JPWO2018190333A1 - Transgenic silkworm and method for producing unnatural amino acid-containing protein using the silkworm - Google Patents

Abstract

A DNA comprising a sequence containing any one of the following amino acid sequences (a) to (c), and encoding a protein having an activity of binding a desired unnatural amino acid to a phenylalanine-specific tRNA; and Transgenic silkworm, which has a promoter that causes silk gland-specific expression. (A) an amino acid sequence having a mutation at amino acid number 432 of the amino acid sequence shown in SEQ ID NO: 1; (b) one or several amino acids are deleted or inserted at a site other than amino acid number 432 in (a). (C) an amino acid sequence having 80% or more identity at a site other than amino acid number 432 in (a)

Description

The present invention relates to a transgenic silkworm and a method for producing an unnatural amino acid-containing protein using the silkworm.
Priority is claimed on Japanese Patent Application No. 2017-79294, filed on April 12, 2017, the content of which is incorporated herein by reference.

Natural resources such as petroleum, metals, and ceramics are feared to be depleted in the future. Silk proteins have been cited as a next-generation material that is not likely to be depleted.
Silk itself has many useful properties, but it is expected that by modifying such properties, it will be used industrially as a more useful material.
For example, the development of a tougher silk material than the existing silk material and the advancement of wearable devices are expected to introduce devices into silk proteins. In addition, application of silk proteins having high biocompatibility to medical materials, cosmetic materials, and the like is expected.

  As a method of modifying the characteristics of silk, there is a method of modifying the characteristics of silk protein using a silkworm genetic recombination technique. In particular, the method of introducing an unnatural amino acid into a protein is attracting attention because an unnatural amino acid can be used as a component and a protein having characteristics not found in nature can be created.

There are two known methods for introducing unnatural amino acids into proteins. One is the “site-specific unnatural amino acid introduction method”. This method requires administration of an unnatural amino acid and modification of the target protein gene sequence, the 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. Another limitation is that the sequence of the target protein gene must be known.
On the other hand, a “residue-specific unnatural amino acid introduction method” is known as a second method. In this method, the efficiency of incorporation of the unnatural amino acid is improved by the forced expression of a mutant aaRS having a mutation introduced into the amino acid binding site in addition to administration of the unnatural amino acid. Since this method for introducing a residue-specific unnatural amino acid can be performed without modifying the sequence of the target protein gene, it is easier to use than the method for introducing a site-specific unnatural amino acid, and the sequence of the target protein gene is unknown. This is a technology of high utility value because it has the advantage that it can be used.

The present inventor has improved the residue-specific unnatural amino acid introduction method, a protein larger structure as a naturally occurring amino acid contains a different unnatural amino certain non-natural amino acids, such as (for example p-N ₃ -Phe) It was successfully produced in silkworm (see Non-Patent Document 1).

Teramoto et al. Azide-Incorporated Clickable Silk Fibroin Materials with the Ability to Photopattern. ACS Biomater. Sci. Eng., 2016, 2 (2), pp 251-258.

  Further, there is a need for a method for producing a protein containing an unnatural amino acid with high efficiency using a small amount of an unnatural amino acid.

  The present invention has been made in view of the above circumstances, a transgenic silkworm capable of efficiently producing an unnatural amino acid-containing protein with a small amount of an unnatural amino acid, and an unnatural amino acid-containing protein using the transgenic silkworm And a method for producing the same.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, have found a transgenic silkworm that specifically expresses a DNA encoding a protein having an activity of binding a desired unnatural amino acid to tRNA in a silk gland. 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 DNA comprising a sequence comprising any one of the following amino acid sequences (a) to (c), and encoding a protein having an activity of binding a desired unnatural amino acid to a phenylalanine-specific tRNA; and And a transgenic silkworm having a promoter for specifically expressing the DNA in a silk gland.
(A) an amino acid sequence having a mutation at amino acid number 432 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 amino acid number 432 in (a),
(C) an amino acid sequence having 80% or more identity at a site other than amino acid number 432 in (a).

