TWI304442B - Expression system for producing collagen - Google Patents

Description

1304442

AL IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a system for producing collagen, and more particularly to a stable transfected insect cell line for simultaneously expressing recombinant collagen and proline 4- Prolyl 4-hydroxylase. [Prior Art]

Collagen belongs to the extracellular matrix protein and contains the Gly-X-Y triamino acid repeat sequence' and this triamino acid repeat allows the folding of the three collagen poly-success bonds (α-bonds) into a triple helix. The guanidine position in the above Gly-XY tribasic acid repeat is usually proline, and the proline can be hydroxylated to stabilize the collagen when the collagen polypeptide chain is post-translationally modified. The triple helix structure. If the hydroxylation of proline is not carried out, the main triple helix of collagen will become unstable at physiologically lower temperatures (Berg, R. A. and Prockop, D. J. (1973) Biochem Biophys Res Commun 52, 115-120; Rosenbloom, J., et al. (1973) Arch Biochem Biophys 158, 478-484). Proline 4-hydroxylase (EC 1.14_11_2) is the key to catalyze the 4-hydroxylation of all collagen's proline residues. The vertebrate-derived proline 4-hydroxylase is α2β2 quaternary. Includes two different subunits (Kivirikko, K. I., et al. (1989) Faseb J3, 1609-1617). The alpha subunits include catalytic and peptide-substrate binding domains, but will not be activated without beta subunit assistance. The beta subunit was found to be equivalent to the protein-disulfide isomerase (Koivu, J., et al. (1987) J Biol Chem 262, 6447-6449; and Pihlajaniemi, T., et al. 1987) Embo J6, 643-649). During collagen biosynthesis, the procollagen alpha chain is transferred to the lumen of the endoplasmic reticulum, where it is subjected to lysine 4-hydroxylase 0648-A20623TWF(N2); P13930006; chiumeow 5 1304442 Hydroxylation reaction. Proline 4-hydroxylase requires Fe2+, 2-oxoglutarate, 02 and ascorbic acid, and activation systems for vertebrate cells to transfer 2-ketoglutarate and ascorbic acid The lumen of the quality net (Kivirikko, Κ·I., et al. (1989) Faseb J3, 1609-1617). Activated recombinant proline 4-hydroxylase has been successfully demonstrated in vitro, for example, by recombinant baculovirus, a pro-acid 4-hydroxylase subunit is co-infected with insect cells such as Spodoptera frugiperda and Trichoplusia ni (y\xoici, K., et al. (1992) Proc Natl Acad Sci USA 89, 7467-7470), or co-transfection of mammalian cell lines such as COS-1 (John, DC) with expression vectors. , &Bulleid, NJ (1996) Biochem J 317 (Pt 3), 659-665) and HEK293 (Wagner, K., et al. (2000) Biochem J352 Pt 3, 907-911), or yeast such as methanol yeast /^Wc^zsXVuorela, A., et al. (1997) Embo J16, 6702-6712) and Saccharomyces cerevisiae (Toman, P. D., et al. (2000) J Biol Chem 275, 23303-23309). Currently, the expression systems for producing recombinant collagen include Escherichia coli (five co., yeast, mammalian cell strain, insect cell strain, and transgenic animal and plant), however, these expression systems have their defects. For example, The E. coli expression system has no post-translational modification, while the yeast expression system lacks proline 4-hydroxylase activity, whereas collagen production in methanol yeast is the highest in these performance systems. The yield is low and limited by the specific cell type, while the activity of the proline acid 4-hydroxylase in the insect cell expression system is low. If it is referred to the transgenic animal, such as silkworm or mouse, or the transgenic plant, such as In tobacco, the collagen product is excessively cross-linked. A baculovirus expression system containing a gene encoding human pro-acid 4-hydroxylase and collagen has been established (U.S. Patent Nos. 5,077,214 and 5,593,859). However, transfected insect cells die within 72 hours, 0648-A20623TWF (N2); P13930006; chiumeow 6 1304442 causes only temporary Recombinant protein production. In addition, it is difficult to recover or purify recombinant collagen due to cytolysis. Moreover, collagen is a secreted or membrane protein, and baculovirus damages endoplasmic reticulum or high-ketite. Production of proteins. Therefore, there is still a need to find a system for producing bioactive collagen in high yield. SUMMARY OF THE INVENTION The present invention relates to a recombinant insect cell, in particular, a stable • performance of human proline 4 - a recombinant insect cell of hydroxylase (Ρ4Η), wherein the recombinant insect cell is a pseudo-squeak and a fruit fly (7) 叩 (6) "melanogaster S2" 另一 another aspect of the invention relates to a method for producing recombinant collagen, in particular The method comprises the steps of providing a recombinant insect cell comprising a transfected gene encoding a proline acid 'hydroxylase, and transfecting a expression vector comprising the recombinant collagen gene into the recombinant insect cell. Yet another aspect relates to a method of producing recombinant collagen, and, in particular, the method comprises A step comprising a transfected recombinant insect gene encoding a recombinant collagen gene and a gene encoding a proline 4-hydroxylase gene. Accordingly, one embodiment of the present invention provides a recombinant insect cell. Transfected gene encoding human proline 4_hydroxylase. The above-described proline 4-hydroxylase gene sequence includes the nucleotide sequence of sequence identification number, 3. 5, or a combination thereof. A DNA molecule comprising a nucleotide sequence of SEQ ID NO: 1. The DNA molecule encodes a collagen triple helix (COL1) region of type XXI collagen and a c-terminal non-collagen protein (NC1) region.蠖 蠖 (7WM noisy / (10) with · ·) cells or fruit flies 0648-A20623TWF (N2); P13930006; chiumeow 7

13044.42 melanogaster) ^ ° Another embodiment of the invention provides a method of producing recombinant collagen. The above method comprises the steps of: providing a recombinant insect cell comprising a transfected gene encoding human proic acid 4-hydroxylase; transfecting a expression vector into said cell, said expression vector comprising a recombinant collagen gene The cells are cultured under conditions suitable for the expression of the recombinant collagen gene described above; and the expressed recombinant collagen is recovered. The above proline 4-hydroxylase gene line includes the nucleotide sequence of the sequence identification number: 3, 5 or a combination thereof. Further, the above cells may be Trichoplusia ni cells or Drosophila melanogaster cells. Yet another embodiment of the present invention provides a method of producing recombinant collagen. The above method comprises the steps of: providing a recombinant insect cell comprising a transfected gene encoding recombinant collagen, and a transfected gene encoding a proline 4_hydroxylase; suitable for the above recombinant collagen The above recombinant insect cells are cultured under the conditions of performance; and the expressed recombinant collagen is recovered. The above proline 4-hydroxylase gene line includes a nucleotide sequence of SEQ ID NO: 3, 5 or a combination thereof. Further, the above-mentioned cells may be Drosophila melanogaster cells or a further embodiment of the present invention. A DNA molecule comprising the nucleotide sequence of Sequence Identification No. 1: The above DNA molecule encodes a collagen triple helix (C0L1) region of the XXI type collagen and a C-terminal non-collagen protein (NC1) region. A recombinant collagen encoding the above DNA molecule is also provided. [Embodiment] The present invention provides a recombinant collagen and a corresponding DNA molecule thereof, comprising a recombinant insect cell transfected with a gene encoding human prolamine hydroxylase, 0648-A20623TWF(N2); P13930006; chiumeow 8 ( Q, <1

1304442 and a method of producing recombinant collagen using the above recombinant insect cells. Accordingly, one embodiment of the present invention provides a recombinant insect cell comprising a transfected gene encoding a proline 4-hydroxylase. The above transfected genes include alpha subunits and/or beta subunits of proline 4-transferase. The alpha subunit of proline 4-hydroxylase includes the nucleotide sequence of SEQ ID NO: 3, and the β subunit of proline 4-hydroxylase includes the nucleotide sequence of SEQ ID NO: 5. The above recombinant insect cell further comprises another transfected gene encoding the collagen triple helix (COL1) region of the XXI type collagen and the C-terminal non-collagen protein (NC1) region, including the nucleotide of the sequence identifier: sequence. In the embodiment of the recombinant insect cell of the present invention, the above cell may be a Trichoplusia ni cell Drosophila melanogaster cell, preferably a Drosophila me/α (10) cell. The above transfected proline 4_hydroxylase and the collagen triple helix (COL1) region encoding the XXI collagen and the C-terminal non-collagen (NC1) region of the Drosophila cell (designated DS2/p4H/mC21) It was deposited in the Food Industry Development Research Institute of the People's Republic of China on September 6, 1994, under the number BCRC 960245. Another embodiment of the invention provides a method of producing recombinant collagen. The method comprises the steps of: providing a recombinant insect cell; transfecting a expression vector into the cell, wherein the expression vector comprises a recombinant collagen gene; and cultivating the cell under conditions suitable for the expression of the recombinant collagen gene; And recovering the expressed recombinant collagen. In an embodiment of the method of producing a recombinant collagen of the present invention, the above recombinant insect cell comprises a transfected gene encoding human proline 4-hydroxylase. In particular, the above transfected gene encoding human proline 4-hydroxylase includes alpha units and/or beta units of niamin 4-hydroxylase. The alpha subunit of the above proline 4_hydroxylase comprises the nucleotide sequence of SEQ ID NO: 3, and the above proline is 0648-A20623TWF(N2); P13930006; chiumeow 9

