Method for producing hedgehog protein

Abstract

To provide a method for producing hedgehog protein having high activity and being animal-free.SOLUTION: The present invention provides a method for producing hedgehog protein, wherein genes encoding respectively a hedgehog protein and an acyltransferase are co-expressed in an endoplasmic reticulum of a non-animal cell, and also provides an expression vector, an expression vector set employed in the method, and the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a hedgehog protein in a non-animal cell and uses thereof. More specifically, the present invention relates to a method for producing a hedgehog protein, which is characterized by coexpressing genes encoding a hedgehog protein and an acyltransferase in the endoplasmic reticulum of the non-animal cell, and uses thereof. [Background technology]

[0002] The Hedgehog signaling pathway regulates morphogenesis and cell proliferation in living organisms. Hedgehog proteins are ligands of this pathway, and three mammalian homologs are known: Sonic hedgehog, Indian hedgehog, and Desert hedgehog.

[0003] Hedgehog protein (HH) is expressed as a precursor protein, and then undergoes C-terminal autocleavage. The resulting N-terminal fragment is modified at the N- and C-termini with palmitoyl and cholesterol, respectively, and secreted as a mature Hedgehog protein. It is known that this N-terminal palmitoylation is important for Hedgehog activity. Hhat has been identified as an acyltransferase that catalyzes the palmitoylation of sonic hedgehog (SHH), and it has been reported that SHH and Hhat are localized in the endoplasmic reticulum and Golgi apparatus (Non-Patent Document 1).

[0004] When SHH is expressed in bacteria such as Escherichia coli, palmitoylation of the N-terminus does not occur, so hydrophobic amino acids such as Ile and Val are introduced into the N-terminus, but this does not result in activity comparable to that of palmitoyl-modified SHH. When SHH is expressed in insect cells or mammalian cells, palmitoyl-modified, highly active SHH is obtained (Patent Document 1), but the resulting SHH is not animal-free, posing safety concerns.

[0005] The inventors have previously described the expression of Hedgehog proteins in plant cells in a patent application (Patent Document 2). However, this application relates to the failure of signal peptide cleavage and is unrelated to palmitoylation of Hedgehog proteins. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO1999 / 028343 (Patent Publication No. 2001-525336) [Patent Document 2] Patent Publication No. 2023-136613 [Non-patent literature]

[0007] [Non-Patent Document 1] Buglino and Resh, THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 32, pp. 22076-22088, August 8, 2008 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for producing a highly active, animal-free hedgehog protein. [Means for solving the problem]

[0009] The inventors attempted to produce N-terminally acylated Hedgehog protein by co-expressing genes encoding acyltransferase and Hedgehog protein in plant cells. In plant cells, enzymes involved in lipid modification are present in the chloroplasts. However, it has been reported that expressing recombinant proteins in chloroplasts significantly inhibits photosynthesis, causing necrosis in the plant (FEBS Journal 274 (2007) 5749-5758). By co-expressing genes encoding Hedgehog protein and acyltransferase in the endoplasmic reticulum, the inventors succeeded in producing highly active Hedgehog protein in non-animal cells without inducing cell necrosis.

[0010] The present invention is based on the above findings and relates to the following [1] to

[21] . [1] A method for producing a hedgehog protein, characterized by co-expressing genes encoding the hedgehog protein and an acyltransferase in the endoplasmic reticulum of a non-animal cell. [2] The method according to any one of [1] to [5], wherein the hedgehog is selected from the group consisting of sonic hedgehog, Indian hedgehog, and desert hedgehog. [3] The method according to [1] or [2], wherein the acyltransferase is hedgehog acyltransferase. [4] The method according to any one of [1] to [3], wherein the obtained hedgehog protein has an acyl group, preferably a palmitoyl group, at the N-terminus. [5] The method according to any one of [1] to [4], wherein the non-animal cells are plant cells. [6] The method according to [5], wherein the plant cells are tobacco cells. [7] An expression vector comprising a gene encoding a hedgehog protein having an endoplasmic reticulum targeting signal sequence and a gene encoding an acyltransferase having an endoplasmic reticulum targeting signal sequence (for example, the gene encoding the hedgehog protein and the gene encoding the acyltransferase are linked via IRES or 2A), or An expression vector set comprising an expression vector containing a gene encoding a hedgehog protein having an endoplasmic reticulum targeting signal sequence, and an expression vector containing a gene encoding an acyltransferase having an endoplasmic reticulum targeting signal sequence. [8] The expression vector or expression vector set according to [7], wherein the hedgehog is any one selected from Sonic hedgehog, Indian hedgehog, and Desert hedgehog. [9] The expression vector or expression vector set according to [7] or [8], wherein the acyltransferase is hedgehog acyltransferase.

[10] The expression vector or expression vector set according to any one of [7] to [9], wherein the non-animal cells are plant cells.

[11] An expression vector or expression vector set according to any one of [7] to

[10] , wherein the endoplasmic reticulum targeting signal sequence is an endoplasmic reticulum targeting signal sequence derived from a non-animal cell that is the host cell (for example, in the case of a plant cell, a signal peptide derived from rice alpha-amylase, a signal peptide derived from tobacco extensin, a signal peptide derived from tobacco PR1 protein, a signal peptide derived from tobacco phylloplanin, a signal peptide derived from radish defensin-like protein 2, or a signal peptide derived from Arabidopsis thaliana chitinase).

[12] The expression vector or expression vector set according to any one of [7] to

[11] , wherein the gene encoding the hedgehog protein is a polynucleotide encoding the following protein 1) or 2): 1) a protein having the amino acid sequence shown in SEQ ID NO: 1; 2) A protein having an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and that functions as a hedgehog protein.

[13] The expression vector or expression vector set according to any one of [7] to

[11] , wherein the gene encoding a hedgehog protein is any one of the following polynucleotides 1) to 3): 1) a polynucleotide having the base sequence shown in SEQ ID NO: 2; 2) a polynucleotide having a nucleotide sequence having 80% or more, preferably 90% or more, more preferably 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, which polynucleotide encodes a protein that functions as a hedgehog protein; 3) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2 and encodes a protein that functions as a hedgehog protein (stringent conditions are as described in the specification).

[14] The expression vector or expression vector set according to any one of [7] to

[13] , wherein the gene for acyltransferase (including an endoplasmic reticulum targeting signal sequence) is a polynucleotide encoding the following protein 1) or 2): 1) a protein having the amino acid sequence shown in SEQ ID NO: 3; 2) A protein having an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and that functions as an acyltransferase.

