (see Tamura, T., et.al., Nature Biotechnology, (2000) Vol. 18, pp. 81-84.).

  As a transposon in the present invention, piggyBac is preferable, but is not limited thereto, and marina, minos, and the like can be used (Shimizu, K., Insect Mol. Biol., (2000). ) Vol.9, pp.277-281.).

  Moreover, a baculovirus vector can also be used for production of the transgenic silkworm of the present invention (see Yamao, M., et.al., Genes Dev. (1999) Vol. 13, pp. 511-516. ).

  The above-described method of injecting the transposon as a vector, the method of using a baculovirus vector, and the other methods of introducing DNA into silkworm eggs are performed on DNA encoding a protein having the amino acid sequence of (a) to (d) above. In addition, in the production of a non-natural amino acid-containing protein using the transgenic silkworm of the present invention, it may be used for DNA encoding any protein produced.

Introduction of DNA into silkworm eggs can be carried out by those skilled in the art by a general method. For example, it can be carried out by the method described in JP 2012-187123 A as follows.
Although it is possible to introduce DNA directly into the egg using a tube for injecting DNA into a silkworm egg, in a preferred embodiment, a hole is physically or chemically formed in advance in the eggshell, and the DNA is removed from the hole. Introduce.

  In the present invention, as a method for physically making a hole in an eggshell, for example, a method for making a hole using a needle, a micro laser, or the like can be mentioned. Among them, a method of making a hole in the eggshell by a method using a needle is preferable. There are no particular restrictions on the material, strength, etc. of the needles used so long as they can make holes in silkworm eggshells. On the other hand, as a method of chemically making a hole in an eggshell, for example, a method of making a hole using a chemical (such as hypochlorous acid) can be mentioned.

  In the present invention, the DNA introduction method includes the steps of physically or chemically making a hole in the silkworm egg, inserting a DNA injection tube into the egg through the hole, and injecting the DNA into a needle and DNA. It is preferable to use a manipulator in which an injection tube is integrated. A device having a manipulator as one of the components can be used.

  Such devices include a dissecting microscope, an illumination device, a movable stage, a coarse manipulator fixed to the microscope with a metal fitting, a micromanipulator attached to this manipulator, and air pressure for injecting DNA. The thing comprised from the injector which adjusts is mentioned. In addition, as an apparatus used, the apparatus of the patent 1654050 gazette or the apparatus which improved this apparatus is mentioned.

  Whether or not DNA has been introduced into silkworm eggs is determined by, for example, extracting the injected DNA again from the eggs (Nagaraju, J., et. Al., Appl. Entomol. Zool, (1996) Vol. 31, pp. 589-598.) And a method for observing the expression of DNA as an injected gene in eggs (Tamura, T., et. Al., (1990) Jpn. J. Genet., Vol. .65, pp. 401-410.) And the like. Whether or not the DNA introduced into the silkworm egg is integrated into the silkworm chromosome and a genetically modified silkworm is obtained is determined, for example, by mating adults raised from eggs injected with DNA (mutually or non-recombinant silkworms). It is possible by obtaining a fertilized egg and observing the expression of a marker gene (for example, EGFP, DsRed, CFP, YFP, etc.) with a fluorescence microscope 6 to 10 days after the egg laying.

  Whether or not DNA introduced into silkworm eggs is expressed in silkworms is confirmed by observing the expression of the marker gene with a fluorescence microscope for eggs (G1 generation) born from individuals that have grown from injected eggs (G0 generation) To do. Note that total RNA may be extracted from silkworms, and the transgene may be confirmed by RT-PCR using the extracted RNA as a template.

  The method of confirming in which part of the silkworm individual the DNA introduced into the silkworm is expressed is, for example, a method in which the tissue is extracted from the larvae and the expression is confirmed for each tissue, CFP, GFP, EGFP, It can be confirmed by a method of observing silkworm individuals using a fluorescent microscope using a known reporter gene such as YFP or DsRED.

<< Production Method of Non-Natural Amino Acid-Containing Protein >>
The method for producing an unnatural amino acid-containing protein of the present invention includes a step of administering an unnatural amino acid to the transgenic silkworm of the present invention. Here, the unnatural amino acid means an amino acid that does not constitute a protein. The amino acids constituting the protein mean 20 standard natural amino acids, selenocysteine, and pyrrolidine. That is, in the present invention, the unnatural amino acid means an analog of these amino acids constituting the protein (modified amino acid) or any amino acid other than these amino acids constituting the protein.