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13044, 42 The β-unit of 4-hydroxylase includes the nucleotide sequence of SEQ ID NO: 5. In the other embodiment of the method for producing recombinant collagen of the present invention, the recombinant cell may encode collagen of type XXI collagen. The protein triple helix (COL1) region and the C-terminal non-collagen protein (NC1) region include the nucleotide sequence of SEQ ID NO: 1. The above expressed recombinant collagen is secreted. Yet another embodiment of the present invention provides a method of producing recombinant collagen. The above method comprises the steps of: providing a recombinant insect cell comprising a transfected gene encoding recombinant collagen, and a transfected gene encoding a proline 4-hydroxylase; suitable for recombinant collagen expression The above recombinant insect cells are cultured under the conditions; and the expressed recombinant collagen is recovered. In the embodiment of the method for producing recombinant collagen of the present invention, the recombinant collagen gene line encodes a collagen triple helix (COL1) region of the XXI type collagen and a C-terminal non-collagen protein (NC1) region, including sequence recognition. No.: 1 nucleotide sequence. The above expressed recombinant collagen is secreted. In another embodiment of the method of producing a recombinant collagen of the present invention, the transfected gene encoding a proline 4-hydroxylase comprises an alpha unit of a proline 4-hydroxylase and/or a beta Subunit. The α-subunit of the above proline 4-hydroxylase includes the nucleotide sequence of SEQ ID NO: 3, and the β-unit of the pro-acid 4-hydroxylase includes the nucleotide sequence of SEQ ID NO: 5 . In still another embodiment of the method of producing a recombinant collagen of the present invention, the above insect cells may be cells of the genus 或/蠖·) or fruit rope cells, preferably fruit rope cells. A further embodiment of the invention provides a DNA molecule comprising sequence recognition 0648-A20623TWF(N2); P13930006; chiumeow 10

1304442 » i nickname: nucleotide sequence of 1. The above DNA molecule encodes the collagen triple helix (COL1) region of the XXI-type collagen protein and the C-terminal non-collagen protein (NC1) region. Also provided is a recombinant collagen encoded by the above DNA molecule. Using the baculovirus infection system, the lepidopteran insect cells, including the Spodoptera frugiperda, were obtained by Trichoplusia ni, which showed two collagen genes and two times encoding proline 4-hydroxylase. In this unit, fully hydroxylated and thermostable collagen has been obtained (Lamberg, A., et al. (1996) J Biol Chem 271, 11988-11995; Hagg, Ρ·M·, et al. (1997) Am J Pathol 150 , 2075-2086; Myllyharju, J., et al. (1997) J Biol Chem 272, 21824-21830; Nokelainen, M., et al. (1998) Matrix Biol 16, 329-338; Pihlajamaa, T., et al. 1999) J Biol Chem 274, 22464_22468). However, one of the disadvantages of the baculovirus system is that the cells are eventually destroyed by the infected virus, resulting in only transient recombinant protein production. In the present invention, non-cytosolic insect expression systems have been established, including the pseudo-scale (7Wc/^p/) and Drosophh S2 cells to promote the downstream process of recombinant collagen production. Since proline 4-hydroxylase is a key enzyme in collagen biosynthesis, there is a high amount of functional proline 4-hydroxylase in these stable transfected insect cell systems, ensuring collagen chains later The binary combination can be successfully performed. The total activity of P4Hcx and Ρ4Ηβ subunits and proline 4-hydroxylase in the Drosophila S2) induction system is more than 3 to 4 times higher than that of the 蠖 蠖 (he) system, which may be due to the insertion of fruit. The recombinant plasmid vector (c〇py number) of the gene of the fly S2) cell is relatively high. The 2-ketoglutamate (2_〇x〇glutamte) enzyme test confirmed that the stable transfection of the fruit fly S2) system could produce a functional recombination of P4H. Subsequent mC21 (small XX! collagen) ternary combination results further demonstrated hydroxylation of the proline in the collagen chain and stable triple helix structure. 0648_A20623TWF(N2); P13930006; chiumeow

1304442 Words, P4H does have functional activity. These results suggest that ρ4Ηα and Ρ4Ηβ can be combined into an activated α2β2 quaternary body in the Drosophila (the (10) 叩 expression system. The present invention is the first to use the three gene expression system to produce recombination in a fruit can (Dros print-like) cell. In addition, the results of the examples of the present invention are also the first biochemical proof of the triple helix formation of recombinant human type XXI small collagen molecules, and proved to be in Drosophila melanogaster (Nai ^ ^ / / / S 2) cells The disulfide-bonded polymer can be formed only in the presence of a sufficient amount of activated proline 4-hydroxylase. Based on the results of mass spectrometry analysis, it was found that even in the presence of P4H, there is still a considerable amount of unoxygenated in mC21. Lysine residues. Since Drosophila (Dr like noisy/π·/α S2) cells cannot be below 2 X 105 cells s/ml (Echalier, G. (1997) cells in culture, Chapters 2 and 3, Academic Press, New York) Survival at cell density, it is difficult to select a single stable strain containing both mC21, Ρ4Ηα and Ρ4Ηβ three gene expression constructs. Therefore, the establishment of stable transfection of multiple cell populations may include Carriers for antibiotic selection, only cells mC21 building body structure. Therefore, the performance of common P4H stable Drosophila S2) cells as purified mC21 will not include hydroxylation of collagen molecules. The presence of anti-pepsin, a non-stranded disulfide-bonded mC21 triple helix structure, indicates that the ternary combination of type XXI collagen may be bound by the COL1 interval and the COL1/NC1 interval. It starts between the disulfide bridges and is not a necessary condition for the formation of a triple helix structure. This result is consistent with previous studies showing that the combination of recombinant small type XII collagen showed that the fold of the triple helix COL1 interval was earlier than the formation of disulfide bonds when using the baculovirus expression system (Mazzorana, M., et al. (2001) J Biol Chem 276, 27989_27998). The results of the examples of the present invention show that the interchain disulfide bond formation of the small XXI type collagen ternary is dependent on the amount of hydroxylated guanamine of the collagen chain, suggesting that 3648-A20623TWF(N2); P13930006; Chiumeow 12 1304442 The combination of helical structures controls the formation of interchain bisulfur bridges.

The present inventors demonstrated the possibility of producing activated recombinant human proline 4-hydroxylase and a collagen molecule having a stable triple helix structure in the Drosophila S2) cell expression system. In addition, these non-lytic insect cell systems also provide a convenient way to study the mechanism of collagen triple helix structure combination, especially for collagens that are under-represented in cells and tissues, which will facilitate protein-level studies. At the same time, it was found that p4H and mC21 can be stably expressed in the Drosophila S2) cell line, which means that the Drosophila 〇Dro(10)S2) cell expression system can be used to produce a variety of recombinant collagen, which can be applied to scientific and medical purposes. The above and other objects, features, and advantages of the present invention will become more apparent and understood. The production of collagen monoclonal antibody is a C-terminal monoclonal antibody producing al(XXI) collagen, and a synthetic peptide (AnaSpec) corresponding to the NCI interval 936-957 residue (CDPSLCFSVIARRDPFRKGPNY) of human XXI type collagen is synthesized. , Inc). 2 mg of the purified peptide was combined with keyhole limpet hemocyanin (Pierce). The combined peptide solution was emulsified with ImmunEasy (Qiagen) and injected intradermally into two mice to produce monoclonal antibodies by standard procedures (Harlow, E., and Lane, D. (1988) Antibodies: a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Fusion tumor 3E2 was selected for subsequent experiments and total IgG was purified from the culture medium by protein G column according to standard procedures. 0648-A20623TWF(N2);P13930006; chiumeow 13