[15] The expression vector or expression vector set according to any one of [7] to

[13] , wherein the gene encoding the acyltransferase (including an endoplasmic reticulum targeting signal sequence) is any one of the following polynucleotides 1) to 3): 1) a polynucleotide having the base sequence shown in SEQ ID NO: 4; 2) A polynucleotide having a nucleotide sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 4, and that encodes a protein that functions as an acyltransferase. 3) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 4 and encodes a protein that functions as an acyltransferase (the stringent conditions are as described above).

[16] The method according to any one of [1] to [6], wherein the hedgehog protein is the following protein 1) or 2): 1) a protein having the amino acid sequence shown in SEQ ID NO: 1; 2) A protein having an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and that functions as a hedgehog protein.

[17] The method according to any one of [1] to [6] and

[16] , wherein the acyltransferase (containing an endoplasmic reticulum targeting signal sequence) is a protein of 1) or 2) below: 1) a protein having the amino acid sequence shown in SEQ ID NO: 3; 2) A protein having an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and that functions as an acyltransferase.

[18] The method for producing a product according to any one of [1] to [6], which is carried out using an expression vector or an expression vector set according to any one of [7] to

[15] .

[19] A non-animal cell, preferably a plant cell, comprising the expression vector or expression vector set according to any one of [7] to

[15] .

[20] An animal-free composition containing a hedgehog protein obtained by the production method according to any one of [1] to [6] and

[16] to

[19] .

[21] An animal-free medium containing a hedgehog protein obtained by the production method according to any one of [1] to [6] and

[16] to

[19] . [Effects of the Invention]

[0011] According to the present invention, highly active hedgehog proteins can be produced simply and animal-free. [Brief explanation of the drawings]

[0012] [Figure 1]Figure 1 shows the results of Western blotting to detect recombinant hSHH (left: hSHH expression alone, right: co-expression of hSHH and hHHAT). Similar levels of protein accumulation were confirmed in both hSHH expression alone and co-expression of hSHH and hHHAT. [Figure 2] Figure 2 shows the ALP activity of recombinant hSHH (left: hSHH expression alone, right: co-expression of hSHH and hHHAT). DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to a method for producing a hedgehog protein, characterized by co-expressing genes encoding the hedgehog protein and an acyltransferase in the endoplasmic reticulum of a non-animal cell. Note that "co-expression in the endoplasmic reticulum" includes expression within or on the endoplasmic reticulum membrane in addition to expression in the lumen of the endoplasmic reticulum, as described below.

[0014] 1. Hedgehog protein A "hedgehog protein" is a ligand of the hedgehog signaling pathway, which controls morphogenesis and cell proliferation in living organisms. The origin of the hedgehog protein according to the present invention is not particularly limited, but mammalian-derived hedgehog proteins (e.g., derived from mice, rats, rabbits, cats, dogs, goats, monkeys, or humans) are preferred, with human-derived hedgehog proteins being more preferred. In the present invention, proteins produced using non-animal cells, even if the gene is derived from an animal, are considered animal-free.

[0015] In mammals, three types of hedgehog proteins are known: Sonic hedgehog, Indian hedgehog, and Desert hedgehog, whose amino acid sequences share high sequence identity with each other. The amino acid sequences and gene sequences of various hedgehog proteins are known and published in public databases such as UniProt and GenBank (see JP 2023-136613 A).

[0016] For example, the UniProt IDs for various hedgehog proteins and their genes include: <h2 style=";text-align:left;direction:ltr">A0A087YBX2、A0A091GJG3、A0A093GGJ2、A0A096NQ75、A0A096NWF2、A0A0D9R 2D0、A0A0D9R7S2、A0A147AUC3、A0A1A8A852、A0A1U7R2A8、A0A286XYV5、A0A2 86XZZ2, A0A2D0R8U5, A0A2I2V005, A0A2I3SFH2, A0A2K5FAB6, A0A2K5KQ34, A0A2K5N1H8, A0A2K5RXL0, A0A2K5S9L3, A0A2K5XF06, A0A2K6DR78, A0A2K6DT M4、A0A2K6EKZ8、A0A2K6KIT0、A0A2K6MG05、A0A2K6RHE6、A0A2K6S9J4、A0A2 R9ATB4、A0A2R9AWY9、A0A2Y9MSV0、A0A2Y9NDZ2、A0A3B1IF35、A0A3B3BD40、A 0A3B3U009, A0A3B4E4T1, A0A3B4FEC5, A0A3B4TU04, A0A3B4YFI5, A0A3B4YYS1, A0A3B5QCS1, A0A3L7H8J6, A0A3P8RHG9, A0A3P8TDM1, A0A3P8UMD8, A0A3P 8ZKH8, A0A3P9D510, A0A3P9M4Y1, A0A3P9NUR4, A0A3Q1CQ78, A0A3Q1FSF6, A0A3Q2DW64, A0A3Q2VNR7, A0A3Q2ZB64, A0A3Q3AKV8, A0A3Q3EWP0, A0A3Q3L86 0、A0A3Q4HLQ4、A0A3S2NT73、A0A452EW38、A0A452FUF0、A0A4U1EZX8、A0A4W 2CR13、A0A4W2G0X3、A0A4W4F687、A0A4W6FHK6、A0A4X2JY06、A0A4X2KP89、A0 A5F5Y3P3, A0A5F7ZJT0, A0A663DB75, A0A6A4SBH0, A0A6D2Y839, A0A6G0HL93, A0A7N9D6X6, A0A8B7EN51, A0A8C0MU20, A0A8C5XJH9, A0A8C8WK42, A0A8C8 XU50, A0A8D0GC64, A0A8D0HRJ9, A0A8I3NRD7, A0A8I3PZB1, A3KNS3, B3LV44, B3P7F8, B4G2I8, B4GKZ8, B4HFB7, B4JTF5, B4K4M0, B4LZT9, B4NJP3, B4NZY8B4PN49, B4Q599, B4R1D8, E6ZJ03, F1LP42, F1MFP2, F1MM97, F1SHU9, F1SJ10, F1SRW6, F6R922, F6RAT8, F6TJJ6, F6UP64, F6XEQ0, F7ER08, G1N0B0 , G1PIE3, G1RZL5, G1TCI1, G1U6S4, G3NIG1, G3RDQ9, G3TYU1, G3V6T0, G3V7Y0, G3WN12, G3WSF2, G5AKL0, H0XAI6, H0YR00, H2M3V5, H2PP55, H2QJG 1, H3D5G4, H9GMG2, I3JF43, M3WT12, O43323, P56674, P91682, P97812, Q02936, Q14623, Q15465, Q29AA9, Q29JX6, Q61488, Q62226, Q63673, Q8AX T0, Q8BMT9, Q90385, Q90419, Q91035, Q91610, Q91611, Q91612, Q92000, Q98862, Q98938, Q9HCP6, Q9VM64, Q9VZU2, U3BBI2, A0A0U5KSU2, A0A1J1 HKS3, A0A1S3AK22, A0A1Y1KPW3, A0A1Y9HAZ5, A0A2K5PCJ7, A0A2M4BP66, A0A2M4CWK7, A0A3B1J4Z1, A0A3B4CS27, A0A3B4TGL1, A0A3B4Y5U4, A0A 3P8U565, A0A3P8YBZ6, A0A3Q1B3K7, A0A3Q1MQ63, A0A447G0W1, A0A4W2E TK0, A0A4X1U583, A0A6M2DUE9, A0A7M7G5X7, A0A7M7KL45, A0A8C9IU73, A0A8D2JXD1, A0A8D8T2N3, A0A8I6RXC4, A0A8R2ASM6, A0A8W7NZE1, D9ZGF9, E3UGU5, Q544P6, Q6BCD0, Q80XI9, Q8C765, T1IHY2, W5MLT6, W5MY62, W5N409, O13234, O13235, O13238, O13241, O13247, O13250, P79682, P79709, P79717, P79838, P79839, P79850, P79858, P79864, P79869, P79915.SEQ ID NOs: 1 and 2 show the amino acid sequence of human Sonic hedgehog (hSHH, UniProt #Q15465) and the nucleotide sequence of its gene (cDNA).