The transgenic silkworm of the present invention has a DNA sequence encoding a protein having an activity of binding a desired unnatural amino acid to a phenylalanine-specific tRNA. Therefore, the non-natural amino acid used is preferably a phenylalanine analog. As the phenylalanine analog, it is preferable that at least one hydrogen atom of the side chain of finilalanine is substituted with a substituent, and that the hydrogen atom of the benzene ring of the finilalanine side chain is substituted. More preferably, the para-position of the benzene ring is particularly preferably substituted. Examples of the substituent include substitution of halogen atoms such as fluorine, chlorine, bromine and iodine; ethynyl group; cyano group; azide group and the like.
That is, as the phenylalanine analog, an amino acid represented by the following general formula (1) is preferable.

[Wherein, R represents a halogen atom, an azide group or an ethynyl group. ]
Among these, R is preferably an azide group or an ethynyl group.

  In producing the unnatural amino acid-containing protein using the transgenic silkworm of the present invention, various methods can be selected as a method for confirming the type and content of the unnatural amino acid contained in the protein. Analysis is preferably performed, and a method of measuring a mass spectrum by a MALDI-TOF-MS method is particularly preferable.

  According to the present invention, the nature and function of the original protein can be improved or modified in the produced non-natural amino acid-containing protein. Furthermore, new properties and functions not found in the original protein can be imparted. For example, by applying a method for introducing an unnatural amino acid to a silk protein, the unnatural amino acid can be contained in the silk protein, and properties and functions not found in the original silk can be imparted.

  By using the method for producing an unnatural amino acid-containing protein of the present invention, problems in conventional modification techniques can be solved. For example, there are advantages such as low concern about toxicity due to chemical reaction, and post-processing, so that functional molecules do not deactivate. By introducing functional groups capable of specific chemical reaction, It is possible to carry out precise and clean chemical modification of proteins that does not depend on the “method”. For example, by incorporating an unnatural amino acid having an azido group or an ethynyl group into a silk protein, any protein, peptide, drug, dye, sugar chain, cross-linking molecule, chelate can be reacted with the ethynyl group or azide group. It is possible to bind molecules such as molecules to silk. By such modification, it is possible to change the physical properties of silk itself. Therefore, the usefulness of the protein that silkworms produce in the silk gland is dramatically increased. In addition, by introducing residues (groups) that exhibit characteristic infrared absorption and nuclear magnetic resonance, such as cyano groups and fluorine, in a residue-specific manner, it is possible to obtain local information on the structure of silk that has been impossible until now. It is done.

  The form of the silk material is not particularly limited, and examples thereof include a powder form, a fiber form, a sponge form, and a film form.

  The unnatural amino acid used in the present invention can be used simultaneously with the natural amino acid, and only one type of unnatural amino acid can be used, or two or more types of unnatural amino acids can be used simultaneously.

  The silkworm breeding method of the present invention can be performed by giving a feed containing an unnatural amino acid to those skilled in the art according to a common silkworm breeding method. For example, breeding can be carried out according to the method described in “Ministry of Education (1978) Soybean Manufacture. Pp193, Jikkyo Publishing Co., Tokyo”.

  In addition, when a non-natural amino acid having a functional group that causes a chemical reaction by light is fed to a silkworm, it is preferable to breed the silkworm under conditions where the non-natural amino acid is not irradiated with light. As a preferred breeding method, there is a method of breeding silkworms in a continuous dark period environment where no light is irradiated. Alternatively, the silkworm may be irradiated with light, and a method of rearing a silkworm under a condition in which light is not irradiated to the unnatural amino acid having a functional group that causes a chemical reaction by the light, For example, the space around the food to be fed is physically shielded from the light source, the unnatural amino acid is given to the silkworm, and when the silkworm produces the protein containing the unnatural amino acid from the silk gland, A method such as rearing silkworms in an environment where no irradiation is applied.

  When a non-natural amino acid is administered (feeding) to the transgenic silkworm of the present invention, the ratio of the natural amino acid (natural amino acid that is presumed to antagonize with the non-natural amino acid) may be reduced. For example, the amount of the natural amino acid is preferably 4/5 to 1/10 of the amount of the natural amino acid contained in the normal composition of the diet, and preferably 2/3 to 1/10. More preferably, the feed is reduced to 2/3 to 1/8, more preferably 1/2 to 1/5.

  The protein production method using the transgenic silkworm of the present invention is a residue-specific unnatural amino acid introduction method. Therefore, according to the present invention, a protein containing any kind of unnatural amino acid can be produced from transgenic silkworms of the same strain only by changing the kind of unnatural amino acid administered to the silkworm.

EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
[Preparation of synthetic feed]
Synthetic feeds of normal composition (feeds that do not contain any protein) used for breeding silkworms have already been reported (Kijiro Watanabe, Yasuhiro Horie, Nissay Zan, (1980) vol. 49, pp. 177-185; Hirayama, C .; , et.al., J. Insect Physiol., (1996) vol.42, pp.983-988). Table 1 shows the composition of the synthetic feed having the normal composition, and Table 2 shows the amino acid composition in the synthetic feed.

  The contents of L-Methionine and L-Phenylalanine represented by * in Table 2 may be changed according to the content of the experiment.

[Selection of recombinant host strain]
As the conditions required for the host used in this example, the following three conditions (1) are silkworms excellent in feeding property of synthetic feed (described later) (2) are limited strains (3) for observation of recombinant markers A suitable white-eye / white egg strain was set.
Particularly in silkworms satisfying the above condition (1), the condition (1) is a particularly preferable condition because it becomes easier to strictly control the ratio of the unnatural amino acid and the natural amino acid to be ingested by the silkworm. The above condition (2) is a preferable condition when it is important to align genders in order to obtain good quality data with little variation, since it is assumed that the amount of food intake and the weight of cocoon differ between males and females. The above condition (3) is not necessarily essential, but is a preferable condition because screening of recombinants can be performed more easily.

  In order to select a line satisfying the condition (1), a synthetic feed produced with the composition shown in Tables 1 and 2 was administered (feeded) to several lines of 5th instar larvae. As a result, white / CS (strain name: MCS601), which was developed, bred and passaged by the genetically modified silkworm research and development unit to which the inventors belong, was most excellent in the feeding property of the synthetic feed. Since the white / CS line also satisfies the conditions (2) and (3), it was selected as the host line.

[Production of recombinant vector]
In accordance with an existing technique that is within the common sense of a person skilled in the art, a piggyBac vector having a DNA encoding the amino acid sequence shown in SEQ ID NO: 1 (BmFRS1 ^* (A450G)) (including a 5 base 5′UTR sequence) (FIG. 1) was produced. In addition, a fibroin light chain (FibL) -derived promoter and 3′UTR sequence were added to the above vector on the 5 ′ and 3 ′ sides of the mutant gene sequence BmFRS1 ^* (A450G), respectively. By adding a promoter sequence derived from FibL, the target gene can be specifically expressed in the posterior silk gland. As a result, it was possible to avoid the larval growth failure that was supposed to occur when the mutant gene was expressed throughout the larvae.

[Production of transgenic silkworms and confirmation of recombinant gene expression]
The injection of piggyBac vector into fertilized eggs and subsequent screening of recombinants were performed according to existing methods that are within the common sense of those skilled in the art. White / CS (line name: MCS601) is a line that lays dormant eggs and cannot be made non-dormant by the above-mentioned low-temperature dark aerating method. . The copy number and insertion pattern of the mutant gene inserted into the genome were analyzed using real-time PCR and Southern blotting, and a transgenic silkworm strain having a mutant gene (BmFRS1 ^* (A450G)) was established. . This transgenic silkworm line was named white H01.

Each part (blood cell, anterior silk gland, middle silk gland, posterior silk gland, middle intestine, testis, Malpighian duct, fat body) was extracted from white H01 strain silkworm larvae (male) on the fifth day of age 4 Total RNA was extracted from. The recombinant gene fragment was amplified by RT-PCR using 1 μg of each extracted RNA as a template. The result of electrophoresis of the amplified DNA fragment is shown in FIG. RT in FIG. 2 represents reverse transcriptase. As the upper result in FIG. 2 shows, BmFRS1 ^* (A450G) is not amplified when RT is not used. The lower part of FIG. 2 is an amplified fragment of endogenous BmFRS1. As is clear from the results shown in the middle of FIG. 2, the recombinant gene (BmFRS1 ^* (A450G)) was expressed specifically in the posterior silk gland.

[Production of silk protein containing p-Cl-Phe]
<Creation of silkworm>
Example 1
The silkworm used in Example 1 is white H01 which is a line of the transgenic silkworm produced using the white / CS line as a host line. After feeding a synthetic feed with a normal composition from the 5th day of aging to the second day, 0.5 equivalents of p-Cl-Phe (represented by the general formula (1)) with respect to the normal amount of Phe after the third day A synthetic feed (containing a normal amount of Phe) containing R = Cl) was fed. The breeding method was performed by placing three males in a plastic container with a lid together with the feed, and placing the container in an incubator set at a temperature of 25 ° C. The feed was changed to a new one once a day, and the average food intake of three animals was calculated from the remaining amount at the time of replacement. At the same time, the body weight of three larvae was measured once a day, and the average body weight was calculated (n = 3).