(2) The transgenic silkworm according to (1), wherein the amino acid number 432 of (a) is alanine, valine, leucine, methionine, threonine, cysteine, or tyrosine.
(3) a DNA comprising a sequence comprising any one of the following amino acid sequences (d) to (h) and encoding a protein having an activity of binding a desired unnatural amino acid to a phenylalanine-specific tRNA; and The transgenic silkworm according to claim 1, further comprising a promoter that specifically expresses the DNA in a silk gland.
(D) an amino acid sequence represented by SEQ ID NO: 2,
(E) the amino acid sequence of SEQ ID NO: 3,
(F) an amino acid sequence represented by SEQ ID NO: 4,
(G) an amino acid sequence in which one or several amino acids have been deleted, inserted, substituted or added at a site other than amino acid number 432 of the amino acid sequence shown in any of SEQ ID NOs: 2 to 4;
(H) an amino acid sequence having 80% or more identity at a site other than amino acid number 432 of the amino acid sequence shown in any of SEQ ID NO: 2 to SEQ ID NO: 4

(4) The transgenic silkworm according to any one of (1) to (3), further having a mutation at amino acid number 407 of (a) to (h).
(5) The transgenic silkworm according to (4), wherein the mutation at amino acid number 407 is alanine or glycine.
(6) The transgenic silkworm according to any one of (1) to (5), wherein the promoter for specifically expressing the DNA in the silk gland is a promoter for specifically expressing the DNA in the rear silk gland.
(7) The transgenic silkworm according to any one of (1) to (6), wherein the promoter for specifically expressing the DNA in a silk gland is a promoter derived from a fibroin L chain.

(8) An F1 hybrid, wherein the transgenic silkworm according to any one of (1) to (7) is crossed with a practical line.
(9) A non-natural amino acid comprising a step of administering an unnatural amino acid to the transgenic silkworm according to any one of (1) to (7) or the F1 hybrid according to (8). A method for producing an amino acid-containing protein.
(10) The method for producing an unnatural amino acid-containing protein according to (9), wherein the unnatural amino acid is a phenylalanine analog.
(11) The method for producing an unnatural amino acid-containing protein according to (9) or (10), wherein the unnatural amino acid is an amino acid represented by the following general formula (1).

[In the formula, R represents a halogen atom, a cyano group, an azido group, or an ethynyl group. ]
(12) The method for producing an unnatural amino acid-containing protein according to (11), wherein R is an azide group or an ethynyl group.

  ADVANTAGE OF THE INVENTION According to this invention, the transgenic silkworm which can manufacture an unnatural amino acid containing protein with high efficiency with a small amount of unnatural amino acid, and the manufacturing method of the unnatural amino acid containing protein using this transgenic silkworm can be provided.

3 is a table summarizing the growth inhibition of each mutant. It is a piggyBac vector map for producing a transgenic silkworm expressing the α subunit gene (BmFRS1) of silkworm PheRS (phenylalanyl-tRNA synthetase) in the posterior silk gland. An asterisk (*) attached to BmFRS1 indicates that it is a mutant gene. Fibroin production per head of silkworms administered an amount of _p-N 3 -Phe (bars), and _{p-N 3 p-N 3} -Phe incorporation of -Phe into fibroin in which silkworms administered is produced Amount (intensity ratio of mass spectrometry peak) (circle plot) is shown. Fibroin production per head of silkworms administered an amount of _p-N 3 -Phe (bars), and _{p-N 3 p-N 3} -Phe incorporation of -Phe into fibroin in which silkworms administered is produced Amount (intensity ratio of mass spectrometry peak) (circle plot) is shown. Fibroin production per head of silkworms administered with _p-N 3 -Phe different amounts (bars), and _{p-N 3 p-N 3} -Phe incorporation of -Phe into fibroin in which silkworms administered is produced Amount (intensity ratio of mass spectrometry peak) (circle plot) is shown. Fibroin production per head of silkworms administered with _p-N 3 -Phe different amounts (bars), and _{p-N 3 p-N 3} -Phe incorporation of -Phe into fibroin in which silkworms administered is produced Amount (intensity ratio of mass spectrometry peak) (circle plot) is shown. Fibroin production per head of silkworms administered with _p-N 3 -Phe different amounts (bars), and _{p-N 3 p-N 3} -Phe incorporation of -Phe into fibroin in which silkworms administered is produced Amount (intensity ratio of mass spectrometry peak) (circle plot) is shown.

