JP5182968B2 - Selenomethionine-containing protein-producing yeast - Google Patents

Description

  The present invention relates to a yeast strain suitable for production of L-selenomethioninated protein used for X-ray crystal structure analysis of protein, and a method for producing L-selenomethionylated protein using the yeast strain.

  Higher-order structure analysis of proteins is mainly performed by X-ray crystal structure analysis and nuclear magnetic resonance spectrum (NMR), and contributes to elucidation of various life phenomena. In cases where a similar molecular structure is already known in X-ray crystal structure analysis, it is possible to model the structure using a molecular substitution method. If it is not clear, heavy atom isomorphous substituted crystals can be obtained by soaking heavy atoms into the crystal. When X-ray analysis is performed with crystals that do not substitute heavy atoms, the phase of diffracted X-rays from known proteins can be determined. This method is called the multiple heavy atom isomorphous substitution method.

  Another method is an anomalous dispersion method. Although X-rays are scattered by atoms, scattered X-rays having the same wavelength as incident X-rays can be used for X-ray analysis, and their phases are shifted by 180 °. This is called normal scattering. On the other hand, the wavelength that is absorbed very well by the atom is called the absorption edge, but when the X-ray near the absorption edge of an atom is irradiated to the atom, the wavelength of the scattered X-ray does not change, but the phase shifts slightly. Produce. Such scattering is called anomalous dispersion or anomalous scattering. A method for determining the phase using this anomalous dispersion is an anomalous dispersion method. When using this anomalous dispersion method, a metal atom originally contained in a protein containing an iron atom such as hemoglobin can be used, but in many cases, it is necessary to incorporate a metal into the protein. As an example, a method of converting a sulfur atom of methionine into a selenium atom can be mentioned (Non-patent Document 1).

  In the case of a host such as Escherichia coli, a protein in which methionine in the expressed protein is substituted with selenomethionine can be produced by adding selenomethionine to the medium. However, selenium atoms are known to be toxic to cells and are very inefficient for expressing selenomethioninated proteins. In order to improve this point, it has been studied to produce selenomethionated proteins using cell-free protein synthesis systems such as E. coli and wheat germ, but in these systems it is possible to prepare a large amount of proteins. There is a problem that it is difficult and costly.

  On the other hand, yeast is used as a large-scale production host for proteins, and production of selenomethionized protein using yeast is also reported in Saccharomyces cerevisiae and Pichia pastoris (Non-patent Documents 2 to 5). Since these are not selenomethionine resistant strains, all of them are grown to a certain extent, and then replaced with a medium containing selenomethionine to express the selenomethionine protein. However, this method requires complicated medium replacement and is complicated. In particular, in the case of a production method using a jar fermenter, it is impossible to completely change the medium with one jar fermenter, and it can be used only for production in a flask. If 5 L of culture is required, it is obvious that in the case of a flask, it is necessary to carry out 20 250 ml flask cultures, which is complicated. Moreover, there is a problem that it is difficult to culture for a long time in a medium containing selenomethionine, and it is difficult to produce a large amount of protein.

From these facts, it is expected that these problems can be solved by imparting selenomethionine resistance to yeast. There are already reported examples of selenomethionine-resistant yeast [Patent Document 1]. In this example, selenomethionine resistance is imparted to improve sulfur and hydrogen sulfide metabolism. However, in this reported example, there is no reason that selenomethionine taken up by yeast is used as an amino acid, and it is highly possible that metabolism is suppressed in the cell.
In protein development and inhibitor development in the post-genome era, it is expected that protein higher-order structure information analysis will be essential in the future. From this point of view, it is considered industrially necessary to produce a selenomethionylated protein.

: JP-A-8-214869

: Noriaki Hirayama, Introduction to Crystal Analysis for Life Science, Maruzen Co., Ltd. : Bushnell et al., Structure, 9, R11-R14 (2001) : Larsson et al., Acta Cryst. D58, 346-348 (2002) : Xu et al., Acta Cryst. D58, 542-545 (2002) : Laurila et al., J. Struct. Biol., 149, 111-115 (2005)

  An object of the present invention is to provide means for mass-producing L-selenomethionylated (selenomethionylated) protein in yeast in view of the above-mentioned present situation.