(Comparative Example 1)
The silkworm used in Comparative Example 1 is the silkworm of the white / CS line. The feeding method and breeding method are the same as in Example 1.
(Comparative Example 2)
The silkworm used in Comparative Example 2 is white H01 which is a transgenic silkworm. As a bait, a synthetic feed having a normal composition not containing p-Cl-Phe was fed. The breeding method is the same as in Example 1.
(Comparative Example 3)
The silkworm used in Comparative Example 3 is a white / CS silkworm. As a bait, a synthetic feed having a normal composition not containing p-Cl-Phe was fed. The breeding method is the same as in Example 1.

  Table 3 shows the silkworm lines used in Example 1 and Comparative Examples 1 to 3, and the non-natural Phe type and the natural Phe amount contained in the feed fed to the silkworm. In addition, the equivalent in the kind of non-natural Phe in Table 3 and the amount of natural Phe show the molar ratio with respect to the amount of natural Phe in the synthetic feed of the said normal composition.

<Measurement of body weight of silkworm larvae upon administration of p-Cl-Phe>
FIG. 3 shows a growth curve of silkworm larvae of Example 1 and Comparative Examples 1 to 3, and FIG. 4 shows a graph of total food intake and cocoon layer weight.

  From the results of FIGS. 3 and 4, the transgenic silkworm of Example 1 to which the unnatural amino acid p-Cl-Phe was administered showed good growth similar to that of the silkworms of Comparative Examples 1 to 3, and the trans It was confirmed that sufficient weight of cocoons can be obtained from transgenic silkworms.

<Analysis of Silk Protein Containing p-Cl-Phe>
The silkworms were obtained from the silkworms of Example 1 and Comparative Examples 1 to 3. Each soot layer was dissolved in an 8M LiBr aqueous solution at a temperature of 35 ° C. for 40 minutes to a concentration of about 50 mg / mL. This lysate is diluted with an 8M aqueous urea solution to a concentration of about 4.5 mg / mL, further mixed with a sample buffer containing a reducing agent to adjust the concentration to about 3 mg / mL, and then at room temperature for 30 The reduction treatment was carried out by standing for more than minutes. The reduced sample solution was separated by SDS-PAGE, the FibL band was cut out, decolorized, washed and dried, and then digested in gel overnight at 37 ° C. with trypsin. The resulting peptide fragment was extracted and solidified by drying, and then dissolved in a 2: 1 mixture of 0.1% TFA aqueous solution and acetonitrile. This solution was mixed with a 2: 1 mixture of 0.1% TFA aqueous solution saturated with HCCA and acetonitrile, and dropped onto a MALDI target plate and dried to measure a MALDI-TOF-MS spectrum. did. FIG. 5 shows MALDI-TOF-MS measurement data in the vicinity of a peptide fragment (SGNFAFFR) containing two residues of Phe generated by digestion of FibL. Weight gain corresponding to replacement of the p-Cl-Phe for Phe only in districts of Example 1 (Found: + 33.97Da, ^{theory: 35 Cl - 1 H = +33.96} Da) was observed. In addition to the 1-substituted product (m / z 889.40), a peak corresponding to the 2-substituted product (m / z 923.37) was observed.

  As is clear from the above results, feeding a transgenic silkworm containing p-Cl-Phe to a silkworm that specifically expresses the silkworm gland PheRSα subunit substituted at the amino acid number 450 position. Thus, FibL protein containing p-Cl-Phe could be obtained.

[Production of p-Br-Phe-containing silk protein]
<Creation of silkworm>
(Example 2)
The silkworm used in Example 2 is white H01 which is a transgenic silkworm. After feeding a synthetic feed having a normal composition from the 5th inception to the second day, 0.5 equivalents of p-Br-Phe (represented by the general formula (1)) from the third day onwards A synthetic feed containing R = Br) was fed. Silkworms were raised in the same manner as in Example 1.
Example 3
The silkworm used in Example 3 is white H01 which is a transgenic silkworm. After feeding a synthetic feed having a normal composition from the 5th inception to the second day, after the third day, it contains 0.5 equivalent of p-Br-Phe to the normal amount of Phe, and the normal amount of Phe Synthetic feed reduced to 1/2 of Phe was fed. The breeding method is the same as in Example 2.
Example 4
The silkworm used in Example 4 is white H01 which is a transgenic silkworm. After feeding a synthetic feed having a normal composition from the 5th inception to the second day, after the third day, it contains 0.5 equivalent of p-Br-Phe to the normal amount of Phe, and the normal amount of Phe The synthetic feed reduced to 1/5 with respect to Phe was fed. The breeding method is the same as in Example 2.