《Transgenic silkworm》
The transgenic silkworm of the present invention comprises a protein comprising a sequence containing any one of the following amino acids (a) to (c) and having an activity of binding a desired unnatural amino acid to a phenylalanine-specific tRNA. A transgenic silkworm comprising a coding DNA and a promoter for specifically expressing the DNA in a silk gland.
(A) an amino acid sequence having a mutation at amino acid number 432 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 amino acid number 432 in (a),
(C) an amino acid sequence having 80% or more identity at a site other than amino acid number 432 in (a).

Aminoacylation of tRNA is catalyzed by the aminoacyl-tRNA synthetase (aaRS) corresponding to each amino acid. aaRS has strict substrate specificity among the 20 amino acids that make up proteins. For example, the aaRS corresponding to phenylalanine is called phenylalanyl-tRNA synthetase (PheRS). The DNA encoding the protein having the amino acid sequence of (a) to (c) 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 phenylalanine-specific tRNA. DNA encoding a protein having
The protein having the activity of binding the desired unnatural amino acid to the phenylalanine-specific tRNA only needs to have the activity, and in addition to any one of the sequences (a) to (c), It may further have an arbitrary sequence such as a protein tag (for example, histidine tag, HA tag, GFP, etc.).
By expressing in a silk gland a DNA encoding a protein having any of the above-mentioned sequences (a) to (c), amino acids (non-natural Amino acids) can be introduced. Furthermore, the unnatural amino acid can be introduced into the protein produced in the silk gland with high efficiency using a small amount of the unnatural amino acid.

  As described later in Examples, in the transgenic silkworm of the present invention, the amino acid number 432 of (a) is preferably alanine, valine, leucine, methionine, threonine, cysteine, or tyrosine, and alanine, valine And methionine are more preferred.

That is, the transgenic silkworm of the present invention comprises a sequence containing any one of the following amino acid sequences (d) to (h), and has an activity of binding a desired unnatural amino acid to phenylalanine-specific tRNA. It is preferable to have a DNA encoding the protein and a promoter for expressing the DNA in a silk gland-specific manner.
(D) an amino acid sequence represented by SEQ ID NO: 2 (Phe432Val),
(E) an amino acid sequence represented by SEQ ID NO: 3 (Phe432Ala),
(F) an amino acid sequence represented by SEQ ID NO: 4 (Phe432Met),
(G) an amino acid sequence in which one or several amino acids have been deleted, inserted, substituted or added at a site other than amino acid number 432 of the amino acid sequence shown in any of SEQ ID NOs: 2 to 4;
(H) an amino acid sequence having 80% or more identity at a site other than amino acid number 432 of the amino acid sequence shown in any of SEQ ID NO: 2 to SEQ ID NO: 4

Here, the number of deleted, inserted, substituted or added amino acids is preferably 1 to 50, more preferably 1 to 20, still more preferably 1 to 10, and particularly preferably 1 to 5, One or two are most preferred.
The identity is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 99% or more.

  Further, the transgenic silkworm of the present invention may have a mutation at amino acid number 407 in addition to the mutation at amino acid number 432. The mutation at amino acid number 407 is preferably alanine or glycine.

  The unnatural amino acid-containing protein produced by the silk gland is not particularly limited, and a silk protein produced by the silkworm in the silk gland is preferable. Further, an 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 out of the silkworm body as a cocoon, 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 toxicity caused by expressing the DNA in the whole transgenic silkworm, a promoter for expressing the DNA in the silk gland is used in the present invention. The silk glands are roughly divided into an anterior silk gland, a middle silk gland, and a posterior silk gland. Here, the promoter for specifically expressing the DNA in the silk gland is not particularly limited as long as it is a promoter capable of specifically expressing the DNA in the silk gland, and may be any one or two of the above three sites. A promoter that can be expressed at a site composed of the above combinations may be used.
Examples of the promoter specifically expressed in the middle silk gland include a promoter of a DNA encoding sericin 1 protein or sericin 2 protein. By using a promoter that specifically expresses in the middle silk gland, an unnatural amino acid can be introduced into a protein produced in the middle silk gland. Sericin is mentioned as a protein produced in the middle silk gland of the silkworm.
In addition, examples of the promoter specifically expressed in the posterior silk gland include a promoter of a DNA encoding a fibroin L chain (FibL) protein. By using a promoter that is specifically expressed in the posterior silk gland, an unnatural amino acid can be introduced into a protein produced in the posterior silk gland. The protein produced in the posterior silk gland of the silkworm is fibroin, and fibroin accounts for the majority of the silkworm cocoon protein, so the use of a promoter that specifically expresses the posterior silk gland controls the properties of the silk protein. Becomes easier.