[0017] As used herein, hedgehog proteins include the following 1) and 2). 1) a protein having the amino acid sequence shown in SEQ ID NO: 1; 2) A protein having an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and that functions as a hedgehog protein.

[0018] As used herein, the hedgehog protein gene includes polynucleotides encoding the following proteins 1) and 2). 1) a protein having the amino acid sequence shown in SEQ ID NO: 1; 2) A protein having an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and that functions as a hedgehog protein.

[0019] As used herein, the hedgehog protein gene includes the following polynucleotides 1) to 3): 1) a polynucleotide having the base sequence shown in SEQ ID NO: 2; 2) a polynucleotide having a nucleotide sequence having 80% or more, preferably 90% or more, more preferably 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, which polynucleotide encodes a protein that functions as a hedgehog protein; 3) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2 and encodes a protein that functions as a hedgehog protein.

[0020] The "stringent conditions" refer to conditions for washing after hybridization, including a salt concentration of 300 to 2000 mM and a temperature of 40 to 75°C, preferably a salt concentration of 600 to 900 mM and a temperature of 65°C. Examples of such conditions include 2×SSC at 50°C. Those skilled in the art can appropriately set stringent conditions by taking into account not only the salt concentration of the buffer and temperature, but also other conditions such as probe concentration, probe length, and reaction time.

[0021] 2. Acyltransferase An "acyltransferase" is a type of transferase that acts on an acyl group. The "acyltransferase" of the present invention is not particularly limited as long as it is an acyltransferase that can acylate, preferably palmitoylate, the N-terminus of a hedgehog protein. The origin of the acyltransferase is not particularly limited, but mammalian acyltransferases (e.g., derived from mice, rats, rabbits, cats, dogs, goats, monkeys, or humans) are preferred, with human-derived acyltransferases being more preferred.