1304442 Example 2: Construction of recombinant plastids The open reading frame of the alpha and beta subunits of the human proline 4-ischemase gene was amplified by RT-PCR from human aortic smooth muscle cells (Clonetics). In order to express proline 4-hydroxylase in a non-lytic insect cell expression system, InsectSelectTM and Fruit Rope Induction (DES8) system (Invitrogen) were used. In the InsectSelectTM system, the cDNA encoding the human proline 4-hydroxylase α and β subunits was transduced in the Kpnl-Xhol and KpnI-Xbal cleavage sites of the expression vector pIZ/V5-His, respectively, and designated as ρΙΖ/ Ρ4Ηα and ρΙΖ/Ρ4Ηβ. The alpha and beta subunits of the human proline 4-hydroxylase gene are under the control of the OpIE2 promoter from the multicapsid nuclear polyhedrosis virus of the baculovirus. The expression cassette of the human valerine 4-transferase gene α-subunit in the ρΙΖ/Ρ4Ηα construct was amplified by PCR with the primers ATCGATTATCATGTCTGGA (SEQ ID NO: 7) and CTTTGAGTGAGCATCGATC (Sequence 歹 U number 8). Transfer to the Clal cleavage site of ρΙΖ/Ρ4Ηβ. The resulting construct was named PIZ/P4H. In the Drosophila-inducible expression system, cDNA encoding human proline 4-hydroxylase α and β-subunits were transfected into the NcoI_XhoI and PstI_XbaI cleavage sites of the expression vector pMT/V5-HisA, and designated as ρΜΤ/Ρ4Ηα and ρΜΤ/Ρ4Ηβ. The expression of the alpha and beta subunits of the human proline 4-hydroxylase gene is controlled by the metallothionein promoter, which can be induced by the addition of copper ions or ions. The DNA construct system of mC21 is expressed in Drosophila S2) cells as follows. The DNA corresponding to nucleotides 2484-2874 of the open reading region is referred to as 5'- TTAGATCTATTCCTGGGCCACCTGGTCCGATAG-31 (the sequence 0J identifies 0648-A20623TWF(N2); P13930006; chiumeow 14 I3Q4442··9) and 5f- AATCTAGACTAATAGTTTGGTCCTTTTCTG-3, (sequence identification number··10) was amplified by PCR. The PCR product was treated with Bglll and Xbal, and then transferred to the same position as the expression vector PMT/BiP-V5-HisA (Invitrogen). The resulting construct was named pMT/BiP-mC21. Example 3: Establishment of a stable transfected insect cell line expressing P4H The steps of expressing the cell in the non-lytic insect cell expression system and the fruit line S2) are as follows.

(1) 拟 蝮wz·) cells In the InsectSelectTM system, pIZ/P4H was transfected into the 蠖π蠖·) cells (High Five, Invitrogen) with Superfect transfection reagent (Qiagan). After transfection, cells were harvested for 4 weeks with antibiotic zeocine at a final concentration of 400 pg/ml. Ten transgenic plants with good resistance to antibiotics were obtained, and Western blot analysis was performed with monoclonal antibodies against Ρ4Ηα and Ρ4Ηβ (purchased from ICN, Ins.) to analyze each stability. The protein expression of P4Hcx and Ρ4Ηβ in the cell lysate of the transgenic strain. Western blot analysis steps are described below. SDS-PAGE was performed using 10% NuPAGE bis-Tris polypropylene guanamine colloid and morpholineethanesulfonic acid (MES) buffer (Invitrogen) and stained with Simple Blue Safestain reagent (Invitrogen). After SDS-PAGE, the protein was transferred to a nitrocellulose membrane. The protein was blocked with 5% skim milk containing 〇.ι〇/0 Tween-20 in PBS and probed with antibodies. The binding anti-system was assayed by peroxidase-conjugated secondary antibody and stained with SuperSignal assay reagent (Pierce). The X-ray film was quantified using a densitometer. Subsequent experiments were performed to select transgenic plants showing high expression of ρ4Ηα. The degree of expression of Ρ4Ηα and Ρ4Ηβ in the cell lysate of the sputum sputum was compared. As shown in Fig. 1, the protein expression of P4Hcx was only about one fifth of that of Ρ4Ηβ. 0648-A20623TWF(N2); P13930006; chiumeow

1304442 (2) Drosophila S2 cells express P4H in the Drosophila-induced expression system, and use the Effectene transfection reagent (Qiagen) to transfer ρΜΤ/Ρ4Ηα, ρΜΤ/Ρ4Ηβ, and pCoHygro (the ratio of the three is 10:10:1) Dyed Drosophila S2) cells. After transfection, cells were harvested for 4 weeks at a final concentration of 300 pg/ml of hygromycin. The expression of Ρ4Ηα and Ρ4Ηβ is controlled by the metallothionein promoter and induced by the addition of copper ions. After adding 0.5 mM copper sulfate to the cultured cells, the cells were allowed to grow for 120 hours, as shown in Fig. 1B, compared with the uninduced control group (Fig. 1B, columns 1, 3), after induction (Fig. 1B). , 2nd and 4th blocks) The protein expression levels of Ρ4Ηα and Ρ4Ηβ were greatly increased. To detect whether the recombinant Ρ4Η produced by the above non-lytic insect system is functionally active, decarboxylation based on 2-oxo[1_14C]glutaric acid is employed (Kivirikko, Κ·I·, and Myllyla, R. (1982) Methods The method of Enzymol. 82, 245-304) tested the activity of P4H enzymes in the cells of Drosophila and Drosophila. The results are shown in Table I. Table I: Test of recombinant proline 4-hydroxylase activity in Drosophila and Drosophila S2 cells Cell lysate proline 4-hydroxylase activity a l00μgb Dpm π·) cells were not transfected into control group 55 soil 15 transfected with P4H 101 soil 24 Drosophila S2) cells were not transfected into control group 172+22 transfected with P4H, uninduced 271 soil 23 transfected with P4H, induced by Cu2+ 356土37 0648-A20623TWF(N2); P13930006; chiumeow 16 13,04442 This test is based on the chemotactic combination of 2 -milk [1 - C] pentanoic acid and de-transformation using synthetic peptide (GPP) 1 () As a quality. The b cell line was disrupted in buffer containing 0.2% Triton X-100 and approximately 100 pg of dissolved cell extract was used per assay. The fruit line cell line transfected with P4H was collected 120 hours after induction.

As shown in Table I, the cells of the genus 7(7>/cA(^/(10)Μ /π·) contain a small amount of endogenous glutamate 4-transferase activity, as disclosed in the previous report (Lamberg, Α·, Et al. (1996) J Biol Chem 271, 11988-11995). P4H activity in stably transfected cell lines increased less than 2 times that of endogenous enzymes in untransfected controls. Interestingly, 'the same Η The endogenous proline 4-hydroxylase activity in the lysed cell protein 'fruit S2) cells was 1.7 times higher than that of the P4H-infected cell line. In the Drosophila S2 cell line that was not transfected with Cu2+-induced stable transfection of P4H, the P4H activity increased by about 1.6 and 2 times, respectively, compared to the untransfected control group. The above results show that the system exhibiting recombinant P4H in the un-cytosolic form, the fruit rope S2) system is better than the ruler system. Therefore, the fruit rope S2) system was selected for subsequent small XXI type collagen expression. Example 4: Recombinant Expression of mC21 The present inventors originally intended to express full-length αΐ (XXI) collagen in the Drosophila and Drosophila cell systems carrying the above-described stably transfected P4H gene, however, anti-αΐ (XXI) collagen antibody was used. Analysis of recombinant collagen products in both systems by Western blot analysis revealed that the full-length al(XXI) collagen molecules degraded into several distinct small fragments (data not shown). Previous studies on the ternary combination of chicken small type XII collagen revealed that the formation of the triple helix was initiated by the C-collagen triple helix region (Mazzorana, Μ·, et al. (2001) J Biol Chem 276, 27989- 27998) 了解 To understand whether the C-terminal collagen triple helix of the al(XXI) collagen molecule is 0648-A20623TWF(N2); P13930006; chiumeow 17 1304442 The startable ternary body is combined into a functional triple helix structure, which will include the complete c The mC21 with no collagen (NC1) and collagen triple helix (COL1) is expressed in the fruit rope S2) cells.