(Comparative Example 4)
The silkworm used in Comparative Example 4 is white H01 which is a transgenic silkworm. After feeding the normal composition of synthetic feed from the 5th day to the 2nd day, after the 3rd day, p-Br-Phe is not included, but the Phe content is reduced to 1/5 of the normal amount of Phe. The fed synthetic feed was fed. The breeding method is the same as in Example 2.
(Comparative Example 5)
The silkworm used in Comparative Example 5 is a white / CS silkworm. After feeding a synthetic feed having a normal composition from the 5th inception to the second day, after the third day, it contains 0.5 equivalent of p-Br-Phe to the normal amount of Phe, and the normal amount of Phe The synthetic feed reduced to 1/5 with respect to Phe was fed. The breeding method is the same as in Example 2.
(Comparative Example 6)
The silkworm used in Comparative Example 6 is a white / CS silkworm. The feed did not contain p-Br-Phe, but was fed a synthetic feed in which the Phe content was reduced to 1/5 of the normal amount of Phe. The breeding method is the same as in Example 2.

  Table 3 shows the silkworm lines used in Examples 2 to 4 and Comparative Examples 4 to 6, and the non-natural Phe types and the amount of natural Phe contained in the feed fed to the silkworms.

<Measurement of body weight of silkworm larvae upon administration of p-Br-Phe>
The growth curves of the silkworm larvae of Examples 2 to 4 and Comparative Examples 4 to 6 are shown in FIG. 6, and the graph of total food intake and cocoon layer weight is shown in FIG.

  As shown in FIG. 6, the transgenic silkworms of Examples 2 to 4 to which the unnatural amino acid p-Br-Phe was administered showed good growth similar to the silkworms of Comparative Examples 4 to 6. Moreover, as shown in FIG. 7, it was confirmed that a cocoon is obtained from the transgenic silkworm of Examples 2-4.

<Analysis of Silk Protein Containing p-Br-Phe>
The silkworms were obtained from the silkworms of Examples 2 to 4 and Comparative Examples 4 to 6. Each soot layer was dissolved in an 8M LiBr aqueous solution at a temperature of 35 ° C. for 40 minutes to a concentration of about 50 mg / mL. This lysate is diluted in an 8M aqueous urea 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 at a room temperature of 30 mg. The reduction treatment was carried out by standing for more than minutes. The reduced sample solution was separated by SDS-PAGE, the FibL band was cut out, decolorized, washed and dried, and then digested in gel overnight at 37 ° C. with trypsin. The resulting peptide fragment was extracted and solidified by drying, and then dissolved in a 2: 1 mixture of 0.1% TFA aqueous solution and acetonitrile. This solution was mixed with a 2: 1 mixture of 0.1% TFA aqueous solution saturated with HCCA and acetonitrile, and dropped onto a MALDI target plate to dry. MALDI-TOF-MS measurement data using this as a sample is shown in FIG. In Examples 2 to 4 in which p-Br-Phe was administered to white H01, mass increase accompanying substitution of Phe to p-Br-Phe (actual value: +77.93 Da, theoretical value: ⁷⁹ Br ⁻¹ H = + 77. 91 Da) was observed. The relative peak intensity of the p-Br-Phe-containing peptide fragment relative to the Phe-containing peptide fragment contains 1/2 and 1/5 the amount of Phe than in Example 2 when fed on a feed containing a normal amount of Phe. Examples 3 and 4 when fed with feed were stronger.

  As is clear from the results of Examples 2 to 4, feeding a silkworm containing p-Br-Phe to a silkworm in which the gene encoding the PheRSα subunit substituted at amino acid position 450 is expressed specifically in the silk gland Thus, FibL protein containing p-Br-Phe could be obtained.