  When the promoter is used, another sequence for controlling the expression of the DNA, subsequent translation into a protein, etc. may be used at the same time. Examples of such a sequence include 3′UTR (untranslated region). ) 5 ′ UTR sequence and the like.

The host silkworm used for producing 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 below. Although it is possible to use strains such as w1-pnd which are widely used for the production of genetic recombination, from the viewpoint of increasing the amount of unnatural amino acids administered as feed to the silkworm individual, the artificial feed intake It is preferable to use silkworms that are excellent in water content. For example, varieties such as day 01, day 509, day 510, day 603, day 604, medium 604, medium 605 and the like are preferable, and the amount of artificial feed containing an unnatural amino acid per animal is preferred. More white / CS (system name: MCS601) is more preferable.
Further, an F1 hybrid obtained by crossing the transgenic silkworm of the present invention with a practical line may be produced. Examples of practical systems include Day 509 x Day 510, Day 603 x Day 604, Middle 509 x Middle 510, Middle 604 x Middle 605, and the like.

  The silkworm used for the production of the transgenic silkworm of the present invention is not particularly limited as described above, and may be either a silkworm having a property of laying a non-diapause egg or a silkworm having a property of laying a dormant egg. 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. during the thoracic limb development stage, particularly after the bristle development stage. This low temperature period requires at least ten days. Keep dark from embryo inversion to head coloring. The silkworm moth obtained by rearing the hatched larvae then produces non-dormant eggs. (Introduction to Silkworm Species Author: Takeo Takami Publisher: National Association of Silkworm Species (May 31, 1969)).

  As a method other than the low-temperature dark blue method, a method of producing non-dormant eggs by controlling the larval stage light conditions (see JP-A-2003-88274) may be adopted.

  In the case of using a silkworm in which a non-dormant egg is not obtained even by using a method such as a low-temperature dark blue method, the egg may be broken by doping the silkworm egg with acid. As a specific method of the acid soaking treatment, a silkworm egg 3 to 4 hours after egg collection is immersed in hydrochloric acid at room temperature for about 30 minutes, washed with water to remove hydrochloric acid, and then air-dried. As the hydrochloric acid, for example, one having a specific gravity of 1.11 can be used (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 of introducing DNA into a silkworm egg. The introduction of DNA into silkworm eggs can be performed, for example, by injecting a transposon as a vector into the early embryo of the silkworm (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 the DNA encoding the transposon transferase (helper vector) introduced into the silkworm egg together with the vector into which the DNA has been 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.).

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

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

  The method of injecting the above-described transposon as a vector, the method of using a baculovirus vector, and the method of introducing DNA into other silkworm eggs may be used for DNA encoding a protein having the amino acid sequence, In the production of the unnatural amino acid-containing protein using the transgenic silkworm of the present invention, the protein 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 eggs of a silkworm egg using a DNA injection tube, a preferred embodiment is to make a hole in the eggshell physically or chemically in advance, and to transfer the DNA from the hole. Introduce.

  In the present invention, examples of a method of physically piercing the eggshell include a method of piercing using a needle, a microlaser, or the like. Among them, a method of making a hole in the eggshell by a method using a needle is preferable. The material used, the strength, etc., of the needle to be used are not particularly limited as long as it can make a hole in the eggshell of the silkworm. On the other hand, as a method of chemically making a hole in the eggshell, for example, a method of making a hole with a chemical (eg, hypochlorous acid) or the like can be mentioned.

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

  Such devices include a dissecting microscope, a lighting device, a movable stage, a coarse manipulator fixed to the microscope with metal fittings, a micromanipulator attached to this manipulator, and a pneumatic pressure for injecting DNA. And an injector that adjusts the pressure. In addition, as an apparatus to be used, an apparatus described in Japanese Patent No. 1654050 or an apparatus obtained by improving the apparatus can be used.

  Whether or not the DNA has been introduced into the silkworm egg is determined, for example, by a method in which the injected DNA is extracted again from the egg and measured (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.). Whether the DNA introduced into the silkworm egg has been integrated into the chromosome of the silkworm and whether the transgenic silkworm has been obtained or not can be determined, for example, by crossing adult grown from the egg injected with the DNA (either mutually or non-recombinant silkworm). And) obtaining a fertilized egg, and observing the expression of a marker gene (eg, EGFP, DsRed, CFP, YFP, etc.) with a fluorescence microscope 6 to 10 days after the egg laying, or using a body color marker gene (eg, Bm- aaNAT) can be visually confirmed.