Figure 2 shows the putative recombination mC21-level sequence. The Drosophila Expression Construct pMT/BiP-mC21 contains the Drosophila BiP signal sequence (shaded area) at the N-terminus of mC21 for secretion, the last 4 amino acid residues in the NC2 region, human ocl (XXI) collagen The complete COL1 and NC1 regions of the protein, as well as a C-terminal histidine tag. The N-terminal line estimated after the signal peptide is removed is indicated by the arrow below. The proline residue at the Y position of the XYG triple sequence is presumed to have been hydroxylated (Kivirikko, Κ·I·, et al. (1992) In Post-Translational Modifications of Proteins (Harding, J. J., and Crabbe, MJ C· , Eds_) CRC Press, Boca Raton, FL·, 1-51) are indicated by black dots. In the FACIT collagen family, the two characteristic GXY瑕疵 and the two cysteine residues associated with the COL1/NC1 junction and the interchain disulfide bridge are indicated in italics and in boldface, respectively. To express mC21, pMT/BiP-mC21 and pCoBlast vector were co-transfected into fruitline S2) cells or stable transgenic plants containing functional P4H. In the fruit rope S2) system, since S2 cells do not survive at a cell density lower than 2 X 1〇5 cells/ml, it is not easy to select a single strain from a plate. Therefore, after three weeks of selection of the antibiotic biastcidine at a final concentration of 30 pg/ml, all of the transgenic plants obtained in the same dish were confluent into a stable cell strain. The cell lysate and the medium were subjected to Western blot analysis using monoclonal antibody 3E2, and the monoclonal antibody 3E2 described above recognized the c-terminal NC1 region of al(XXI) collagen. The result is as shown in Figure 2. The medium obtained from the stably transfected fruit rope cells was electrophoresed 120 hours after Cu2+ induction, and 1%% of SDS/Bis-Tris polypropylene guanamine colloid and MES buffer were used, which was not reduced (1st to 0648- A20623TWF(N2);P13930006; chiumeow 18

In the state of 1304442 and 2櫊) and the reduction (line 3), the monoclonal antibody 3Ε2 of the C-terminal of αΐ (XXI) collagen was identified for immunoblotting analysis. It was found that mC21 was expressed in the medium alone, i.e., in a dissolved form, and the Drosophila BiP signal sequence successfully guided the secretion of the protein. When mC21 is stably expressed in Drosophila, two main electrophoresis bands can be detected corresponding to mC21 monomer and interchain disulfide bond, which are molecular weights of 20 and 40 kD, respectively, as shown in Figure 2B. As shown in the column. However, in Drosophila cell lines stably transfected with P4H, mC21 can be combined into an interchain disulfide homogenous ternary (60 kD), as shown in column 2. Reduction of mC21 produced by a D4 fly-transfected Drosophila cell line showed that both the obtained binary and ternary bodies were reduced (column 3). These results represent that in the Drosophila cell expression system, a sufficient amount of activated recombinant proline 4-hydroxylase is required for the combination of the disulfide triplet mC21 molecules. Example 5: Stable transfection of Drosophila S2 The three human genes are continuously expressed in the cells to test whether the three genes are commonly transfected: Ρ4Η(χ,Ρ4Ηβ, and mC21 Drosophila transgenic plants can continuously produce recombinant proteins, and the inventors performed a 6-day time-lapse experiment using each protein. Corresponding antibodies were analyzed for each protein with Western blots. At the beginning of the experiment, Drosophila S2 cells expressing stably transfected mC21 and/or P4H genes were cultured in shake flasks containing serum-free medium (Hyclone). The density of X 106 cells/ml was seeded in a culture flask and maintained at 1 X 107 cells/ml. 0.5 mM copper sulfate was added to the medium to induce the expression of the recombinant protein, and growth was carried out for different times. Sodium ascorbate was added daily (80 pg /ml) to the medium to supply the transgenic strain containing the mC21 cDNA construct. Western blot analysis results are shown in Fig. 3. Column 1 shows that the control group was not induced. The cell lysate obtained by the day has an increased amount of Ρ4Ηα and Ρ4Ηβ. Stable transfection in Drosophila 0648-A20623TWF(N2); P13930006; chiumeow 19

1304442

The Ρ4Ηβ produced by the strain reached its maximum amount on the second day after induction, while ρ4Ηα took 5 days to reach the maximum amount. The intensity of the three types of mC21 reached its maximum on the 5th day after induction. The interchain disulfide triplet (T) form of mC21 was measured at the first hour after induction and continued to increase until the maximum amount was reached on the 5th day after induction. In particular, the degree of expression of Ρ4Ηα and mC2l ternary bodies increased with time, indicating that a significant amount of catalytic ρ4Ηα was required in Drosophila cells to assist in the formation of interchain disulfide triplets of the first paternal type 1 collagen. About 1 month after induction, the contents of Ρ4Ηα, Ρ4Ηβ, and mC21 in stable transfectants of different passages were again compared. It was found that the intensity of the three genes was about the same in the same passage, indicating that the stable transgenic strain of Drosophila stably produced three recombinant proteins (data not shown). Example 6: Characterization of purified recombinant small collagen XXI Recombination in fruit rope S2 cells was purified from the medium by the following column chromatography step, whether or not it was performed together with P4H. The stably transfected Drosophila S2 cells were expressed as mC21 and P4H with 0.5 mM copper sulfate, and the cells were cultured in conditioned medium at 27 °C for 120 hours. Approximately 300 ml of the filtered medium containing the C-terminal His-楳-small collagen was loaded into a pre-loaded His-Bind with 20 mM sodium acetate buffer, pH 6.0, at a flow rate of 60 ml/h. Fractogel column (1·5 X 8 cm, Novagen). After washing with the same buffer, the small collagen was flushed out in the same buffer containing 0.1 M imidazole. The UV absorbance at a wavelength of 280 nm was monitored, peaks containing fractions of small collagen were collected, pooled together and dialyzed overnight at 4 ° C with 20 mM sodium acetate buffer, pH 6.0. The dialysis product was loaded with a HlghTrap sulfopropyl column (1-ml empty bed volume) previously equilibrated with 20 mM sodium acetate buffer, pH 6.0, at a flow rate of 60 ml/h. The column was first washed with 50 mM 2 NaCl, and the bound small collagen was eluted with the above buffer containing 〇·25 M NaCl. 0648-A20623TWF(N2); P13930006; chiumeow 20 1304442 t t came out. The peak fraction was collected and a chelating agarose HighTrap column (Ι-ml) previously equilibrated with ZnS04 at a flow rate of 60 ml/h in 50 mM sodium acetate buffer, pH 6.0 containing 0.1 M NaCl. Empty bed volume). The column was first washed with 25 mM of imidazole, and the bound small collagen was eluted with the above buffer containing 0·25 imidazole. Finally the product was dialyzed against 50 mM sodium acetate, pH 6.0. Whether or not co-expressed with P4H, mC21 expression in Drosophila S2 cells was approximately ~3 mg/1 after 5 days of copper sulfate induction, comparable to the estimated amount of purified and recovered mC21 obtained (data not shown). The protein concentration was determined using a bovine serum albumin using a standard procedure using the Lowry assay. The purified recombinant mC21 sample was analyzed by electrophoresis using 1% SDS/Bis-Tris polypropylene guanamine and MES buffer. As shown in Fig. 4, the reduction state was column 1 and column 3, and the non-reduction state was 2nd. 4 columns. The gum system was stained with Simple Blue Safestain reagent. The three main dye bands correspond to the interchain disulfide triplet (T), the binary (D), and the disulfide free monomer molecule (M) of mC21 (columns 2 and 4 of Figure 4). In the unreduced state in SDS-PAGE, the paired dyeing bands were observed, as observed by Western blot analysis (Fig. 2B). The individual proteins of the paired dye bands of each of the ternary, binary, and monomeric molecules were cleaved from the SDS-polyacrylamide colloid, using a sequence of trypsin (Promega) according to established procedures. deal with. The recovered peptide was analyzed by a MALDI_TOF (Matrix-Assisted Laser Desorption Ionization Time-of-Flight) analyzer (ABI 4700 Proteomics analyzer). The results confirmed that there was no molecular weight difference between the mC21 peptide fragments between each pair of proteins, indicating that these paired proteins were caused by some residual secondary structure, even if SDS, or the type of non-chain disulfide bond was used (data Not shown) has not been eliminated. These mC21 can be reduced to a pair of monomers in the reduced state (columns 1 and 3 of Figure 4). To directly demonstrate the presence of hydroxylated proline in recombinant mC21, such as stable transfection 0648-A20623TWF (N2); P13930006; chiumeow 21 1304442