[0022] For example, the UniProt IDs of various acyltransferases and their genes are as follows: <h2 style=";text-align:left;direction:ltr">A0A1S2Z9U0, Q02936, Q5VTY9, Q62226, Q8BMT9, Q9HCP6, Q9VZU2, A0A1S2ZMC7, A0A1S3A0I7, A0A1S3WHL4, Q9D1G3, A0A1S3AJ88, A0A2I2Y4W5, A0A2I3TTU 4, A0A2J8V6G4, A0A2K5DCZ8, A0A2K5LN46, A0A2K5R3T3, A0A2K6CMQ4, A0A7N9CA19, A0A8I6GIY5, F1QYT4, F7I7Y8, G1QPJ0, M0R7X5, Q6P3I5, A0A087XTN0, A0A0A6YWJ4, A0A0D9RRZ4, A0A131YI18, A0A1A8AMW8, A0A1A8LCY7, A0A2K5K8I6, A0A2K5K8I8, A0A2K5K8J3, A0A2K5K8J6, A0A2K5M440, A0A2K5M475, A0A 2K5QSA2, A0A2K5R3S8, A0A2K5R3T9, A0A2K5R3U7, A0A2K5WBU1, A0A2K5WBX7, A0A2K6AD54, A0A2K6F8A9, A0A2K6JPH2, A0A2K6LHB2, A0A2K6RHV8, A0A2K6V 314, A0A2K6V327, A0A2R9CD97, A0A2R9CLE3, A0A3B3DGL6, A0A3B3DGN7, A0A3B3QFZ7, A0A3B3UXN8, A0A3B3UY68, A0A3B3YWU2, A0A3B3ZG34, A0A3B4C493 A0A3B4F6R3, A0A3B4FB43, A0A3B4H4T4, A0A3B4UX27, A0A3B4V8Y8, A0A3B4YKN6, A0A3B5AVZ5, A0A3B5MDK0, A0A3P8R891, A0A3P8RRA4, A0A3P8ULD8, A0A 3P8WJJ0, A0A3P8WMA2, A0A3P8Z849, A0A3P8Z854, A0A3P8Z9S3, A0A3P9CVS5, A0A3P9DJA0, A0A3P9DQE8, A0A3P9IDW9, A0A3P9JNF3, A0A3P9KWL4, A0A3P9N 2Z0, A0A3Q0RNS8, A0A3Q1AVX3, A0A3Q1C8P8, A0A3Q1GPB4, A0A3Q1GRR0, A0A3Q1I5Q8, A0A3Q1NEJ8, A0A3Q2CK92, A0A3Q2CT98, A0A3Q2LFA5, A0A3Q2QCN5A0A3Q2VZW3、A0A3Q2YLL3、A0A3Q3B0Y0、A0A3Q3G158、A0A3Q3G1A1、A0A3Q3G2P8、A0A3Q3G754、A0A3Q3R8B9、A0A3Q3SY61、A0A3Q3WC76、A0A452H1M7、A0A452ID54、A0A493SS13、A0A493T0Z9、A0A493TNZ2、A0A493TVC3、A0A4W2C4L1、A0A4W2C7Y1、A0A4W2C7Z1、A0A4W2DXA8、A0A4W2E5I1、A0A4W2EGJ8、A0A4W2GN69、A0A4W5QG11、A0A4W5QG19、A0A4W6F872、A0A4W6FBD7、A0A4W6G6Z9、A0A4X1TFN5、A0A4X1THC0、A0A4X1TJ77、A0A4X1TJB7、A0A4X1TJG4、A0A4X1TJI8、A0A4X1TJJ7、A0A5F5PZW2、A0A5F9CCF1、A0A663EMG8、A0A663LVQ7、A0A665TAC8、A0A668RIC1、A0A668RXL0、A0A669P2W3、A0A670I4F3、A0A670I541、A0A670I550、A0A670XN39、A0A670XUY3、A0A671WPD5、A0A672TZ41、A0A672ZVP2、A0A672ZWA3、A0A673T2Z3、A0A673V9A5、A0A673V9C3、A0A673VLB3、A0A674BEQ7、A0A6G1R6T5、A0A6G1RXZ5、A0A8B9CD37、A0A8B9DAV2、A0A8B9DBD2、A0A8B9DD72、A0A8B9F1X0、A0A8B9GHI9、A0A8B9IF15、A0A8B9LHK8、A0A8B9NJT2、A0A8B9P1H4、A0A8B9QBE3、A0A8B9RSB7、A0A8B9RSC3、A0A8B9TVG1、A0A8B9UBH0、A0A8B9UEE0、A0A8B9UWU3、A0A8C0B854、A0A8C0B9H6、A0A8C0EDJ2、A0A8C0NJS9、A0A8C0T147、A0A8C0T1E6、A0A8C0T1J2、A0A8C0T2B7、A0A8C0T4A4、A0A8C0UYC7、A0A8C0Z194、A0A8C1DIM4、A0A8C1DQ20、A0A8C1LX71、A0A8C1M2D9、A0A8C1RCK7、A0A8C1VRY4、A0A8C1VTY7、A0A8C1W950、A0A8C1Z6L8、A0A8C2DZ22、A0A8C2DZH4、A0A8C2IL36、A0A8C2PAA3、A0A8C2PT80、A0A8C2QXR4、A0A8C2RKI9、A0A8C2RKV5、A0A8C2RKW2、A0A8C2RL38、A0A8C2RL48、A0A8C2RLR7、A0A8C2RMB2、A0A8C2RMN3、A0A8C2RMX2、A0A8C2XXT9、A0A8C3AS42、A0A8C3ATH2、A0A8C3BYJ2、A0A8C3GF32、A0A8C3HPT0、A0A8C3KPY6、A0A8C3KQ25、A0A8C3KRQ4、A0A8C3KXU3、A0A8C3KZ69、A0A8C3T157、A0A8C3WEP6、A0A8C3XEI4、A0A8C3XXM0、A0A8C4R1D0、A0A8C4R3B1、A0A8C4R3U1、A0A8C4U4Z4、A0A8C5J1Q3、A0A8C5J3V9、A0A8C5NNE1、A0A8C5TB82、A0A8C6UEP0、A0A8C6UK07、A0A8C7QKC4、A0A8C7YTQ8、A0A8C7YTT5、A0A8C8BFG0、A0A8C8HJ47、A0A8C8SDI1、A0A8C8T5M0、A0A8C9FDV6、A0A8C9MU30、A0A8C9TXT1、A0A8C9V0F0、A0A8D0EMD2、A0A8D0EP67、A0A8D0IBU5、A0A8D0IGU3、A0A8D0IIU7、A0A8D0IIV7、A0A8D0LHM7、A0A8D0MPB7、A0A8D0MRQ8、A0A8D0XWH8、A0A8D0Y1S5、A0A8D1FIQ8、A0A8D1FMN1、A0A8D1TN57、A0A8D1VT40、A0A8D1YCI2、A0A8D2EUI6、A0A8D2EWS0、A0A8D2EY99、A0A8D2JZ13、A0A8D2PRY1、A0A9J7X4G7、A0A9J7XJI0、A0A9J7Y4G9、A0A9J8A1Z0、A0A9J8BPB5、A0A9J8C6R4、A0A9J8DLZ1、C9JEF1、C9JHL0、C9JJ25、C9JKQ1、C9JL39、G3PA28、G9K491、G9K492、G9K493、H3CPS9、H7C1Q3、M4ARK1, S4RUF4, U3IJL2, W5K4Q5, W5NEX7, W5NEX9. As an example, SEQ ID NOs: 3 and 4 show the amino acid sequence and cDNA sequence of human hedgehog acyltransferase (hHHAT, UniProt #Q5VTY9, including an endoplasmic reticulum targeting signal sequence).

[0023] As used herein, acyltransferases include the following 1) and 2). 1) a protein having the amino acid sequence shown in SEQ ID NO: 3; 2) A protein having an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and that functions as an acyltransferase.

[0024] As used herein, acyltransferase genes include polynucleotides encoding the following proteins 1) and 2). 1) a protein having the amino acid sequence shown in SEQ ID NO: 3; 2) A protein having an amino acid sequence that has 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and that functions as an acyltransferase.

[0025] As used herein, the acyltransferase gene includes the following polynucleotides 1) to 3): 1) a polynucleotide having the base sequence shown in SEQ ID NO: 4; 2) a polynucleotide having a nucleotide sequence having 80% or more, preferably 90% or more, more preferably 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 4, which polynucleotide encodes a protein that functions as an acyltransferase; 3) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having the base sequence shown in SEQ ID NO: 4 and encodes a protein that functions as an acyltransferase (the stringent conditions are as described above).

[0026] 3. Non-animal cells The "non-animal cells" according to the present invention are not particularly limited as long as they are cells other than animals, and examples thereof include plant cells, yeast cells, algae cells, etc. Preferably, the non-animal cells are plant cells.

[0027] Examples of plant cells include cells of Solanaceae plants (tobacco, tomato, potato, etc.), Gramineae plants (rice, wheat, barley, corn, etc.), Brassicaceae plants (Arabidopsis thaliana, Brassica napus, etc.), Asteraceae plants (lettuce, etc.), and bryophytes (Marchantia polymorpha, Physcomitrella patens, etc.), and among these, cells of Nicotiana plants are preferred.

[0028] Examples of Nicotiana plants include, but are not limited to, Nicotiana benthamiana, N. tabacum, and N. excelsior. Examples of cultured cell lines include tobacco BY-2 cells.

[0029] As yeast cells, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, etc. can be used.

[0030] 4. Expression Vector Co-expression of a hedgehog protein and an acyltransferase in the endoplasmic reticulum of a non-animal cell is achieved by incorporating the genes encoding the hedgehog protein and the acyltransferase into an appropriate vector, which is then introduced into a non-animal host cell for expression.