[Production of pN ₃ -Phe-containing silk protein]
<Creation of silkworm>
(Example 5)
The silkworm used in Example 5 is white H01 which is a transgenic silkworm. After feeding a synthetic feed having a normal composition from the 5th inception to the second day, 0.5 equivalents of p-N ₃ -Phe (general formula (1)) with respect to the normal amount of Phe after the third day. A synthetic feed containing R = N ₃ ) was fed. The silkworms were bred up to the third day in the same manner as in Example 1 above, and the silkworms were bred under the light-shielding conditions in order to avoid the photoreaction of p-N ₃ -Phe. .
(Example 6)
The silkworm used in Example 6 is white H01 which is a transgenic silkworm. After feeding the normal composition of synthetic feed from the 5th day to the 2nd day, after the 3rd day, it contains 0.5 equivalent of p-N ₃ -Phe to the normal amount of Phe, and the Phe content is usually Synthetic feed reduced to 1/2 of the amount of Phe was fed. The breeding method is the same as in Example 5.
(Example 7)
The silkworm used in Example 7 is white H01 which is a transgenic silkworm. After feeding the normal composition of synthetic feed from the 5th day to the 2nd day, after the 3rd day, it contains 0.5 equivalent of p-N ₃ -Phe to the normal amount of Phe, and the Phe content is usually The synthetic feed reduced to 1/5 with respect to the amount of Phe was fed. The breeding method is the same as in Example 5.

(Comparative Example 7)
The silkworm used in Comparative Example 7 is white H01 which is a transgenic silkworm. After feeding a synthetic feed having a normal composition from the 5th day of rising to the 2nd day, after the 3rd day, it does not contain p-N ₃ -Phe, but the Phe content is reduced to 1/5 of the normal amount of Phe. Reduced synthetic feed was fed. The breeding method is the same as in Example 5.
(Comparative Example 8)
The silkworm used in Comparative Example 8 is a white / CS silkworm. After feeding the normal composition of synthetic feed from the 5th day to the 2nd day, after the 3rd day, it contains 0.5 equivalent of p-N ₃ -Phe to the normal amount of Phe, and the Phe content is usually The synthetic feed reduced to 1/5 with respect to the amount of Phe was fed. The breeding method is the same as in Example 5.
(Comparative Example 9)
The silkworm used in Comparative Example 9 is a white / CS silkworm. After feeding the synthetic feed of the normal composition from the 5th day of aging to the second day, after the third day, it does not contain p-N ₃ -Phe, but the Phe content is reduced to 1/5 of the normal amount of Phe. Reduced synthetic feed was fed. The breeding method is the same as in Example 5.

  Table 3 shows the silkworm lines used in Examples 5 to 7 and Comparative Examples 7 to 9, and the non-natural Phe type and the natural Phe amount contained in the feed fed to the silkworm.

<Measurement of body weight of silkworm larvae at the time of p-N ₃ -Phe administration>
The growth curves of silkworm larvae of Examples 5 to 7 and Comparative Examples 7 to 9 are shown in FIG. 9, and the graph of total food intake and cocoon layer weight is shown in FIG.

As shown in FIG. 9, the transgenic silkworms of Examples 5 and 6 to which the unnatural amino acid pN ₃ -Phe was administered showed good growth similar to the silkworms of Comparative Examples 7-9. Although the growth of the silkworm of Example 7 was slightly poor, it was confirmed that cocoons were obtained from any of the transgenic silkworms of Examples 5 to 7, as shown in FIG.

<Analysis of _p-N 3 -Phe containing silk protein>
The silkworms were obtained from the silkworms of Examples 5 to 7 and Comparative Examples 7 to 9. Each soot layer was dissolved in an 8M LiBr aqueous solution at a temperature of 35 ° C. for 40 minutes to a concentration of about 50 mg / mL. This lysate is diluted in an 8M aqueous urea 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 at a room temperature of 30 mg. The reduction treatment was carried out by standing for more than minutes. The reduced sample solution was separated by SDS-PAGE, the FibL band was cut out, decolorized, washed and dried, and then digested in gel overnight at 37 ° C. with trypsin. The resulting peptide fragment was extracted and solidified by drying, and then dissolved in a 2: 1 mixture of 0.1% TFA aqueous solution and acetonitrile. This solution was mixed with a 2: 1 mixture of 0.1% TFA aqueous solution saturated with HCCA and acetonitrile, and dropped onto a MALDI target plate to dry. MALDI-TOF-MS measurement data using this as a sample is shown in FIG. In Examples 5 to 7 in which p-N ₃ -Phe (0.5 equivalent) was administered to white H01, the mass increase accompanying substitution of Phe to p-NH ₂ -Phe (actual value: +15.06 Da, theoretical value: ^{14 N 1 H 1 H - 1} H = +15.01 Da) was observed. The N ₃ group is considered to have been reduced to the NH ₂ group by the reduction treatment during the preparation of the SDS-PAGE sample. The relative peak intensity of the p-N ₃ -Phe-containing peptide fragment relative to the Phe-containing peptide fragment is 1/2 and 1/5 the amount of Phe compared to Example 5 when fed with a feed containing a normal amount of Phe. Examples 6 and 7 were stronger when they were bred with the feed they contained.