The purified sample was subjected to amino acid composition and mass spectrometry after treatment with proline 4-hydroxylase in Drosophila S2 cells. For amino acid analysis, purified recombinant mC21 was dialyzed against 50 mM acetic acid, hydrolyzed with 6 N of HCl at 110 °C for 24 hours, and subjected to amino acid analysis using a Beckman System 6300 Amino Acid Analyzer. The results are shown in Table II. Table II: Analysis of Amino Acids of Purified Recombinant Human Small Collagen XXI in Drosophila S2 Cells Transfected Individually or with Human Proline 4-Hydroxygenase

Amino acid recombinant human small collagen XXI calculation result a untransfected P4H co-transfected P4Hb (from cDNA) residue e Asx 9.4+1.9 10.6+0.2 10 Glx 14.4+4.3 11·6±0·4 9 Hyp 2.4+ 0.1 5.5 soil 0·2 - Ser 8.5 soil 1_8 9_9 soil 0·2 10 Gly 29_8 soil 2.9 27.1+0.5 37 His 10.0+1.2 9.5+0.5 7 Arg 8.5+0.5 8.9+0.2 9 Thr 1.6+0.4 2.0+0.2 0 Ala 3.0+0.5 3.5+0.3 1 Pro 23.2 Soil 1.3 20.3 Earth 2.2 31 Tyr 2.9+0.3 3·0 Soil 0.3 2 Val 5.6 Soil 0.3 5.3+0.1 5 Met 0.5+0.3 0.4+0.1 0 Cys 1.4+0.2 1.6+0.6 2 0648 -A20623TWF(N2);P13930006; chiumeow 22

1304442 lie 5.0+0.6 5.3 Soil 0·1 6 Leu 7_8 Soil 1·9 8·9 Soil 0.2 8 Phe 4.7+0.6 4.1+0.2 3 Lys 7.4+1.2 8.3+0.1 6 Hyp/Pro = 0.10 0.27 % 9 21 Hyp/ (Hyp+P ro )= a Calculate the amino acid residue based on the mC21 amino acid sequence after presumably removing the Drosophila BiP signal sequence, as shown in Figure 2A. b Purified mC21 is a cell 120 hours after induction by copper. The number listed in e is the mean soil standard deviation, η = 2. As shown in Table II, the amino acid composition of mC21 purified from Drosophila S2 cells, whether or not co-transfected with P4H, was consistent with the putative composition based on the cDNA amino acid sequence. The degree of hydroxylation in mC21 (the ratio of hydroxylated proline residues to total proline residues plus hydroxylated valerine residues) was 9 and 21%, respectively, either alone or in combination with P4H. The theoretical enthalpy of complete hydroxylation of the proline residue is 17 (Fig. 2A) and the degree of hydroxylation is 54.8%. The results showed that the proline residues of the mC21 molecule co-expressed in the D2 cells stably transfected with P4H were not completely hydroxylated, but the hydroxylation amount of the XXI collagen in nature was also unknown. To further examine the degree of hydroxylation of the mC21 polypeptide chain proline, the purified mC21 was digested with trypsin and analyzed by MALDI-TOF mass spectrometry. The trypsin digested peptide fragments obtained by mC21 of Drosophila S2 cells alone (Fig. 5A) or with P4H (Fig. 5B) were selected for alignment. Recombinant mC21 with 10% SDS/Bis-Tris polypropyl 0648-A20623TWF(N2); P13930006; chiumeow 23 whether or not it is expressed in Drosophila S2 cells simultaneously with PH4

1304442 ^ >

The decylamine colloid and MES buffer were subjected to electrophoresis analysis under reduced conditions. The protein staining bands were stained with Simple Blue Safestain reagent, trypsin for colloidal enzyme treatment, and analyzed by MALDI-TOF mass spectrometry. The trypsin-treated fragments of mC21 (mC21/P4H, Figure 5B), which were separately expressed as mC21 (mC21AP4H, Figure 5A) and mC21 (mC21/P4H, Figure 5B), which were co-expressed with stably transfected P4H, were selected. It is speculated that the hydroxyl group of the Y position in the XYG three sequence is hydroxylated and is indicated by a black dot. The results showed that the peptide fragments of Drosophila S2 cells alone or in combination with P4H still had substantially unhydroxylated molecular fragments of molecular weights of 1531.86 and 1583.86, respectively. Based on the molecular weight of the oxygen atom added by each peptide, it was found that the trypsin-digested fragment also had a mono- and di-hydroxylated peptide. In S2 cells stably transfected with P4H, the relative intensity of hydroxylation for unhydroxylated peptide molecules was substantially higher than that of untransfected P4H cells (see Figure 5, comparing A and B), and this result was correlated with small collagen molecules. The results of the analysis of the total amino acid composition were consistent. Example 7 · Triple helix structure of small collagen XXI The collagen triple helix structure is resistant to pepsin degradation. In order to confirm whether the COL1 interval of mC21 is a triple helix structure, purified mC21 expressed alone or in combination with P4H is cultured at 0.5 ° C for 15 minutes at 37 ° C for 15 minutes to reach pH 2.5, followed by pepsin. (25 pg/ml) was digested for 2 hours, and its helical structure stability was tested. If mC21 has a triple helix structure, pepsin should only remove the NC1 interval, resulting in a COL1 interval with two cysteines bounded by COL1/NC1. The small collagen, which was digested by pepsin, was analyzed in an unreduced state by electrophoresis of 10% SDS/Bis-Tris polypropylene guanamine colloid and MES buffer, and then the protein was colored in Coomass blue. The results are shown in Fig. 6. Columns 1 and 4 are untreated recombinant small collagen XXI, which are mC21AP4H and mC21/P4H, respectively. Pepsin is indicated by an arrow. (aCOLl)3 0648-A20623TWF(N2);P13930006; chiumeow 24 1304442 «

It is a ternary disulfide bond COL1 interval; (ocCOLl) is an isolated chain derived from the ternary non-disulfide bond COL 1 interval. When mC21 is expressed alone in Drosophila cells (mC21AP4H), after the pepsin digestion treatment, the intact interchain disulfide binary and most of the monomers disappear regardless of the ambient temperature (columns 2 and 3). The proline residue representing most of the mC21 molecule is unhydroxylated, so no triple helix structure is formed. Conversely, after pepsin digestion with mC21 (mC21/P4H), which is co-expressed with P4H, some anti-pepsin fragments from interchain disulfide triplet (cxCOL1)3 and monomer (aCOL1) remain. Column 5). In addition, these fragments are still present in mC21 pre-warmed at 37 °C (column 6) prior to treatment with pepsin, representing some of the mC21 molecules that are expressed in Drosophila cells stably transfected with P4H containing sufficient hydroxylated proline Stable triple helix structure. These pepsin treatment results showed that mC21 and P4H together exhibited in Drosophila cells to form a thermostable triple helix structure. An anti-pepsin monomer fragment (aCOL1) is still present after pepsin treatment (Fig. 6, columns 5 and 6), indicating that these monomers may be derived from a non-interchain disulfide triplet of a triple helix structure, which may It is broken down into single strands during SDS-PAGE. In order to confirm that mC21 co-expressed in the fruit transfected S2 cells stably transfected with P4H contained a non-bonded disulfide triplet, a different mC21 fragment was separated by a colloidal filter column. The purified recombinant mC21 expressed in Drosophila S2 cells stably transfected with P4H can be superdex 200 (HR 10/30) colloidal filter tube previously equilibrated with 0.1 M NaCl, 50 mM sodium acetate (pH 6.00). The column is separated without first denaturation (Fig. 7A a) and does not need to be heat treated with 5 mM DTT for 15 minutes (Fig. 7A, b). The column flow rate was 0.4 ml/min. The mc21 exhibited in conjunction with P4H can be extracted in a natural environment to obtain two peaks (Fig. 7A a). Different peak fractions (numbered 1 to 3 in Figure 7A) were analyzed by SDS-PAGE in an unreduced state. The colloid is colored in silver nitrate. The results show that the earlier peak fraction (the first peak) contains the chain 0648-A20623TWF (N2); P13930006; chiumeow 25 1304442 I di-sulfur bond ternary, binary and non-chain disulfide monomer The mixture, while the later peak fraction (second peak) contains only non-interchain disulfide monomer (Fig. 7B, panels 1 and 2). The monomers in the earlier peak fractions that are simultaneously conjugated to the interchain disulfide triplets represent these monosystems from non-chain disulfide triplets. After heat treatment with DTT, the tri-chain will decompose and be eluted to the later peak (b) in Fig. 7A, b, and only the monomer (Fig. 7B, column 3). From the above, it is known that the mC21 triple helix structure which is expressed simultaneously with P4H in the Drosophila S2 cell system contains a mixture of interchain and non-chain disulfide triplets.