  Whether or not the DNA introduced into the silkworm egg was expressed in the silkworm was confirmed by observing the expression of the marker gene with a fluorescence microscope for the egg (G1 generation) laid by the individual grown from the injected egg (G0 generation). I do. In addition, the total RNA may be extracted from the silkworm, and the transgene may be confirmed by RT-PCR using the extracted RNA as a template.

  In addition, a method of confirming at which site of a silkworm individual the DNA introduced into the silkworm is expressed is, for example, a method of extracting tissues from larvae and confirming expression of each tissue, or a method of CFP, GFP, EGFP, or the like. It can be confirmed by using a known reporter gene such as YFP or DsRED or the like and observing an individual silkworm using a fluorescence microscope.

<< Method for producing unnatural amino acid-containing protein >>
The method for producing an unnatural amino acid-containing protein of the present invention has 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 that make up proteins refer to the twenty standard natural amino acids, selenocysteine, and pyrrolidine. That is, in the present invention, the unnatural amino acid means an analog (modified amino acid) of these amino acids constituting the protein 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 unnatural amino acid used is preferably a phenylalanine analog. As the phenylalanine analog, at least one hydrogen atom of the side chain of phenylalanine is preferably substituted by a substituent, more preferably the hydrogen atom of the benzene ring of the phenylalanine side chain is substituted, It is particularly preferred that the para position of the benzene ring is substituted. Examples of the substituent include substituents such as a halogen atom such as fluorine, chlorine, bromine and iodine; an ethynyl group; a cyano group; and an azide group.
That is, as the phenylalanine analog, an amino acid represented by the following general formula (1) is preferable.

[In the formula, R represents a halogen atom, an azide group, or an ethynyl group. ]
Among them, R is preferably an azide group or an ethynyl group.

  In producing an 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 the MALDI-TOF-MS method is particularly preferable.

  ADVANTAGE OF THE INVENTION According to this invention, the property and function of the original protein can be improved or modified in the produced protein containing an unnatural amino acid. Furthermore, new properties and functions that are not present 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 provided 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 the conventional modification method can be solved. For example, there are advantages such as less concern about toxicity due to chemical reaction, and the post-processing does not inactivate functional molecules. It is possible to carry out precise and clean chemical modification of proteins without relying on "methods". For example, by incorporating an unnatural amino acid having an azide group or an ethynyl group into a silk protein, via a reaction with an ethynyl group or an azide group, any protein, peptide, drug, dye, sugar chain, cross-linking molecule, chelate It is possible to bind molecules such as molecules to the silk. By such a modification, it is possible to change the physical properties of the silk itself. Therefore, the usefulness of the protein that the silkworm produces in the silk gland is greatly increased. In addition, by introducing atoms (groups) exhibiting characteristic infrared absorption and nuclear magnetic resonance, such as cyano groups and fluorine, in a residue-specific manner, local silk structural information that has been impossible until now can be obtained. Can be

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

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

  The breeding method of the silkworm in the present invention can be carried out by giving a feed containing an unnatural amino acid according to a general breeding method of a silkworm in a person skilled in the art. For example, breeding can be carried out according to the method described in "Ministry of Education (1978) Silkworm Species Production. Pp193, Jikkyo Publishing Company, Tokyo".

  When feeding unnatural amino acids having a functional group that causes a chemical reaction by light to silkworms, it is preferable to breed the silkworms under conditions where the unnatural amino acids are not irradiated with light. A preferred breeding method is a method of breeding silkworms in a continuous dark environment without any light irradiation. Alternatively, the silkworm may be irradiated with light, but a method of breeding the silkworm under conditions where the unnatural amino acid having a functional group that causes a chemical reaction by the light is not irradiated with light, For example, the space around the feed to be fed is physically shielded from the light source, the unnatural amino acid is given to the silkworm, and the silkworm produces a protein containing the unnatural amino acid from the silk gland. Breeding silkworms in an environment where is not irradiated.