[0031] Many acyltransferases are endoplasmic reticulum membrane enzymes that are not completely inserted into the endoplasmic reticulum lumen but exist in a state that penetrates the endoplasmic reticulum membrane multiple times. Therefore, in this specification, "co-expressed in the endoplasmic reticulum" includes not only expression in the endoplasmic reticulum lumen but also expression within (on) the endoplasmic reticulum membrane.

[0032] Examples of "vectors" that can be used include plasmid vectors and viral vectors. Vectors may be either vectors for transient expression or vectors for constitutive expression. Those skilled in the art will be able to appropriately select the vector to be used depending on the type and purpose of the host into which the vector is to be introduced. Examples of plasmid vectors that can be used include pRI vectors (pRI101, pRI909, pRI910, pRI201, etc.). Examples of viral vectors that can be used include TMV vectors, PVX vectors, CPMV vectors, CMV vectors, PPV vectors, AIMV vectors, Geminivirus vectors, and ZYMV vectors.

[0033] When constructing a vector, the codons of the genes encoding the hedgehog protein and the acyltransferase may be optimized to suit the host cell to be used.

[0034] The genes encoding the hedgehog protein and acyltransferase each have an "endoplasmic reticulum targeting signal sequence" (also referred to as an endoplasmic reticulum targeting signal peptide or endoplasmic reticulum localization signal sequence) that enable the protein to be translocated to the endoplasmic reticulum after translation. Cleavage of the signal peptide at the endoplasmic reticulum membrane is important for the acylation (preferably palmitoylation) of the hedgehog protein by the acyltransferase. The endoplasmic reticulum targeting signal sequence is not particularly limited, as long as this cleavage occurs accurately. In one embodiment, an endogenous endoplasmic reticulum targeting signal sequence is used as is. In another embodiment, the endogenous endoplasmic reticulum targeting signal sequence is replaced with an exogenous endoplasmic reticulum targeting signal sequence. For example, when a plant cell is used as the host, the endoplasmic reticulum targeting signal sequence can be replaced with a signal peptide derived from rice α-amylase, tobacco extensin, tobacco PR1 protein, tobacco phylloplanin, Arabidopsis chitinase, or radish defensin-like protein 2.

[0035] The genes encoding the hedgehog protein and the acyltransferase may be expressed monocistronically or bicistronically using IRES, 2A, or the like.

[0036] In addition to the genes encoding the hedgehog protein or acyltransferase, cis elements such as enhancers, splicing signals, polyA addition signals, ribosome binding sequences (SD sequences), selectable marker genes, reporter genes, etc. can be linked to the expression vectors as desired.

[0037] Examples of selectable marker genes include dihydrofolate reductase genes, ampicillin resistance genes, neomycin resistance genes, etc. Examples of reporter genes include genes such as green fluorescent protein (GFP) or its variants (fluorescent proteins such as EGFP, BFP, and YFP), luciferase, alkaline phosphatase, and LacZ.

[0038] The gene encoding the hedgehog protein and the gene encoding the acyltransferase may be incorporated into the same vector and introduced into host cells for co-expression, or they may be incorporated into separate vectors and introduced into host cells for co-expression. When incorporated into the same vector for co-expression, the hedgehog protein and the acyltransferase may be expressed monocistronically, or bicistronically using virus-derived factors such as IRES and 2A that enable polycistronic translation and processing. From the viewpoint of ease of control of expression levels, it is preferable to incorporate the genes for the hedgehog protein and the acyltransferase into separate vectors for "co-expression."

[0039] Vectors can be introduced into host cells by methods well known in the art, depending on the host cell.

[0040] For example, in the case of plant cells, the Agrobacterium method, particle gun method, PEG method, electroporation method, etc. can be used, while in the case of yeast cells, the electroporation method, spheroplast method, lithium acetate method, etc. can be used.

[0041] 5. Production of Hedgehog Protein Hedgehog proteins can be produced by transforming non-animal cells with the expression vector or expression vector set described in "4." above, and culturing or culturing the resulting transformants.

[0042] The genes (vectors) encoding the hedgehog proteins and acyltransferases may or may not be integrated into the genome of the non-animal host cells, and the introduced genes (vectors) may be maintained, for example, in episomes or plasmids.

[0043] As already described, preferred transformants are plant cells. The plant cells may be a plant body or a part thereof. Examples of "parts of a plant body" include callus, plant tissue, plant tissue culture, seeds, fruits, leaves, stems, trunks, roots, and flowers.

[0044] Vectors can be introduced into plant cells using known methods, such as methods using viral vectors, agroinfiltration, calcium phosphate, microinjection, particle gun, DEAE-dextran, electroporation, and cationic lipid methods, with the methods using plant viral vectors and agroinfiltration being preferred.

[0045] The agroinfiltration method will be briefly explained below. First, a vector containing a gene of interest is introduced into Agrobacterium by electroporation or other methods to transform the Agrobacterium. Examples of Agrobacterium include, but are not limited to, GV3101, LBA4404, EHA101, EHA105, and AGL1 strains.

[0046] Next, the transformed Agrobacterium is infected into plants (plant cells) or the like. Methods for infecting plants or the like with Agrobacterium include, for example, vacuum infiltration, syringe infiltration, leaf disk method, and foliar spray. When using vacuum infiltration, for example, the cultivated plant is inverted and immersed in a beaker containing a solution of Agrobacterium bacteria so that all leaves are completely submerged in the solution. The beaker is then placed in a vacuum desiccator and left to stand for several minutes (e.g., 1 minute) to reduce the pressure. The valve is then suddenly opened to restore the pressure. After the pressure is restored, the plant is returned to an upright position and placed in a climate chamber. After infection, the plant is cultivated in the climate chamber for 1 to 14 days (e.g., 6 days) using, for example, the DFT method. The environmental conditions are the same as those described above, and those skilled in the art can adjust these conditions appropriately depending on the plant's growth conditions.

[0047] The above procedure can be used to produce transformed plants (plant cells), etc. When infecting a plant with Agrobacterium, multiple Agrobacterium strains each containing a vector containing a hedgehog protein gene and a vector containing an acyltransferase gene can be simultaneously infected.

[0048] If the obtained plant (plant cell) or the like is cultivated or cultured, the genes encoding the hedgehog protein and the acyltransferase are co-expressed. Cultivation or culture can be carried out according to known methods.