  As a result of diligent research to solve the above-mentioned problems, the present inventors confer resistance to a yeast that is originally sensitive to selenomethionine by natural mutation, and newly produce a yeast that produces L-selenomethionylated protein, In addition to demonstrating that the yeast can efficiently produce L-selenomethionated protein, the transformant obtained by introducing the methionine aminoacyl tRNA synthetase gene into yeast has good L-selenomethionylated protein productivity. As a result, the present invention has been completed.

That is, the present invention is as follows.
(1) A yeast strain belonging to the genus Pichia, which is resistant to L-selenomethionine and has an ability to produce L-selenomethionine protein.
(2) The yeast strain according to (1) above, which is further resistant to selenate.
(3) The yeast strain according to (1) or (2) above, wherein the yeast strain belongs to Pichia pastoris .
(4) A transformed yeast strain belonging to the genus Pichia, wherein a gene encoding a methionine aminoacyl tRNA synthetase having the ability to bind L-selenomethionine to tRNA is introduced.
(5) The transformed yeast strain according to (4) above, wherein the yeast strain is a strain belonging to Pichia pastoris .
(6) The transformed yeast according to (4) above, wherein the yeast strain is the strain described in any of (1) to (3) above.
(7) The yeast strain according to (6) above, wherein the methionine aminoacyl tRNA synthetase having the ability to bind L-selenomethionine to tRNA is derived from Escherichia coli.
(8) The yeast strain according to (1) to (7) above, wherein a gene of a target protein for X-ray crystal structure analysis is introduced.
(9) The yeast strain according to any one of (1) to (6) above is cultured in an L-selenomethionine-containing medium, and L-selenomethionine protein is collected from the resulting culture. And a method for producing a selenomethionylated protein.

  According to the present invention, L-selenomethionylated protein can be mass-produced efficiently and inexpensively using yeast. The technology of the present invention enables protein production in X-ray crystal structure analysis of proteins, and can greatly contribute to the development of various pharmaceuticals.

It is a figure which shows the result of having culture | cultivated each selenomethionine resistant strain in the YPD culture medium, the L-selenomethionine containing medium, and the selenate containing medium. It is a figure which shows the result of having analyzed the selenomethionine-ized protein which selenomethionine resistant strain SMR-1, SMR-37, and SMR-94 produce using a mass spectrometer. The arrow is a peptide containing selenomethionine. It is a figure which shows the measurement result of XAFS of the selenomethioninated protein. It is a figure which shows the result of having measured the selenomethionine content rate of the protein to produce with and without the case where a MetG gene is introduce | transduced into the selenomethionine resistant strain SMR-1. Strain that left is not introduced metG gene, right metG gene introduced strain. 1 and 3 are peptides containing methionine, and 2 and 4 are peptides containing selenomethionine.

The present invention relates to a yeast strain belonging to the genus Pichia which is resistant to L-selenomethionine and has the ability to produce L-selenomethionylated protein, and also has the ability to bind L-selenomethionine to tRNA. gene encoding synthase is related to the introduced Pichia (Pichia) transformed yeast strain belonging to the genus.
The former yeast strain was obtained by culturing Pichia yeast in an L-selenomethionine-containing medium, selecting a strain that produces colonies, and further testing the ability to produce L-selenomethionine protein. . The L-selenomethionine resistance in the properties of the yeast strain refers to the property of being able to grow on a medium containing 100 μg / ml of L-selenomethionine. Strains that only survive in media containing L-selenomethionine are excluded.
Specific yeast strains include Pichia pastoris SMR-1, SMR-37 and SMR-94. These strains are incorporated by the National Institute of Advanced Industrial Science and Technology patent biological deposit center. Are sequentially deposited under the deposit numbers FERM P-20807, FERM BP-10787, and FERM BP-10788, respectively. Among these strains, SMR-94 is excellent in selenomethionine uptake and is particularly excellent in protein production for phase determination during X-ray crystal structure analysis.