While the invention has been described above by way of a preferred embodiment, it is not intended to limit the invention, and the invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application. 0648-A20623TWF(N2) ;P 13930006; chiumeow 26 13.04442 * , [Simple description of the diagram] Figures 1A and 1B show the pseudo-scales (Fig. 1) and Drosophila S2 with stable transfected P4Hoc and Ρ4Ηβ expression constructs. Western blot analysis of soluble protein extracts from cells (Fig. 1).

Figures 2 and 2 show the combination of human proline 4-hydroxylase-assisted type XXI small collagen in Drosophila S2 cells. Figure 2 shows the putative recombination mC21-level sequence. Figure 2 shows Western blot analysis of mC2l molecules expressed in Drosophila S2 cells. The first column is the medium for the stable expression of mC21 (mC21AP4H) in Drosophila cells; the second and third columns are the cells of Drosophila cells which simultaneously express Ρ4Ηα, Ρ4Ηβ, and mC21 (mC21/P4H). T is an interchain disulfide bond ternary; D is an interchain disulfide double dimer; Μ is a monomer. Figure 3 shows a time course test of protein expression of Drosophila cells stably transfected with Ρ4Ηα, Ρ4Ηβ, and small collagen XXI genes. Specific monoclonal antibodies are listed on the left side of the figure. In column 1, the stably transfected Drosophila S2 cells were not induced by Cu2+. Τ is an interchain disulfide bond ternary; D is an interchain disulfide bond binary; Μ is a monomer. Figure 4 shows a small collagen protein purified from stably transfected Drosophila S2 cells. When the first and third blocks are in the restored state, the second and fourth columns are in the unreduced state. Τ is an interchain disulfide bond ternary; D is an interchain disulfide bond binary; Μ is a monomer. Figures 5 and 5 show the results of MALDI-TOF mass spectrometry analysis of trypsin-treated fragments of small collagen expressed by Drosophila S2 cells. Figure 5 shows that Drosophila S2 cells express mC21 (mC21AP4H) alone, and Figure 5B shows trypsin-treated fragments of mC21 (mC21/P4H) co-expressed with stably transfected P4H. It is speculated that the hydroxyl group at the Y position on the XYG three sequence is hydroxylated and is indicated by a black dot. Figure 6 shows the characteristics of the small collagen XXI triple helix. Columns 1 and 4 are untreated recombinant small collagen XXI, mC21AP4H and mC21/P4H, respectively. Pepsin is indicated by an arrow. (aCOLl) 3 is a ternary disulfide 0648-A20623TWF (N2); P13930006; chiumeow 27 13.04442 bond COL1 interval; (aCOLl) is a separated chain derived from the ternary non-disulfide bond COL 1 interval. Figures 7A and 7B show the separation of small collagen XXI by a colloidal filter column. Figure 7A shows recombinant mC21 co-expressed with P4H in purified Drosophila S2 cells. A is undenatured and b is treated with 5 mM DTT (dithiothreitol) 'heated for 15 minutes. Figure 7B shows the results of SDS-PAGE analysis in the unreduced state for different peak fractions (numbered from Figure 7A. 1 to 3). T is a chain double bismuth bond ternary body; D is an interchain disulfide bond binary body; Μ is a monomer.

[Main component symbol description] No 0

<140&gt 160> 10

<210> 1 <211> 492 <212> DNA <213>Artificial sequence <220><221> CDS <222> 1-492 <223> Recombinant mC21 (XXI) nucleotide sequence < 400> 1 atgaagttat gcatattact ggccgtcgtg gcctttgttg gcctctcgct 50 cgggagatct attcctgggc cacctggtcc gataggccca gagggtccca 100 gaggattacc tggtttgcca ggaagagatg gtgttcctgg attagtgggt 150 gtccctggac gtccaggtgt cagaggatta aaaggcctac caggaagaaa 200 tggggaaaaa gggagccaag ggtttgggta tcctggagaa caaggtcctc 250 ctggtccccc aggtccagag ggccctcctg gaataagcaa agaaggtcct 300 ccaggagacc caggtctccc tggcaaagat ggagaccatg gaaaacctgg 350 1304442

t A s - aatccaaggg caaccaggcc ccccaggcat ctgcgaccca tcactatgtt 400 ttagtgtaat tgccagaaga gatccgttca gaaaaggacc aaactatagt 450 ctagacgaca gcagccatca tcaccatcac catagcagcg gc 492 <210> 2 <211>

<212> PRT <213> Artificial sequence <220><221> PEPTIDE <222> 1-164 <223> Recombinant mC21 (XXI) amino acid sequence <400>

Met Lys Leu Cys lie Leu Leu Ala Val Val 10 Ala Phe Val Gly Leu Ser Leu Gly Arg Ser 20 lie Pro Gly Pro Pro Gly Pro lie Gly Pro 30 Glu Gly Pro Arg Gly Leu Pro Gly Leu Pro 40 Gly Arg Asp Gly Val Pro Gly Leu Val Gly 50 Val Pro Gly Arg Pro Gly Val Arg Gly Leu 60 Lys Gly Leu Pro Gly Arg Asn Gly Glu Lys 70 Gly Ser Gin Gly Gly Gly Tyr Pro Gly Glu 80 Gin Gly Pro Pro Gly Pro Pro Gly Pro Glu 90

Gly Pro Pro Gly lie Ser Lys Glu Gly Pro 100 1304442 164

Pro Gly Asp Pro Gly Leu Pro Gly Lys Asp 110 Gly Asp His Gly Lys Pro Gly lie Gin Gly 120 Gin Pro Gly Pro Pro Gly lie Cys Asp Pro 130 Ser Leu Cys Phe Ser Val lie Ala Arg Arg 140 Asp Pro Phe Arg Lys Gly Pro Asn Tyr Ser 150 Leu Asp Gly Ser Ser His His His His His 160 His Ser Ser Gly

<210> 3 <211> 1602 <212> DNA <213> homo sapien <220><221> gene <222> 1-1602 <223> Human proline 4-hydroxylase α subunit < 400 > 3 atggtctggt atatattaat tataggaatt ctgcttcccc agtctttggc 50 tcatccaggc ttttttactt caattggtca gatgactgat ttgatccata 100 ctgagaaaga tctggtgact tctctgaaag attatattaa ggcagaagag 150 gacaagttag aacaaataaa aaaatgggca gagaagttag atcggctaac tagtacagcg acaaaagatc cagaaggatt tgttgggcat ccagtaaatg 250 cattcaaatt aatgaaacgt ctgaatactg agtggagtga 200 gttggagaat 300 1304442

ctggtcctta aggatatgtc agatggcttt atctctaacc taaccattca 350 gagaccagta ctttctaatg atgaagatca ggttggggca gccaaagctc 400 tgttacgtct ccaggatacc tacaatttgg atacagatac catctcaaag 450 ggtaatcttc caggagtgaa acacaaatct tttctaacgg ctgaggactg 500 ctttgagttg ggcaaagtgg cctatacaga agcagattat taccatacgg 550 aactgtggat ggaacaagcc ctaaggcaac tggatgaagg cgagatttct 600 accatagata aagtctctgt tctagattat ttgagctatg cggtatatca 650 gcagggagac ctggataagg cacttttgct cacaaagaag cttcttgaac 700 tagatcctga acatcagagg gctaatggta acttaaaata ttttgagtat 750 ataatggcta aagaaaaaga tgtcaataag tctgcttcag atgaccaatc 800 tgatcagaaa actacaccaa agaaaaaagg ggttgctgtg gattacctgc 850 cagagagaca gaagtacgaa atgctgtgcc gtggggaggg tatcaaaatg 900 acccctcgga gacagaaaaa actcttttgc cgctgccatg atggaaaccg 950 taatcctaaa tttattctgg ctccagctaa acaggaggat gaatgggaca 1000 agcctcgtat tattcgcttc catgatatta tttctgatgc agaaattgaa 1050 atcgtcaaag acctagcaaa accaaggctg aggcgagcca ccatttcaaa 1100 cccaatagca ggagacttgg agacggtaca ttacagaatt Agcaaaagt g 1150 cctggctctc tggctatgaa aatcctgtgg tgtctcgaat taatatgaga 1200 atacaagatc taacaggact agatgtttcc acagcagagg aattacaggt 1250 agcaaattat ggagttggag gacagtatga accccatttt gactttgcac 1300 ggaaagatga gccagatgct ttcaaagagc tggggacagg aaatagaatt 1350 gctacatggc tgttttatat gagtgatgtg tctgcaggag gagccactgt 1400 ttttcctgaa gttggagcta gtgtttggcc caaaaaagga actgctgttt 1450 1304442 f. mol. tctggtataa tctgtttgcc agtggagaag gagattatag tacacggcat 1500 gcagcctgtc Cagtgctagt tggcaacaaa tgggtatcca ataaatggct 1550 ccatgaacgt ggacaagaat ttcgaagacc ttgtacgttg tcagaattgg 1600 aa 1602