[0049] For example, seeds are sown in seedling trays containing fertilizer, and the sown plants are grown in an artificial climate chamber under a controlled light cycle for several days. If liquid fertilizer is used, the liquid fertilizer can be soaked into a hydroponic urethane mat and placed in the seedling tray. Next, the raised plants are transplanted to a panel for cultivation (early stage), and the panel after transplantation is set in an artificial climate chamber, where they are cultivated for several days using, for example, the deep flow technique (DFT method). The plants are then removed from the early cultivation panel and transplanted into the late cultivation panel. The transplanted late cultivation panel is placed in an artificial climate chamber and cultivated for several days using the DFT method to obtain the plants.

[0050] The fertilizer may be, but is not limited to, a liquid fertilizer. When using liquid fertilizer, the liquid fertilizer can be soaked into a urethane mat for hydroponic cultivation and placed in the seedling tray.

[0051] The liquid fertilizer can be any combination of commercially available products and is not limited to these. The liquid fertilizer can be dissolved in dechlorinated water. The electrical conductivity and pH of the liquid fertilizer can be adjusted before use, and those skilled in the art can adjust these using known methods.

[0052] The environmental conditions can be set, for example, as follows: temperature of 10 to 40°C (e.g., 28°C), relative humidity of 60 to 80%, CO2 concentration of 300 to 5000 ppm (e.g., 400 ppm, 500 ppm), and the number of days for cultivation is 0 to 35 days (e.g., 9 days) in the early cultivation period and 0 to 35 days (e.g., 7 days) in the late cultivation period; however, the conditions are not limited to these, and a person skilled in the art can adjust these conditions appropriately depending on the growth status of the plant, etc.

[0053] The main hydroponic cultivation methods that can be used are the deep flow technique (DFT) and the nutrient film technique (NFT).

[0054] Hedgehog proteins can also be isolated from plants and the like by conventionally known techniques. For example, leaves from a transformed plant are harvested and AFP is extracted using an extraction buffer. The amount of plant leaves harvested varies depending on the plant species. The leaves can be frozen and stored at -80°C until extraction. Examples of extraction buffers include, but are not limited to, phosphate buffer, Tris buffer, and acetate buffer. The pH is typically adjusted to between 2 and 11, which includes the range in which the above buffers function appropriately.

[0055] Next, the hedgehog protein contained in the extract is purified. The method of the present invention facilitates purification because impurities such as protein degradation products are reduced. Purification can be performed using conventional methods, such as aqueous two-phase partitioning, ammonium sulfate fractionation, affinity chromatography, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and reverse-phase chromatography, either alone or in combination. For example, when a His-tag (His x 6 tag) is attached upstream of the protein, plant leaves can be disrupted with a Polytron homogenizer in a phosphate buffer solution at pH 7 to 9, and the resulting protein extract can be easily purified by treating it with a Ni-NTA affinity column.

[0056] The identity of the purified substance obtained as the target protein can be confirmed by conventional methods, such as SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis, Western blotting, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, etc.

[0057] 6. Hedgehog proteins and their uses The hedgehog protein obtained by the present invention is a highly active hedgehog protein having an acyl group, preferably a palmitoyl group, at the N-terminus. The hedgehog protein of the present invention, which is produced using non-animal cells, is animal-free and can be safely used in human cell culture, medical treatment, etc.

[0058] The animal-free composition containing the hedgehog protein of the present invention is free from the risk of contamination with pathogens that infect humans, and is therefore useful as a reagent or culture medium (a culture medium for human cells, for example, human stem cells) used in medical fields such as regenerative medicine. In addition to the hedgehog protein, the composition may contain known additives such as physiological saline, buffer solutions, and excipients.

[0059] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples. [Example]

[0060] Construction of Sonic hedgehog expression vector (1) Artificial gene synthesis The amino acid sequence (SEQ ID NO: 5) of human Sonic hedgehog protein (hSHH, UniProt #Q15465) was deleted from cysteine 198 to serine 462, and a His6 tag (HHHHHH) was added to the resulting C-terminus. The signal peptide (23 amino acid residues from the N-terminus) was then removed and replaced with the signal peptide (23 amino acid residues from the N-terminus: SEQ ID NO: 6) derived from Nicotiana tabacum phylloplanin (UniProt #Q56S59) (SEQ ID NO: 7). The nucleotide sequence encoding this polypeptide was artificially synthesized using GeneArt Gene Synthesis (Thermo Fisher Scientific). The gene sequence was optimized to match the codon frequency of Nicotiana benthamiana using GeneArt GeneOptimizer software provided by Thermo Fisher Scientific (SEQ ID NO: 8).

[0061] The amino acid sequence (SEQ ID NO: 3) of human hedgehog acyltransferase (hHHAT, UniProt #Q5VTY9), including the signal peptide, was reverse-translated using SnapGene (GSL Biotech LLC) to match the codon frequency of Nicotiana benthamiana. The predicted introns contained in the resulting sequence were searched for using NetGene2 (https: / / services.healthtech.dtu.dk / services / NetGene2-2.42 / ), and silent mutations were introduced to prevent splicing (SEQ ID NO: 9). The polynucleotide sequence was artificially synthesized by Eurofins Genomics.

[0062] (2) Construction of expression vector The hSHH synthetic gene (SEQ ID NO: 8) was inserted into the Sal I site of pBYR2HS (WO2021 / 020421), and the hHHAT synthetic gene (SEQ ID NO: 9) was inserted between the Nde I and Sal I sites of the pRI201-AN vector (Takara Bio Inc.) by in-fusion cloning. [Example]

[0063] Host plant cultivation and agroinfiltration 1. Host Plant Cultivation In this example, Nicotiana benthamiana, a plant of the genus Nicotiana, was used as the host plant. (1) Seeding Liquid fertilizer for sowing (0.78 g / L from Otsuka House S1 (Otsuka Agritechno Co., Ltd.), 0.25 g / L from Otsuka House 2 (Otsuka Agritechno Co., Ltd.), pH 5.0) was soaked into a hydroponic urethane mat (Ematsu Kasei, W587.5 mm × D282 mm × H28 mm: 12 × 2 grids, hole diameter φ9 mm), placed in a seedling tray (W600 mm × D300 mm × H300 mm), and Nicotiana benthamiana seeds were sown. (2) Seedling raising After sowing, the plants were grown in an artificial climate chamber (NC-410HC) (Nihon Medical and Chemical Instruments Manufacturing Co., Ltd.) at a room temperature of 28°C, a relative humidity of 60-80%, and a light cycle of 16 hours day / 8 hours night for 12 days.