Examples of the methionine aminoacyl tRNA synthetase gene having the ability to bind L-selenomethionine, which is a gene to be introduced into the latter transformed yeast, with tRNA include, for example, a gene encoding methionine aminoacyl tRNA synthetase of Escherichia coli. . The sequence of the gene is known (Genbank, etc .; see SEQ ID NO: 1), and the gene can be obtained by PCR using primers prepared on the basis of the sequence of the gene using, for example, a commercially available chromosomal DNA of E. coli. It can be obtained by amplification. However, as long as it has the ability to bind L-selenomethionine to tRNA, it is not particularly limited to those derived from Escherichia coli.
In order to introduce the methionine aminoacyl tRNA synthetase gene into the yeast of the genus Pichia , normal gene recombination means may be used. For example, the gene encoding the methionine aminoacyl tRNA synthetase is used as an expression vector such as a plasmid. Insert and transform the Pichia strain with the vector. Examples of the host to be used include Pichia pastoris . Moreover, although these Pichia spp. Can produce selenomethioninated proteins even if they are not L-selenomethionine resistant strains, it is more preferable that they are L-selenomethionine resistant strains. The above SMR-1, SMR-37 and SMR-94 strains are all suitable as hosts.

The strain or transformed strain of the genus Pichia of the present invention described above incorporates L-selenomethionine, and selenomethionine is produced by converting methionine residues among the constituent amino acid residues of the protein produced by the strain to produce selenomethionylated proteins. However, according to the present invention, proteins derived from other organisms such as humans can be selenomethionylated in addition to proteins originally produced by Pichia strains.
For this purpose, a gene DNA encoding a protein to be subjected to X-ray crystal structure analysis is introduced into the Pichia strain or transformant of the present invention. The introduction of the gene DNA of the protein to be analyzed may be performed in accordance with ordinary gene recombination means. The transformant thus obtained expresses the protein to be analyzed, and this expressed protein is selenomethionylated. In order to obtain a selenomethionine protein, the transformed yeast of the present invention obtained as described above is cultured in an L-selenomethionine-containing medium, and the selenomethionine protein is recovered from the culture.
Protein recovery and purification can be performed in the same manner as when expressing a protein that is not selenomethionylated. The selenomethionylated protein thus obtained can be subjected to X-ray crystal structure analysis by anomalous dispersion method.
Examples of the present invention are shown below, but the present invention is not limited to the examples.

[Example 1] Breeding of selenomethionine-resistant yeast
Although P. pastoris SMD1168 is sensitive to selenomethionine, a selenomethionine resistant strain was obtained by the following method.
Specifically, SMD1168 was inoculated into 5 mL of YPD (Difco) liquid medium, cultured at 30 ° C overnight, collected by centrifugation, added with 5 mL of sterilized water, stirred well, and centrifuged again. The cells were washed. This operation was repeated twice. The obtained washed cells were again suspended in 5 mL of sterilized water. This bacterial cell 0.42 OD ₆₀₀ minutes was apply | coated to the SD-Met agar medium containing 3.13 microgram / mL L-selenomethionine, and the colony was obtained by carrying out stationary culture at 30 degreeC for 6 days. Further, the obtained colonies were inoculated into the following SD (-Met) agar medium containing 100 μg / ml L-selenomethionine as a selective medium and cultured again to obtain a grown strain.
(SD (-Met) agar medium)
2% agar, 2% glucose, 0.67% Yeast Nitrogen Base w / o amino acid (Difco), 20 mg / L L-arginine hydrochloride, L-histidine hydrochloride-hydrate, L-methionine, L-tryptophan, uracil, 30 mg / L L-isoleucine, L-lysine hydrochloride, L-tyrosine, 50 mg / L L-phenylalanine, 100 mg / L adenine hemisulfate, L-leucine, 150 mg / L L-valine, 200 mg / L-methionine is excluded from the composition above ml L-threonine. SMD1168, the yeast before mutation treatment, cannot grow on SD (-Met) agar containing 100 μg / ml L-selenomethionine, but the resulting yeast is From the fact that it was able to grow, it was confirmed that the acquired strain was an L-selenomethionine resistant strain.

[Example 2] Screening of yeast producing selenomethionine-containing glycoprotein using selenic acid From the 160 L-selenomethionine resistant strains obtained in Example 1, the following method using sodium selenate was further performed. Secondary screening was performed.
Specifically, the 160 L-selenomethionine resistant strains obtained were applied to a YPD (Difco) agar medium and cultured at 30 ° C. overnight. The grown cells were suspended in sterilized water, adjusted to 1.0 OD ₆₀₀ , and then diluted 5 times in 5 stages. The adjusted bacterial cell solution was added dropwise in an amount of 3 μl each to a YPD agar medium containing 10 mM selenate, and cultured at 30 ° C. for 2 days. As a result, the original SMD1168 strain showed selenate resistance, whereas most L-selenomethionine resistant strains including SMR-1 were selenate sensitive. However, among 160 L-selenomethionine resistant strains, SMR-37 showed selenate supersensitivity and SMR-94 showed selenate resistance (FIG. 1).
Among the above L-selenomethionine resistant strains, Pichia pastoris SMR-1, SMR-37, and SMR-94, which have good properties, are registered with the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary, respectively. Deposited as FERM P-20807, deposit number FERM BP-10787, deposit number FERM BP-10788.