As is clear from the results of Examples 5 to 7, a silkworm in which a gene encoding a PheRSα subunit substituted at amino acid number 450 is expressed specifically in the silk gland is fed with a feed containing p-N ₃ -Phe. it _is, it could be obtained FibL protein containing _p-N 3 -Phe.

[Click reaction to p-N ₃ -Phe introduced into silk protein]
Each cocoon layer obtained from the silkworms of Examples 5 to 7 and Comparative Examples 7 to 9 was dissolved in 8M LiBr, and click reaction with a biotin reagent having an ethynyl group (Biotin-PEG4-Alkyne) was performed. The reaction scheme is shown in FIG. FIG. 13 shows the result of Western blotting using HRP-modified streptavidin after transferring FibL and FibH separated by SDS-PAGE after the click reaction onto a PVDF membrane. For FibL, signals were observed only in Examples 5 to 7 in which p-N ₃ -Phe was administered to white H01, and for FibH only in Examples 6 and 7. In addition, a stronger signal was observed as the Phe content decreased.
From the results shown in FIG. 13, it was confirmed that biotin was added to the silk proteins obtained from the silkworms of Examples 5 to 7 by a click reaction.

Although the introduction amount of p-N ₃ -Phe was considered to be relatively small from the result of mass spectrometry of silk (FIG. 11), the signal of bound biotin (FIG. 13) was very strong. . This suggests that the click reaction in the silk molecule proceeds with high efficiency. Phe is Phe residue for a hydrophobic amino acid are likely positioned inside silk molecules, the _p-N 3 -Phe residue be replaced with this _p-N 3 -Phe residue Although it was assumed that it was difficult to react with biotin, the p-N ₃ -Phe-containing silk produced using the transgenic silkworm of the present invention has a pN ₃ -Phe residue in the silk molecule. It has been confirmed that the click reaction with respect to the p-N ₃ -Phe-containing silk with extremely high utility value can be obtained.

[Production of p-Eth-Phe-containing silk protein]
<Creation of silkworm>
(Example 8)
The silkworm used in Example 8 is white H01 which is a transgenic silkworm. After feeding a synthetic feed having a normal composition from the 5th inception to the second day, 0.5 equivalent of p-Eth-Phe (represented by the general formula (1)) from the third day onward. A synthetic feed containing R = C≡CH) was fed. Silkworms were raised in the same manner as in Example 1.

(Comparative Example 10)
The silkworm used in Comparative Example 10 is a white / CS silkworm. The feeding method and breeding method are the same as in Example 8.
(Comparative Example 11)
The silkworm used in Comparative Example 11 is white H01 which is a transgenic silkworm. As a bait, a synthetic feed having a normal composition not containing p-Eth-Phe was fed. The breeding method is the same as in Example 8.
(Comparative Example 12)
The silkworm used in Comparative Example 12 is a white / CS silkworm. As a bait, a synthetic feed having a normal composition not containing p-Eth-Phe was fed. The breeding method is the same as in Example 8.

  Table 3 shows the silkworm lines used in Example 8 and Comparative Examples 10 to 12, and the non-natural Phe type and the natural Phe amount contained in the feed fed to the silkworm.

<Measurement of body weight of silkworm larvae upon administration of p-Eth-Phe>
FIG. 14 shows a growth curve of silkworm larvae of Example 8 and Comparative Examples 10 to 12, and FIG. 15 shows a graph of total food intake and cocoon layer weight.

  As shown in FIGS. 14 and 15, although silkworm weight and average cocoon weight tended to decrease in the silkworm of Example 8, it was confirmed that the silkworm of Example 8 could obtain a sufficient weight of cocoon. It was done.

<Analysis of Silk Protein Containing p-Eth-Phe>
The silkworms were obtained from the silkworms of Example 8 and Comparative Examples 10-12. Each soot layer was dissolved in an 8M LiBr aqueous solution at a temperature of 35 ° C. for 40 minutes to a concentration of about 50 mg / mL. This lysate is diluted in an 8M aqueous urea 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 at a room temperature of 30 mg. The reduction treatment was carried out by standing for more than minutes. The reduced sample solution was separated by SDS-PAGE, the FibL band was cut out, decolorized, washed and dried, and then digested in gel overnight at 37 ° C. with trypsin. The resulting peptide fragment was extracted and solidified by drying, and then dissolved in a 2: 1 mixture of 0.1% TFA aqueous solution and acetonitrile. This solution was mixed with a 2: 1 mixture of 0.1% TFA aqueous solution saturated with HCCA and acetonitrile, and dropped onto a MALDI target plate to dry. In Example 8 in which p-Eth-Phe (0.5 equivalent) was administered to white H01, the mass increase accompanying the replacement of Phe with p-Eth-Phe (actual value: +24.01 Da, theoretical value: ¹² C ¹² C ^{1 H - 1 H = +24.00 Da} ) was observed.