[0064] (3) Cultivation (first half) The urethane mats used for raising seedlings were separated into individual squares and transplanted into cultivation panels (600 mm wide x 300 mm deep, 30 holes). After transplantation, the cultivation panels (early stage) were placed in an artificial climate chamber (LH-410SP) (Nihon Medical and Chemical Machinery Manufacturing Co., Ltd.) and cultivated using the deep flow technique (DFT) for 9 days. The environmental conditions and liquid fertilizer conditions were controlled as follows: <Environmental conditions> Temperature: 28℃ Relative humidity: 60-80% Illumination: Average photosynthetic photon flux density (PPFD): 140 μmol / m 2 10 seconds, 24-hour continuous irradiation, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation) <Liquid fertilizer conditions> Liquid fertilizers were prepared by dissolving fertilizer A (Otsuka House S1, 150 g / L; Otsuka House No. 5, Otsuka Agritechno Co., Ltd., 2.5 g / L) and fertilizer B (Otsuka House No. 2, 100 g / L) in dechlorinated water and mixing them in equal amounts. pH was adjusted using pH adjuster Down (Otsuka Agritechno Co., Ltd.) and 4% KOH aqueous solution. The electrical conductivity (EC) and pH of the liquid fertilizer were adjusted to EC: 2.3 mS / cm and pH: 6.0 using the "Easy Fertilizer Management Machine 3" (Sem Corporation).

[0065] (4) Cultivation (late stage) The plants were removed from the early cultivation panel and transplanted into the late cultivation panel (600 mm W × 300 mm D, 6 holes). After transplantation, the late cultivation panel was placed in an artificial climate chamber (LH-410SP) (Nihon Medical and Chemical Instruments Manufacturing Co., Ltd.) and cultivated using the DFT method for 7 days (28 days after sowing). The environmental conditions were controlled as follows: <Environmental conditions> Temperature: 28℃ Relative humidity: 40-60% Illumination: Average photosynthetic photon flux density (PPFD): 140 μmol / m 2 10 seconds, 24-hour continuous irradiation, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation)

[0066] 2. Agroinfiltration (1) Infection by Vacuum Infiltration The vectors constructed in Example 1 were each introduced into Agrobacterium strain AGL1 by electroporation, and Nicotiana benthamiana was infected by agroinfiltration with either the Sonic hedgehog protein expression vector alone or both the Sonic hedgehog protein expression vector and the hedgehog acyltransferase expression vector.

[0067] Specifically, Nicotiana benthamiana plants 28 days after sowing, obtained in "1." above, were inverted and submerged in the Agrobacterium solution in a beaker so that all leaves were completely submerged. The beaker was then placed in a vacuum desiccator (FV-3P) (Tokyo Glass Instruments Co., Ltd.) and left to stand at -0.09 MPa for 1 minute to reduce the pressure. The valve was then quickly opened to restore the pressure. After the pressure had been restored, the plants were returned to an upright position and planted in an artificial climate chamber (LH-410SP) (Nippon Medical and Chemical Instruments Manufacturing Co., Ltd.).

[0068] (2) Cultivation of infected leaves (expression process) After infection, the plants were cultivated using an artificial climate chamber (LH-410SP) (Nihon Ika Kikai Seisakusho). They were cultivated for 3 days using the DFT method. The environmental conditions were controlled as follows: <Environmental conditions> Temperature: 20℃ Relative humidity: 60-80% Illumination: Average photosynthetic photon flux density (PPFD): 140 μmol / m 2 10 seconds, 24-hour continuous irradiation, three-wavelength fluorescent lamp "Lupica Line" (Mitsubishi Electric Corporation) [Example]

[0069] Protein extraction The leaves of Nicotiana benthamiana agroinfiltrated in Example 2 were frozen at -80°C, and then an extraction buffer solution twice the weight of the leaves was added. The leaves were then homogenized using a Polytron homogenizer PT2500E (Kinematica). The extraction buffer used was a solution of 100 mM potassium phosphate, 400 mM arginine hydrochloride, and 1% (w / v) CHAPS, to which sodium pyrosulfite was added at 0.4 mg / mL, and the pH was adjusted to 8.0. The homogenate was centrifuged at 15,000 x g at 4°C for 15 minutes, and the supernatant was collected as the extract. [Example]

[0070] Western blotting An equal volume of 2x Laemmli sample buffer (Bio-Rad) was added to the protein extract obtained in Example 3 and heated at 95°C for 5 minutes. SDS-PAGE was performed using a 4-20% TGX gel (Bio-Rad) and transferred to a PVDF membrane using a Transblot Turbo transfer system (Bio-Rad). Anti-human / mouse sonic hedgehog goat polyclonal antibody (R&D Systems) was used as the primary antibody, followed by anti-goat HRP-conjugated rabbit antibody (R&D Systems) as the secondary antibody. Clarity Western ECL Substrate (Bio-Rad) was used as the luminescence reagent, and signals were detected using an ImageQuant LAS 500 (Cytiva).

[0071] The results of the detection are shown in Figure 1. As shown in Figure 1, coexpression of hSHH and hHHAT resulted in protein accumulation at the same level as that observed with hSHH expression alone. These results demonstrate that coexpression of hedgehog acyltransferase does not inhibit the expression of Sonic hedgehog protein.

[0072] Purification of Sonic Hedgehog (1) Clarification The protein extract was prepared by the method of Example 3 from 40 g of N. benthamiana leaves agroinfiltrated in Example 2. While stirring, the protein extract was adjusted to pH 5.5 by adding 1 M hydrochloric acid dropwise. The protein from N. benthamiana was precipitated by centrifugation at 15,000 × g for 15 minutes. The resulting supernatant was filtered through a 0.22 μm Nalgene bottle-top filter 291-4520 (Thermo Fisher Scientific) pre-coated with Celpure P300 (Sigma-Aldrich Fine Chemicals) to obtain a clarified solution.

[0073] (2) Purification by cation chromatography and nickel affinity chromatography The clarified solution prepared in (1) was loaded onto a 1 mL HiTrap SP HP column (Cytiva) equilibrated with column buffer (20 mM potassium phosphate, 150 mM NaCl, 1% (w / v) CHAPS, 10% (w / v) glycerol, pH 5.5) at a retention time of 1 min to adsorb the protein. The column was then washed with 5 mL of column buffer, and finally, the column was loaded with increasing NaCl concentration up to 1000 mM. The fractions with an absorbance peak at 280 nm were collected to obtain a crude solution containing hSHH.