[Example 3] Expression of selenomethionylated human lysozyme Expression of selenomethionylated protein was performed by the following method.
The human lysozyme gene was templated using a chemically synthesized gene designed to use codons suitable for expression in yeast (Muraki et al., Agric. Biol. Chem., 50, 713-723 (1986)). And Jigami et al., Gene, 43, 273-279 (1986)). The resistant strain obtained in Example 2 was constructed by substituting the secretion signal of human lysozyme with the secretion signal of the α factor of S. cerevisiae and expressing it with the glyceraldehyde-3-phosphate dehydrogenase ( TDH3 ) promoter. SMR-1, SMR-37, and SMR-943 were each transformed. Lithium chloride method was used for gene transfer into these resistant strains. That is, each of these resistant strains was cultured in 50 ml of YPD, and the culture solution grown to about 10 ⁸ cells / ml was collected by centrifugation, washed sequentially with sterile water and 100 mM lithium chloride solution, and finally 400 μl of The cells were suspended in 100 mM lithium chloride solution. Lysozyme expression vector treated with restriction enzyme in 240 μl of 50% polyethylene glycol (PEG) # 3350, 36 μl of 1 M lithium chloride, 25 μl of 2 mg / ml salmon sperm DNA, 50 μl of Stu I in 50 μl of this cell suspension The mixture was further stirred well and allowed to stand at 30 ° C. for 30 minutes and at 42 ° C. for 20 minutes. The supernatant was removed from the cell solution by centrifugation, suspended in 1 ml of sterile water, applied to the following SD (-His) agar medium, cultured at 30 ° C for 3 days, and then a transformant was selected.
(SD (-His) agar medium)
2% agar, 2% glucose, 0.67% Yeast Nitrogen Base w / o amino acid (Difco), 20 mg / L L-arginine hydrochloride, L-histidine hydrochloride-hydrate, L-methionine, L-tryptophan, uracil, 30 mg / L L-isoleucine, L-lysine hydrochloride, L-tyrosine, 50 mg / L L-phenylalanine, 100 mg / L adenine hemisulfate, L-leucine, 150 mg / L L-valine, 200 mg / Excluding L-histidine hydrochloride-hydrate from the composition of ml L-threonine or higher. Secretion expression of human lysozyme was performed with these transformants. Specifically, each transformant was inoculated into 5 mL of YPD liquid medium and cultured overnight at 30 ° C. The grown transformant was washed and inoculated to a medium containing 50 mL / ml L-selenomethionine in 200 mL of the following expression liquid medium at an OD ₆₀₀ of 0.01 for 5 days at 30 ° C. Shaking culture was performed.
(Liquid medium for expression)
1.34% Yeast Nitrogen Base w / o amino acid (Difco), 100 mM potassium phosphate buffer (pH 6.0), 2% glucose, 10 mg / L myo-inositol, 90 mg / L adenine hemisulfate, uracil, L-arginine hydrochloride, L-histidine hydrochloride-hydrate, L-tryptophan, L-isoleucine, L-lysine hydrochloride, L-tyrosine, L-leucine, 120 mg / L L-cysteine, 150 mg / L L -Phenylalanine, 200 mg / L L-proline, L-alanine, 300 mg / L L-aspartic acid, L-glutamic acid, L-glutamine, succinic acid, 340 mg / L thiamine, 450 mg / L L-valine, 600 After culturing mg / L L-threonine, 1200 mg / L L-serine, the culture supernatant was collected from the culture solution by centrifugation, concentrated by ultrafiltration, and desalted by dialysis. The crude enzyme solution after dialysis was subjected to anion exchange chromatography to obtain a purified human lysozyme enzyme.