  As is apparent from the results of Example 8, by giving a feed containing p-Eth-Phe to a silkworm in which a gene encoding a PheRSα subunit substituted at amino acid number 450 was expressed specifically in the silk gland, FibL protein containing p-Eth-Phe could be obtained.

[Click reaction to p-Eth-Phe introduced into silk protein]
Each cocoon layer obtained from the silkworms of Example 8 and Comparative Examples 10 to 12 was dissolved in 8M LiBr, and click reaction with a biotin reagent having an azide group (Biotin-PEG4-Azide) was performed. The reaction scheme corresponds to that obtained by exchanging the azide group and the ethynyl group in FIG. FIG. 17 shows the result of transferring the FibL separated by SDS-PAGE after the click reaction onto a PVDF membrane and performing Western blotting using HRP-modified streptavidin. A signal was observed only in Example 8 in which p-Eth-Phe was administered to white H01.
From the results shown in FIG. 17, it was confirmed that the silk protein obtained from the silkworm of Example 8 was modified with biotin by a click reaction.

  As is apparent from the results of FIG. 16 and FIG. 17, feeding a silkworm that contains p-Eth-Phe to a silkworm in which the gene encoding the PheRSα subunit substituted at amino acid position 450 is expressed specifically in the silk gland Thus, FibL protein containing p-Eth-Phe could be obtained.

  Although the introduction amount of p-Eth-Phe was considered to be relatively small from the mass spectrometry result of silk (FIG. 16), the signal of bound biotin (FIG. 17) could be clearly observed. . This suggests that the click reaction in the silk molecule proceeds with high efficiency. Since Phe is a hydrophobic amino acid, it is highly possible that the Phe residue is located inside the silk molecule. Even if this is substituted with p-Eth-Phe residue, the p-Eth-Phe residue is replaced with biotin. Although it was assumed that the reaction was difficult, the p-Eth-Phe-containing silk produced using the transgenic silkworm of the present invention had a highly efficient click reaction for p-Eth-Phe residues. It was confirmed that a p-Eth-Phe-containing silk with extremely high utility value can be obtained.

  The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.

  Non-natural amino acid-containing proteins produced using the transgenic silkworms of the present invention, such as unnatural amino acid-containing silk proteins, are widely applicable in the fields of textiles, medical fields, functional materials, etc., and are more suitable for each application. Have the potential to provide modified silk. In addition, there is a possibility that new silk applications can be developed by the present invention.

Claims (7)

(A) the amino acid sequence shown in SEQ ID NO: 1,
(B) the amino acid sequence shown in SEQ ID NO: 2,
(C) an amino acid sequence in which one to several amino acids have been deleted, inserted, substituted or added at a site other than the amino acid number 450 position of the amino acid sequence shown in SEQ ID NO: 1, and desired for phenylalanine-specific tRNA Amino acid sequence of a protein having an activity of binding an unnatural amino acid, and (d) deletion, insertion, substitution or addition of one to several amino acids at a site other than amino acid number 407 of the amino acid sequence shown in SEQ ID NO: 2 A DNA encoding a protein having any one amino acid sequence selected from the group consisting of amino acid sequences of proteins having an activity of binding a desired unnatural amino acid to a phenylalanine-specific tRNA, and Has a promoter that expresses the DNA specifically in the silk gland Transgenic silkworm that characterized the door.   The transgenic silkworm according to claim 1, wherein the promoter that specifically expresses the DNA is a promoter that specifically expresses the DNA in the posterior silk gland.   The transgenic silkworm according to claim 1 or 2, wherein the promoter that specifically expresses the DNA is a fibroin L chain-derived promoter.   A method for producing a non-natural amino acid-containing protein, comprising the step of administering a non-natural amino acid to the transgenic silkworm according to any one of claims 1 to 3.   The method for producing an unnatural amino acid-containing protein according to claim 4, wherein the unnatural amino acid is a phenylalanine analog. The method for producing an unnatural amino acid-containing protein according to claim 4 or 5, wherein the unnatural amino acid is an amino acid represented by the following general formula (1).
[Wherein, R represents a halogen atom, an azide group or an ethynyl group. ]   The method for producing an unnatural amino acid-containing protein according to claim 6, wherein R is an azide group or an ethynyl group.