<210> 4 <211> 534 <212> PRT <213> homo sapien <220><221> gene <222> 1-534 <223> Human proline 4-hydroxylase次次单位<400> 4

Met Val Trp Tyr lie Leu lie lie Gly lie 10 Leu Leu Pro Gin Ser Leu Ala His Pro Gly 20 Phe Phe Thr Ser lie Gly Gin Met Thr Asp 30 Leu lie His Thr Glu Lys Asp Leu Val Thr 40 Ser Leu Lys Asp Tyr lie Lys Ala Glu Glu 50 Asp Lys Leu Glu Gin lie Lys Lys Trp Ala 60 Glu Lys Leu Asp Arg Leu Thr Ser Thr Ala 70 Thr Lys Asp Pro Glu Gly Phe Val Gly His 80 Pro Val Asn Ala Phe Lys Leu Met Lys Arg 90 1304442

Leu Asn Thr Glu Trp Ser Glu Leu Glu Asn 100 Leu Val Leu Lys Asp Met Ser Asp Gly Phe 110 lie Ser Asn Leu Thr lie Gin Arg Pro Val 120 Leu Ser Asn Asp Glu Asp Gin Val Gly Ala 130 Ala Lys Ala Leu Leu Arg Leu Gin Asp Thr 140 Tyr Asn Leu Asp Thr Asp Thr lie Ser Lys 150 Gly Asn Leu Pro Gly Val Lys His Lys Ser 160 Phe Leu Thr Ala Glu Asp Cys Phe Glu Leu 170 Gly Lys Val Ala Tyr Thr Glu Ala Asp Tyr 180 Tyr His Thr Glu Leu Trp Met Glu Gin Ala 190 Leu Arg Gin Leu Asp Glu Gly Glu lie Ser 200 Thr lie Asp Lys Val Ser Val Leu Asp Tyr 210 Leu Ser Tyr Ala Val Tyr Gin Gin Gly Asp 220 Leu Asp Lys Ala Leu Leu Leu Thr Lys Lys 230 Leu Leu Glu Leu Asp Pro Glu His Gin Arg 240 Ala Asn Gly Asn Leu Lys Tyr Phe Glu Tyr 250 lie Met Ala Lys Glu Lys Asp Val Asn Lys 260 Ser Ala Ser Asp Asp Gin Ser Asp Gin Lys 270 Thr Thr Pro Lys Lys Lys Gly Val Ala Val 280 Asp Tyr Leu Pro Glu Arg Gin Lys Tyr Glu 290 Met Leu Cys Arg Gly Glu Gly He Lys Met 300 Thr Pro Arg Arg Gin Lys Lys Leu Phe Cys 310 Arg Cys His Asp Gly Asn Arg Asn P Ro Lys 320

1304442

Phe lie Leu Ala Pro Ala Lys Gin Glu Asp 330 Glu Trp Asp Lys Pro Arg lie lie Arg Phe 340 His Asp lie lie Ser Asp Ala Glu lie Glu 350 lie Val Lys Asp Leu Ala Lys Pro Arg Leu 360 Arg Arg Ala Thr lie Ser Asn Pro lie Ala 370 Gly Asp Leu Glu Thr Val His Tyr Arg lie 380 Ser Lys Ser Ala Trp Leu Ser Gly Tyr Glu 390 Asn Pro Val Val Ser Arg lie Asn Met Arg 400 lie Gin Asp Leu Thr Gly Leu Asp Val Ser 410 Thr Ala Glu Glu Leu Gin Val Ala Asn Tyr 420 Gly Val Gly Gly Gin Tyr Glu Pro His Phe 430 Asp Phe Ala Arg Lys Asp Glu Pro Asp Ala 440 Phe Lys Glu Leu Gly Thr Gly Asn Arg lie 450 Ala Thr Trp Leu Phe Tyr Met Ser Asp Val 460 Ser Ala Gly Gly Ala Thr Val Phe Pro Glu 470 Val Gly Ala Ser Val Trp Pro Lys Lys Gly 480 Thr Ala Val Phe Trp Tyr Asn Leu Phe Ala 490 Ser Gly Glu Gly Asp Tyr Ser Thr Arg His 500 Ala Ala Cys Pro Val Leu Val Gly Asn Lys 510 Trp Val Ser Asn Lys Trp Leu His Glu Arg 520 Gly Gin Glu Phe Arg Arg Pro Cys Thr Leu 530

Ser Glu Leu Glu 1304442 <210> 5 <211> 1524 <212> DNA <213> homo sapien <220><221> gene <222>

≪ 223 > human-proline-4-hydroxylase β subunit < 400 > · 5 atggtgcgcc gcgctctgct gtgcctgccg tggaccgccc tggtgcgcgc 50 cgacgccccc gaggaggagg accacgtcct ggtgctgegg aaaagcaact 100 tcgcggaggc gctggcggcc cacaagtacc tgctggtgga gttctatgcc 150 ccttggtgtg gccactgcaa ggctctggcc cctgagtatg ccaaagccgc 200 tgggaagctg aaggcagaag gttccgagat caggttggcc aaggtggacg 250 ccacggagga gtctgaccta gcccagcagt acggcgtgcg cggctatccc 300 accatcaagt tcttcaggaa tggagacacg gcttccccca aggaatatac 350 agctggcaga gaggctgatg acatcgtgaa ctggctgaag aagcgcacgg 400 gcccggctgc caccaccctg cctgacggcg cagctgcaga gtccttggtg 450 gagtccagcg aggtggctgt catcggcttc ttcaaggacg tggagtcgga 500 ctctgccaag cagtttttgc aggcagcaga ggccatcgat gacataccat 550 ttgggatcac ttccaacagt gacgtgttct ccaaatacca gctcgacaaa 600 gatggggttg tcctctttaa gaagtttgat gaaggccgga acaactttga 650 Aggggaggtc accaaggaga acctgctgga ctttatcaaa cacaaccagc 700 1304442

tgccccttgt catcgagttc accgagcaga cagccccgaa gatttttgga 750 ggtgaaatca agactcacat cctgctgttc ttgcccaaga gtgtgtctga 800 ctatgacggc aaactgagca acttcaaaac agcagccgag agcttcaagg 850 gcaagatcct gttcatcttc atcgacagcg accacaccga caaccagcgc 900 atcctcgagt tctttggcct gaagaaggaa gagtgcccgg ccgtgcgcct 950 catcaccctg gaggaggaga tgaccaagta caagcccgaa tcggaggagc 1000 tgacggcaga gaggatcaca gagttctgcc accgcttcct ggagggcaaa 1050 atcaagcccc acctgatgag ccaggagctg ccggaggact gggacaagca 1100 gcctgtcaag gtgcttgttg ggaagaactt tgaagacgtg gcttttgatg 1150 aaaaaaaaaa cgtctttgtg gagttctatg ccccatggtg tggtcactgc 1200 aaacagttgg ctcccatttg ggataaactg ggagagacgt acaaggacca 1250 tgagaacatc gtcatcgcca agatggactc gactgccaac gaggtggagg 1300 ccgtcaaagt gcacagcttc cccacactca agttctttcc tgccagtgcc 1350 gacaggacgg tcattgatta caacggggaa cgcacgctgg atggttttaa 1400 gaaattcctg gagagcggtg gccaagatgg ggcagggggt gatgacgatc 1450 tcgaggacct ggaagaagca gaggagccag acatggagga agacgatgat 1500 cagaaagctg tgaaagatga actg <210> 6 < 211 > 508 <212> PRT <213> homo sapien 9 <220> 1304442 <221>gene <222> 1-508 <223> Human proline 4-hydroxylase beta subunit < 400> 6