[Example 4] Confirmation of selenium element contained in protein Confirmation that the obtained lysozyme contained selenomethionine was performed using a mass spectrometer. That is, peptide fragments were obtained by digesting the purified enzyme with trypsin, and mass spectrometry was performed by the peptide mass fingerprinting method. In the peptide fragment containing methionine, a peak was observed at a position where the mass difference between sulfur element and selenium element increased by 48 from the expected mass. This result was observed in all three strains (SMR-1, SMR-37, SMR-94) into which the lysozyme expression vector was introduced, and the results are shown in FIG.
Furthermore, amino acid composition analysis was performed on lysozyme obtained from the above three strains. 5 μg or 10 μg of lysozyme was acid-hydrolyzed and then dissolved in 200 μl of 20 mM hydrochloric acid, 50 μl of which was quantitatively analyzed with an amino acid analyzer (Hitachi L8500 type amino acid analyzer). When methionine is replaced with selenomethionine, the content of methionine is reduced by that amount, and the reduced amount of methionine can be converted into the content of selenomethionine. When the ratio of methionine was calculated from the amino acid composition other than cysteine, tryptophan, and proline, it was 1.9 mol / mol protein for lysozyme expressed in wild-type yeast, whereas 1.4 mol / mol protein, SMR-37 for SMR-1. Was 0.5 mol / mol protein and SMR-94 was 0.7 mol / mol protein (Table 1).

This reveals that all three strains have the ability to transfer L-selenomethionine to protein, with 65% methionine in SMR-94 and 75% methionine in SMR-37. It was suggested that selenomethionine was substituted.
The mass difference was confirmed to be selenium by qualitative analysis by X-ray absorption fine structure (XAFS). In other words, the purified enzyme was concentrated from the SMR-94 strain and irradiated with X-rays using the synchrotron beamline (BL-5A) of the High Energy Accelerator Research Organization, and a K-shell absorption edge peculiar to selenium atoms was observed (Fig. 3 ). From the above, it was clarified that the obtained yeast is resistant to L-selenomethionine and has the ability to transfer L-selenomethionine to protein.

[Example 5] Cloning of Escherichia coli methionine aminoacyl tRNA synthetase gene Expression of a selenomethionine-containing protein in Escherichia coli is a general method, and the selenomethionine content of the expressed protein is almost 100%. As shown in Table 1, it is presumed that the selenomethionylated protein expressed in yeast still contains a lot of methionine. Therefore, the methionine aminoacyl tRNA synthetase gene of E. coli was cloned for the purpose of improving the selenomethionine content.
Based on the information described in the E. coli genome database (http://genolist.pasteur.fr/Colibri/), a gene ( metG ) encoding methionyl tRNA synthetase was amplified by PCR and isolated. The base sequence of isolated metG is shown in SEQ ID NO: 1. Specifically, chromosomal DNA of Escherichia coli BL21 strain (Novagen) was prepared and used as a PCR template. As primers for amplifying metG , 5′-gcgcTTCGAAatgactcaagtcgcgaag-3 ′ (SEQ ID NO: 2) and 5′-gcgcTCTAGActaGCTAGCtttcacctgatgacccg-3 ′ (SEQ ID NO: 3) were used.
The amplified DNA fragment of about 2k bp was digested with Bst BI and Xba I was treated as vectors: obtain a pGZ-metG-C by introducing in (PGAPzarufaei Invitrogen). A vector pGZ-metG-CHA expressing a protein in which the HA tag was fused to the C-terminal side of the MetG protein was obtained by introducing a DNA fragment encoding the HA tag into the obtained vector.