[0074] The pH of this crude solution was adjusted to 7.5 with 1N NaOH and loaded onto a 1 mL HisTrap HP column (Cytiva) with a 1-minute retention time to adsorb the protein. The column was then washed with 5 mL of column buffer (20 mM potassium phosphate, 250 mM NaCl, 1% (w / v) CHAPS, 10% (w / v) glycerol, pH 7.5). Finally, the column was loaded with a stepwise increase in imidazole concentration up to 500 mM. The fractions with an absorbance peak at 280 nm were collected to obtain a purified protein solution containing hSHH. The buffer solution was then replaced with 20 mM MES, 500 mM NaCl, 0.5% CHAPS, pH 6.65, by ultrafiltration using an Amicon Ultra-15 Ultracel-10K (Merck). The purified protein was subjected to SDS-PAGE and stained with Bio-Safe Coomassie Stain (Bio-Rad). Electrophoretic images were captured using an ImageQuant LAS 500 (Cytiva). The concentration of hSHH protein was calculated by densitometric comparison with a bovine serum albumin standard (ThermoFisher). [Example]

[0075] Activity evaluation The physiological activity of the hSHH purified in Example 4 was evaluated by the following procedure. Mouse embryonic C3H10T1 / 2 cells were seeded at 10,000–25,000 cells / well in 10% serum-containing growth medium and cultured for 1 day at 37°C in a 5% CO2 environment. hSHH solutions were serially diluted in growth medium and treated with a medium exchange method. The cells were then treated twice with similarly prepared hSHH-containing medium every 1 and 2 days. After 2 days of culture, the cells were harvested, and alkaline phosphatase (ALP) activity was measured in each cell lysate using a kit for measuring alkaline phosphatase activity (Fujifilm Wako Co., Ltd.). Protein content of the cell lysates was measured using a Micro BCA Protein Assay Kit (ThermoFisher Scientific) to calculate ALP activity per μg. This confirmed the dose-dependent activity of hSHH depending on the treatment concentration, and the 50% effective concentration (ED) was calculated by fitting a four-parameter curve using GraphPad Prism 8 (GraphPad Software). 50 ) was calculated.

[0076] The evaluation results are shown in Figure 2 and Table 1. The Sonic hedgehog protein obtained by co-expressing hedgehog acyltransferase exhibited approximately 10 times stronger activity than that obtained by sole expression.

[0077] [Table 1]

[0078] These results demonstrate that the sonic hedgehog protein produced by the production method of the present invention has higher activity than that produced by sole expression. [Industrial Applicability]

[0079] According to the present invention, a highly active, animal-free hedgehog protein can be provided. Because hedgehog is involved in the regulation of morphogenesis and cell proliferation in living organisms, the present invention is useful in medical fields including gene therapy and regenerative medicine.

[0080] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0081]

Claims

1. A method for producing a hedgehog protein, characterized by co-expressing genes encoding the hedgehog protein and an acyltransferase in the endoplasmic reticulum of a non-animal cell.

2. 2. The method according to claim 1, wherein the hedgehog is any one selected from the group consisting of Sonic hedgehog, Indian hedgehog, and Desert hedgehog.

3. The method according to claim 1, wherein the acyltransferase is hedgehog acyltransferase.

4. The method of claim 1, wherein the resulting hedgehog protein has an acyl group at the N-terminus.

5. The method according to any one of claims 1 to 4, wherein the non-animal cells are plant cells.

6. The method of claim 5, wherein the plant cells are tobacco cells.

7. an expression vector comprising a gene encoding a hedgehog protein having an endoplasmic reticulum targeting signal sequence and a gene encoding an acyltransferase having an endoplasmic reticulum targeting signal sequence; An expression vector set comprising an expression vector containing a gene encoding a hedgehog protein having an endoplasmic reticulum targeting signal sequence, and an expression vector containing a gene encoding an acyltransferase having an endoplasmic reticulum targeting signal sequence.

8. 8. The expression vector or expression vector set according to claim 7, wherein the hedgehog is any one selected from the group consisting of Sonic hedgehog, Indian hedgehog, and Desert hedgehog.

9. The expression vector or expression vector set according to claim 7 , wherein the acyltransferase is hedgehog acyltransferase.

10. The expression vector or expression vector set according to any one of claims 7 to 9, wherein the non-animal cells are plant cells.

11. The expression vector or expression vector set according to claim 10, wherein the endoplasmic reticulum targeting signal sequence is any one selected from the group consisting of a signal peptide derived from rice alpha-amylase, a signal peptide derived from tobacco extensin, a signal peptide derived from tobacco PR1 protein, a signal peptide derived from tobacco phylloplanin, a signal peptide derived from radish defensin-like protein 2, and a signal peptide derived from Arabidopsis thaliana chitinase.

12. The expression vector or expression vector set according to claim 7, wherein the hedgehog protein gene is a polynucleotide encoding the following protein 1) or 2): 1) a protein having the amino acid sequence shown in SEQ ID NO: 1; 2) A protein having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and that functions as a hedgehog protein.

13. The expression vector or expression vector set according to claim 7, wherein the gene encoding a hedgehog protein is any one of the following polynucleotides 1) to 3): 1) a polynucleotide having the base sequence shown in SEQ ID NO: 2; 2) a polynucleotide having a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, which encodes a protein that functions as a hedgehog protein; 3) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2 and encodes a protein that functions as a hedgehog protein.

14. The expression vector or expression vector set according to claim 7, wherein the acyltransferase gene comprising an endoplasmic reticulum targeting signal sequence is a polynucleotide encoding the following protein 1) or 2): 1) a protein having the amino acid sequence shown in SEQ ID NO: 3; 2) A protein having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and that functions as an acyltransferase.

15. The expression vector or expression vector set according to claim 7, wherein the gene encoding an acyltransferase containing an endoplasmic reticulum targeting signal sequence is any one of the following polynucleotides 1) to 3): 1) a polynucleotide having the base sequence shown in SEQ ID NO: 4; 2) a polynucleotide having a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 4, which encodes a protein that functions as an acyltransferase; 3) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 4 and encodes a protein that functions as an acyltransferase.

16. The method according to claim 1, wherein the hedgehog protein is the following protein 1) or 2): 1) a protein having the amino acid sequence shown in SEQ ID NO: 1; 2) A protein having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and that functions as a hedgehog protein.

17. The method according to claim 1, wherein the acyltransferase containing an endoplasmic reticulum targeting signal sequence is the following protein 1) or 2): 1) a protein having the amino acid sequence shown in SEQ ID NO: 3; 2) A protein having an amino acid sequence that has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and that functions as an acyltransferase.

18. The method of claim 1, which is carried out using the expression vector or expression vector set of claim 7.

19. A non-animal cell comprising the expression vector or expression vector set according to claim 7.

20. An animal-free composition comprising a hedgehog protein obtained by the production method of claim 1.

21. An animal-free medium containing a hedgehog protein obtained by the production method of claim 1.

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