Met Val Arg Arg Ala Leu Leu Cys Leu Pro 50 Trp Thr Ala Leu Val Arg Ala Asp Ala Pro 100 Glu Glu Glu Asp His Val Leu Val Leu Arg 150 Lys Ser Asn Phe Ala Glu Ala Leu Ala Ala 200 His Lys Tyr Leu Leu Val Glu Phe Tyr Ala 250 Pro Trp Cys Gly His Cys Lys Ala Leu Ala 300 Pro Glu Tyr Ala Lys Ala Ala Gly Lys Leu 350 Lys Ala Glu Gly Ser Glu lie Arg Leu Ala 400 Lys Val Asp Ala Thr Glu Glu Ser Asp Leu 450 Ala Gin Gin Tyr Gly Val Arg Gly Tyr Pro 500 Thr lie Lys Phe Phe Arg Asn Gly Asp Thr 550 Ala Ser Pro Lys Glu Tyr Thr Ala Gly Arg 600 Glu Ala Asp Asp lie Val Asn Trp Leu Lys 650 Lys Arg Thr Gly Pro Ala Ala Thr Thr Leu 700 Pro Asp Gly Ala Ala Ala Glu Ser Leu Val 750 Glu Ser Ser Glu Val Ala Val lie Gly Phe 800 Phe Lys Asp Val Glu Ser Asp Ser Ala Lys 850 Gin Phe Leu Gin Ala Ala Glu Ala lie Asp 900 Asp lie Pro Phe Gly lie Thr Ser Asn Ser 950 10 1304442

Asp Val Phe Ser Lys Tyr Gin Leu Asp Lys 1000 Asp Gly Val Val Leu Phe Lys Lys Phe Asp 1050 Glu Gly Arg Asn Asn Phe Glu Gly Glu Val 1100 Thr Lys Glu Asn Leu Leu Asp Phe lie Lys 1150 His Asn Gin Leu Pro Leu Val lie Glu Phe 1200 Thr Glu Gin Thr Ala Pro Lys lie Phe Gly 1250 Gly Glu lie Lys Thr His lie Leu Leu Phe 1300 Leu Pro Lys Ser Val Ser Asp Tyr Asp Gly 1350 Lys Leu Ser Asn Phe Lys Thr Ala Ala Glu 1400 Ser Phe Lys Gly Lys lie Leu Phe lie Phe 1450 lie Asp Ser Asp His Thr Asp Asn Gin Arg 1500 lie Leu Glu Phe Phe Gly Leu Lys Lys Glu 1550 Glu Cys Pro Ala Val Arg Leu lie Thr Leu 1600 Glu Glu Glu Met Thr Lys Tyr Lys Pro Glu 1650 Ser Glu Glu Leu Thr Ala Glu Arg lie Thr 1700 Glu Phe Cys His Arg Phe Leu Glu Gly Lys 1750 lie Lys Pro His Leu Met Ser Gin Glu Leu 1800 Pro Glu Asp Trp Asp Lys Gin Pro Val Lys 1850 Val Leu Val Gly Lys Asn Phe Glu Asp Val 1900 Ala Phe Asp Glu Lys Lys Asn Val Phe Val 1950 Glu Phe Tyr Ala Pro Trp Cys Gly His Cys 2000 Lys Gin Leu Ala Pro lie Trp Asp Lys Leu 2050 Gly Glu Th r Tyr Lys Asp His Glu Asn lie 2100 1304442 Μ ♦ • Val lie Ala Lys Met Asp Ser Thr Ala Asn 2150 %

Glu Val Glu Ala Val Lys Val His Ser Phe 2200 Pro Thr Leu Lys Phe Phe Pro Ala Ser Ala 2250 Asp Arg Thr Val lie Asp Tyr Asn Gly Glu 2300 - Arg Thr Leu Asp Gly Phe Lys Lys Phe Leu 2350 Glu Ser Gly Gly Gin Asp Gly Ala Gly Gly 2400 Asp Asp Asp Leu Glu Asp Leu Glu Glu Ala 2450 Glu Glu Pro Asp Met Glu Glu Asp Asp Asp 2500

Gin Lys Ala Val Lys Asp Glu Leu <210> 7 <211> 19 <212> DNA <213>Artificial Sequence <220>

<222> 1-19 <223> Augmentation of human proline 4-hydroxylase alpha subunit positive primer <400> 7 atcgattatc atgtctgga 19 <210> 8 <211> 19 12 1304442 <212> DNA <213>Artificial sequence<220><221>.<222> 1-19 Reverse inversion of α subunits <223> Augmented human proline 4-hydroxylase <400> 8 ctttgagtga gcatcgatc 19

<210> 9 <211> 33 <212> DNA <213> artificial sequence <220><221>

<222> 1-33 <223> Increasing the forward index of COL21A1 <400> 9 33 ttagatctat tcctgggcca cctggtccga tag

<210> 10 <211> 30 <212> DNA

<S 13 Ί304442 4 * • <213>Manual sequence % <220><221><222> 1-30 . <223> Increasing COL21A1 reverse introduction "<400> 10 30 aatctagact Aatagtttgg tccttttctg

14

Claims (1)

I 1304442 No. 94141441 Application for Amendment of Patent Scope Amendment Date: 97.6.16 The revised version of the June ¥ ¥ Japanese Patent Application: 1. A recombinant insect cell, including a transfected alpha, beta subunit gene encoding a proline-hydroxylase, encoding a type XXI collagen Collagen snail • Cyclone (C〇Ll·) interval and C-terminal non-collagen protein (NC1) interval, wherein the coding 脯. Amino acid 'hydroxylase α subunit is the SEQ ID NO: 3 nucleoside The acid sequence, the "the beta unit of the proline hydroxylase is the nucleotide sequence of the sequence identification number: 5, the collagen triple helix (c〇L1) region encoding the XXI type collagen and C The non-collagen (NC1) region is the nucleotide sequence of the sequence identifier. Φ _2. The recombinant insect cell of claim 1, wherein the cell is a Trichoplusia ni cell. 3. The recombinant insect cell of claim 1, wherein the cell is a Drosophila me丨anogaster cell. 4. A method of producing recombinant collagen, comprising the steps of: providing a recombinant insect cell comprising a transfected gene encoding a proline 4'-enzyme, wherein the proline-hydroxylase gene Is a nucleotide sequence of sequence identification numbers: 3 and 5; • transfecting a expression vector into the cell, the expression vector comprising a recombinant collagen gene, wherein the recombinant collagen gene line is a sequence identifier. a nucleotide sequence; culturing the cell under conditions suitable for expression of the recombinant collagen gene; and recovering the expressed recombinant collagen, wherein the expressed recombinant collagen is secreted. 5. The method of claim 4, wherein the nucleotide sequence of the sequence identifier is encoding a unit of valeric acid 4-hydroxylase. 6. The method of claim 4, wherein the nucleotide sequence of the sequence identification number: 4 1304442 is a beta subunit encoding a proline 4-hydroxylase. > 7. The method of claim 4, wherein the cell is a Trichoplusia ni cell. 8. The method of claim 4, wherein the cell is pure {Drosophila melanogaster, each of which is 9. The method of claim 4, wherein the recombinant collagen gene encoding is XXI Collagen collagen triple helix (c〇L1) interval and C-terminal non-collagen protein (NC1) interval. 10. A method for producing recombinant collagen comprising the steps of: providing a recombinant insect cell comprising a transfected recombinant collagen gene encoding and a transfected methionine-transferase gene; Wherein the proline acid 4-hydroxylase gene sequence is a nucleotide sequence of sequence identification numbers: 3 and 5, and the recombinant collagen gene line is a nucleotide sequence of sequence identification number: i; suitable for the recombination The recombinant insect cells are cultured under conditions in which the collagen is expressed; and the expressed recombinant collagen is recovered, wherein the expressed recombinant collagen is secreted. The method of claim 1, wherein the cell is a Tyichoplusia ni cell. The method of claim 10, wherein the cell is a fruit (Drosophila) Melanogaster) cells. The method of claim 1, wherein the recombinant collagen gene encodes a collagen triple helix (〇〇11) region of type XXI collagen and a C-terminal non-collagen protein (NC1) interval. . 14. The method of claim 1, wherein the nucleotide sequence of the sequence identifier: 3 encodes a unit of valeric acid 4-hydroxylase. The method of claim 10, wherein the nucleotide sequence of the sequence identifier: 5 encodes a beta unit of the proline 4-hydroxylase.