[Example 6] Breeding of yeast introduced with Escherichia coli methionyl tRNA synthetase gene The gene was introduced into yeast using the lithium chloride method. That is, in the same manner as in Example 1, after the Pichia pastoris (Pichia pastoris) SMR-1 strain was suspended in lithium chloride solution, is brought into contact with pGZ-metG-CHA was treated with restriction enzymes Avr II to the cells. After contact, the supernatant was removed from the bacterial cell solution by centrifugation, 1 ml of YPD medium was added, and shaking culture was performed at 30 ° C. for 4 hours. The culture solution was applied to a selective medium (YPD medium containing 100 μg / ml Zeocin, 2% agar), and the transformant was selected after culturing at 30 ° C. for 3 days.
The expression of MetG protein in the obtained transformant was confirmed. That is, the transformant was inoculated into 5 ml YPD medium and cultured with shaking at 30 ° C. for 24 hours. And the cells were disrupted by subsequent recovered glass beads ¹⁰⁹ cells fraction by centrifugation. Using the disrupted solution as a sample, SDS-polyacrylamide electrophoresis and transfer to a PVDF membrane were performed, and immunostaining was performed using an anti-HA antibody. As a result, a signal peculiar to the anti-HA antibody was obtained, so that the expression of MetG protein in the cells was confirmed.
About the transformant which can confirm MetG protein expression, the lysozyme expression vector was introduce | transduced similarly to Example 3, and the expression and purification of the selenomethionine containing protein were performed.
As shown in Example 4, the purified sample was confirmed to contain selenomethionine with a mass spectrometer. As a control experiment, lysozyme obtained from a host not expressing the MetG protein was analyzed in the same manner. As a result, as shown in FIG. 4, the peak intensity indicating the peptide containing selenomethionine increased by about 10% with respect to the peptide containing methionine, so that selenomethionine was transferred to protein by E. coli tRNA synthetase. It was confirmed that

[Example 7] Breeding of yeast into which E. coli methionyl tRNA synthetase gene and E. coli methionine tRNA were introduced In order to further improve the efficiency, E. coli-derived methionine tRNA serving as a substrate for E. coli methionine aminoacyl tRNA synthetase was cloned.
Based on the information described in the E. coli genome database (http://genolist.pasteur.fr/Colibri/), a gene ( metG ) encoding methionyl tRNA synthetase was amplified by PCR and isolated. Specifically, chromosomal DNA of DH5α strain (TOYOBO) was prepared and used as a PCR template. As primers for amplifying metG , 5′-gcgcCTCGAGatgactcaagtcgcgaag-3 ′ (SEQ ID NO: 4) and 5′-gcgcTCTAGAttatttcacctgatgaccc-3 ′ (SEQ ID NO: 5) were used. The amplified DNA fragment of about 2 kbp was digested with XbaI and XhoI, and introduced into pBluescriptIISK (+) (Stratagene) similarly treated to obtain pBS-metG. Using this pBS-metG as a template, the metG gene was amplified by performing a PCR reaction using the primer (SEQ ID NO: 2) and SEQ ID NO: 5 used in Example 5. This was digested with BstBI and XbaI and introduced into pGAPZαA (manufactured by Invitrogen) similarly treated to obtain pGZ-metG. Furthermore, a DNA fragment containing tRNA was inserted into this plasmid. Specifically, pUC-EctRNA was treated with HidIII and EcoRI and introduced into pBluscriptIISK (+) treated in the same manner to prepare pBS-EctRNA. This was digested with BamHI , a DNA fragment containing tRNA ^met was purified, and inserted into the BglII recognition site of pGZ-metG to create pGZ-EctRNA-metG. The obtained plasmid was introduced into Pichia pastoris SMR-37 and SMR-94 strains in the same manner as in Example 6 to obtain transformants SMR37tR and SMR-94tR.

According to the present invention, selenomethioninated protein can be mass-produced efficiently and inexpensively. INDUSTRIAL APPLICABILITY The present invention enables protein production in protein X-ray crystal structure analysis and can be used for various pharmaceutical developments such as the development of inhibitors for tumor marker proteins such as cancer.

Claims (4)

Pichia, which is a strain according to any one of the following (1) to (3), wherein a gene encoding a methionine aminoacyl tRNA synthetase having the ability to bind L-selenomethionine to tRNA has been introduced. A transformed yeast strain belonging to the genus.
(1) A yeast strain belonging to the genus Pichia, which is resistant to L-selenomethionine and has an ability to produce an L-selenomethionine protein.
(2) The yeast strain according to (1) above, which is further resistant to selenate.
(3) The yeast strain according to (1) or (2), wherein the yeast strain belongs to Pichia pastoris. The yeast strain according to claim 1 , wherein the methionine aminoacyl tRNA synthetase having the ability to bind L-selenomethionine to tRNA is derived from E. coli. Furthermore, the yeast strain of Claim 1 or 2 by which the gene of the target protein of X-ray crystal structure analysis is introduce | transduced. The yeast strain according to any one of claims 1 to 3 , wherein the yeast strain is cultured in an L-selenomethionine-containing medium, and L-selenomethioninated protein is collected from the resulting culture. A method for producing a protein.

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