  The method for producing a protein using the transgenic silkworm of the present invention is a method for introducing a residue-specific unnatural amino acid. Therefore, according to the present invention, a protein containing any type of unnatural amino acid can be produced from the same strain of transgenic silkworm simply by changing the type of unnatural amino acid administered to the silkworm.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
[Preparation of mutant silkworm PheRS]
To generate mutant silkworm PheRS superior in recognition of _p-N 3 -Phe, structural analysis of human PheRS (Structure of human cytosolic phenylalanyl- tRNA synthetase: evidence for kingdom-specific design of the active sites and tRNA binding patterns. From the information of Finarov I, Moor N, Kessler N, Klipcan L, Safro MG. Structure. 2010 Mar 10; 18 (3): 343-53.), Asn404 → Ala, Pro405 → Gly8 → Ayr40 → Gly, Tyr406 → Gly8 → Tyr406 , Phe432 → Ala, and produced these mutants. It was examined or has an activity as heRS, to have the activity of all as PheRS was found.
These variants silkworm PheRS, or Thr407Gly, or a mutant silkworm PheRS of Thr407Ala, and yeast ^{tRNA Phe,} E. coli _{co-expressing,} were cultured in medium containing _p-N 3 -Phe. As long observed growth inhibition of E. coli, it can be determined that p-N ₃ -Phe was incorporated into protein synthesis. Among the produced mutants, remarkable growth inhibition was observed in Thr407Gly, Thr407Ala, and Phe432Ala. Furthermore, mutants in which Phe432X (X = 20 amino acids) and Thr407X ′ (X ′ = Thr, Gly, Ala) were combined were prepared, and the growth inhibition of each mutant was examined. The results are shown in FIG. In FIG. 1, the darker the color, the stronger the growth inhibition was observed.
As shown in FIG. 1, Phe432Val, Phe432Ala, Thr407Ala / Phe432Val, four Thr407Ala / Phe432Met it was confirmed to have excellent recognition of _p-N 3 -Phe.

[Preparation of recombinant vector]
According to an existing method which is within the common knowledge of those skilled in the art, using a DNA (SEQ ID NO: 7) encoding the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 (Phe432Val), SEQ ID NO: 3 (Phe432Ala), No. 5 (Thr407Ala / Phe432Val) and DNA (BmFRS1 ^* ) encoding the amino acid sequence shown in SEQ ID NO: 6 (Thr407Ala / Phe432Met); (SEQ ID NO: 8: DNA encoding the amino acid sequence shown in SEQ ID NO: 2 (Phe432Val)) SEQ ID NO: 9: DNA encoding the amino acid sequence shown in SEQ ID NO: 3 (Phe432Ala); SEQ ID NO: 10: SEQ ID NO: 5 (Thr407Ala / Phe432Val); SEQ ID NO: 11: SEQ ID NO: 6 (Thr407Ala / Phe432Met) piggyBac vector having a DNA)) encoding the amino acid sequence (5 comprises a base 5'UTR sequence) (Figure 2) were produced. In addition, a promoter derived from the fibroin L chain (FibL) and a 3′UTR sequence were added to the 5 ′ and 3 ′ sides of the BmFRS1 ^* , respectively, to the above vector. 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 larval growth dysfunction, which is assumed to be caused by expression of the mutant gene throughout the larva.

[Construction of transgenic silkworm and confirmation of expression of recombinant gene]
Injection of the piggyBac vector into a fertilized egg and subsequent screening of a recombinant were performed according to an existing method which is within the common knowledge of those skilled in the art. Since white / CS (strain name: MCS601) is a strain that produces a dormant egg and cannot be made non-dormant by the above-described low-temperature dark blue method, the dormancy of the egg was broken by the above-described acid treatment. . The copy number of the mutant gene inserted into the genome was analyzed using real-time PCR to establish a transgenic silkworm line homozygous for the mutant gene. The lines of these transgenic silkworms were named H06 (Phe432Val), H07 (Phe432Ala), H08 (Thr407Ala / Phe432Val), and H09 (Thr407Ala / Phe432Met), respectively.

Production of _p-N 3 -Phe containing silk protein]
(Experimental example 1)
The transgenic silkworm H06～H09, after fed the artificial diet of normal composition from fifth instar Okoshiko day 2, _p-N 3 -Phe the third day after, 0.1% (dry diet equivalent) ( An artificial feed containing the amino acid represented by the general formula (1), R = N ₃ ) was fed. Of the third day after the silkworm breeding, in order to avoid the light reaction of p-N ₃ -Phe, it was carried out at the light-shielding conditions. H03 (Thr407Ala) was used as a control. Fibroin production per head of silkworms and p-N ₃ -Phe intake amount (intensity ratio mass spectrometry peaks) are shown in FIGS. 3-4.
In Figure 3-4, the vertical axis (left) shows the fibroin production per head of silkworms represented by a bar graph, the vertical axis (right), p-N ₃ in represented by a circle plots fibroin - The ratio of the peak intensity of Phe is shown.

Peak intensity of _p-N 3 -Phe in fibroin was calculated by the following method.
The cocoon layer of the transgenic silkworm was dissolved in an 8M LiBr aqueous solution by heating at 35 ° C. for 40 minutes to a concentration of about 50 mg / mL. This solution is added to an 8 M aqueous urea solution at 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 allowed to stand at room temperature for 30 minutes or more to perform a reduction treatment. The reduced sample solution was separated by SDS-PAGE, FibL band was cut out, decolorized, washed, and dried, and then digested with trypsin at 37 ° C. in a gel overnight. The resulting peptide fragment was extracted, solidified by drying, and then dissolved in a 2: 1 mixture of a 0.1% TFA aqueous solution and acetonitrile. This solution was mixed with a 2: 1 mixture of a 0.1% TFA aqueous solution saturated with HCCA and acetonitrile, dropped on a MALDI target plate, and dried to obtain measurement data.
In transgenic silkworm treated with _p-N 3 -Phe, mass increase (theoretical ^{value: 14 N 1 H 1 H- 1} H = +15.01 Da) with the substitution of _p-NH 2 -Phe from Phe observed Is done. It is considered that the N ₃ group was reduced to an NH ₂ group during the reduction treatment at the time of preparing the SDS-PAGE sample and / or the MALDI-TOF-MS measurement. From the measured data according sought relative peak intensities of _p-N 3 -Phe-containing peptide fragment to Phe-containing peptide fragment.

As shown in FIG. 4, production of fibroin in H09 gave artificial diet containing 0.1% of _p-N 3 -Phe was comparable to production of fibroin in H03. Furthermore, as shown in FIGS. 3 and 4, the incorporation amount of p-N ₃ -Phe in the fibroin produced by the silkworm was higher in H06 to H09 than in H03.

(Experimental example 2)
Confirmed in transgenic silkworm H06, H07, fibroin production per unit amount of the silkworm when changing the content of _p-N 3 -Ph of the artificial diet for feeding, and the uptake of _p-N 3 -Phe did. The results are shown in FIGS. In FIGS. 5 and 6, the horizontal axis indicates the content ratio (in dry diet) of p-N ₃ -Phe in the artificial feed, and the vertical axis (left) indicates fibroin per unit amount of silkworm represented by a bar graph. shows the production, the vertical axis (right) shows the ratio of the peak intensity of the p-N ₃ -Phe in represented by circles plot fibroin.

5, the H06, synthesized in feed by containing 0.05% _{p-N 3 -Phe, p-} N 3 -Phe can about 100mg production the introduced fibroin was taken the proportion of the peak intensity of the _p-N 3 -Phe was about 0.15. Compared with the results of H03 in Non-Patent Document 1, H06, the content ratio of _p-N 3 -Phe necessary, requires only one-tenth of H03, and _the introduction efficiency of _p-N 3 -Phe is, H03 It was three times as large as

Similarly, in FIG. 6, in H07, fibroin production was reduced to about 80 mg by containing 0.05% of p-N ₃ -Phe in the synthetic feed, but the incorporated p-N _3- The ratio of the signal intensity of Phe was about 0.16.

(Experimental example 3)
The transgenic silkworm H06 was crossed with a general variety day 509 × day 510 to obtain a hybrid H06 × (day 509 × day 510). To H06 × (day 509 × day 510), respectively male larvae and female larvae after fed the artificial diet of normal composition from fifth instar Okoshiko day 2, day 3 or later, different _p-N 3 -Phe The artificial feed was fed in the content ratio. For each condition, fibroin production per unit quantity of silkworm, and to confirm the uptake of p-N ₃ -Phe. FIG. 7 shows the results. In FIG. 7, the horizontal axis indicates the content of p-N3-Phe in the artificial feed (in terms of dry diet), and the vertical axis (left) indicates the amount of fibroin produced per unit amount of the silkworm represented by the bar graph. shown, vertical axis (right) shows the ratio of the peak intensity of the p-N ₃ -Phe in represented by circles plot fibroin.
As shown in FIG. 7, fibroin production in H06 × (day 509 × day 510) fed with an artificial diet containing 0.05% of p-N ₃ -Phe was approximately 170 mg for male larvae and approximately 170 mg for female larvae. 160 mg, which was about 1.6 to 1.7 times the amount of fibroin produced in H06. On the other hand, uptake of _p-N 3 -Phe in fibroin about 0.14 male larvae, and about 0.16 in the female larvae, is equivalent to H06, decreased uptake by hybridization was observed .
From the above experimental results, by general varieties crossed transgenic lines shown in the present invention were shown to be to improve the fibroin production without reducing the uptake of p-N ₃ -Phe.

  Each configuration and the combination thereof in each embodiment described above is an example, and addition, omission, substitution, and other changes of the configuration are possible without departing from the gist of the present invention. The present invention is not limited by each embodiment, but is limited only by the scope of the claims.

  The unnatural amino acid-containing protein produced using the transgenic silkworm of the present invention, for example, an unnatural amino acid-containing silk protein, is widely applicable to fields such as textiles, medical fields, and functional materials, and is more suitable for each use. Has the potential to provide modified silk. Further, there is a possibility that a new use of silk can be developed by the present invention.

Claims (12)

A DNA comprising a sequence comprising any one of the following amino acid sequences (a) to (c), and encoding a protein having an activity of binding a desired unnatural amino acid to a phenylalanine-specific tRNA; and Transgenic silkworm, which has a promoter that causes silk gland-specific expression.
(A) an amino acid sequence having a mutation at amino acid number 432 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 amino acid number 432 in (a),
(C) an amino acid sequence having 80% or more identity at a site other than amino acid number 432 in (a).   The transgenic silkworm according to claim 1, wherein the amino acid number 432 in (a) is alanine, valine, leucine, methionine, threonine, cysteine, or tyrosine. A DNA comprising a sequence containing any one of the following amino acid sequences (d) to (h), and encoding a protein having an activity of binding a desired unnatural amino acid to a phenylalanine-specific tRNA; and 3. The transgenic silkworm according to claim 1, further comprising a promoter that specifically expresses silkworm gland.
(D) an amino acid sequence represented by SEQ ID NO: 2,
(E) the amino acid sequence of SEQ ID NO: 3,
(F) the amino acid sequence represented by SEQ ID NO: 4, and
(G) an amino acid sequence in which one or several amino acids have been deleted, inserted, substituted or added at a site other than amino acid number 432 of the amino acid sequence shown in any of SEQ ID NOs: 2 to 4;
(H) an amino acid sequence having 80% or more identity at a site other than amino acid number 432 of the amino acid sequence shown in any of SEQ ID NO: 2 to SEQ ID NO: 4   The transgenic silkworm according to any one of claims 1 to 3, further having a mutation at amino acid number 407 of (a) to (h).   The transgenic silkworm according to claim 4, wherein the mutation at amino acid number 407 is alanine or glycine.   The transgenic silkworm according to any one of claims 1 to 5, wherein the promoter that specifically expresses the DNA in the silk gland is a promoter that specifically expresses the DNA in the posterior silk gland.   The transgenic silkworm according to any one of claims 1 to 6, wherein the promoter for specifically expressing the DNA in the silk gland is a promoter derived from a fibroin L chain.   An F1 hybrid, wherein the transgenic silkworm according to any one of claims 1 to 7 is crossed with a practical line.   A method for producing an unnatural amino acid-containing protein, comprising a step of administering an unnatural amino acid to the transgenic silkworm according to any one of claims 1 to 7 or the F1 hybrid according to claim 8.   The method for producing an unnatural amino acid-containing protein according to claim 9, wherein the unnatural amino acid is a phenylalanine analog. The method for producing an unnatural amino acid-containing protein according to claim 9 or 10, wherein the unnatural amino acid is an amino acid represented by the following general formula (1).

[In the formula, R represents a halogen atom, an azide group, or an ethynyl group. ]   The method according to claim 11, wherein R is an azide group or an ethynyl group.

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