KR100414182B1 - Molecular cloning and characterization of a gene encoding protein disulfide isomerase from Bombyx mori Bm5 cell line - Google Patents

Abstract

The present invention relates to a nucleotide sequence of a cDNA gene encoding protein disulfide isomerase isolated from silkworm cultured cells and an amino acid sequence deduced therefrom.

Description

Molecular cloning and characterization of a gene encoding protein disulfide isomerase from Bombyx mori Bm5 cell line} encoding cDNA gene encoding protein disulfide isomerase isolated from silkworm culture cells

The present invention relates to a base sequence of cDNA encoding protein disulfide isomerase (PDI) isolated from silkworm cultured cells and an amino acid sequence deduced therefrom.

Synthesis of mRNA from DNA and protein synthesis from mRNA is a fundamental concept of genetic engineering. In this case, the protein synthesized in the cytoplasm has a protein primary structure of peptide strands in which amino acids are linearly linked. Here, the primary structure of a protein means only the sequence of amino acids connected in linear form. The primary structure of these proteins is secondary to the protein, due to the nature of the amino acids (charge, hydrophobicity, hydrophilicity, amino acid size, etc.) and interactions between molecules in the amino acid sequence (hydrogen bonds, non-covalent bonds, disulfide bonds, etc.). Structures (some peptide sequences formed α-helix structures, β-sheet structures, etc.) and tertiary structures (one strand of peptides formed structures) are formed. In addition, the formed peptide strands form the quaternary structure of the protein by interaction with each other. By this process, the proteins form a higher order structure, thereby producing the structure and activity of the protein, such as enzyme active site, enzyme activity control site, ligand binding site.

Conventionally, such protein folding is understood as a process of simply changing to the most thermodynamically stable state. However, it was found that most of the denatured proteins did not restore their original activity when the original environment was reset after applying heat to the structured protein in an active form. These findings suggest that protein structuring is not just a thermodynamic stabilization process, and that there is a second material that helps protein structuring.

Many studies have been conducted to find such a second substance, and as a result, a protein called protein disulfide isomerase was discovered. Protein disulfide isomerase is an enzyme involved in disulfide bonds in peptides or between different peptides that play an important role in the structure of proteins. Most glycoproteins have disulfide bonds (SS), and proper disulfide bonds are essential biochemical reactions for higher order structure formation and function expression (Raina Missiaks, Annu. Rev. Microbiol., 51, 179-202, 1997; Rietsch Beckwith, Annu. Rev. Genet., 32, 163-184, 1998). Therefore, when disulfide bonds are thermodynamically only or disulfide bonds are abnormally caused by external stress or the like, protein disulfide isomerase normally turns these abnormal disulfide bonds, thereby structuring and activating a protein It makes the function appropriate.

In other words, protein disulfide isomerase (PDI, EC 5.3.4.1), which is involved in protein disulfide binding, functions as a kind of protein-folding editor, improperly structured. Disulfide bonds in proteins are appropriately bound to isomerize to functional proteins.

At the time of the discovery of the protein disulfide isomerase, it was only recognized as an oxidoreductase, but is now involved in the structure, assembly and post translational modification of numerous proteins with disulfide bonds. As a multifunctional protein of Freedman et al ., Trends Biochem., 19, 331-336, 1994; Creighton et al ., Trends Biochem., 13, 18-23, 1995), about 10 ³ in the ER lumen It has been reported to be present at high concentrations of M and play an important role in protein structuring, which accounts for about 12% of the vesicle total protein weight (Zapun et al ., Proteins, 14, 10-15, 1992). Protein disulfide isomerase has also been reported to function as a molecular chaperone that promotes the formation of protein higher structures (LaMantia Lennarz, Proc. Natl. Acad. Sci. USA, 88, 4453-4457, 1991; Wang; , Biochemistry, 63, 407-412, 1997; Cai et al ., J. Biol. Chem., 269, 24550-24552, 1994).

As a result of primary structural analysis of protein disulfide isomerase cloned in vertebrates, protein disulfide isomerase is an active site of protein-thiol oxidoreductase such as thioredoxin. 2 motifs of Cys-Gly-His-Cys- (Novia Lennarz, J. Biol. Chem., 267, 3553-3556, 1992; Darby Creighton, Biochemistry, 34, 11725-11735, 1995) The C-terminus has Lys-Asp-Glu-Leu, an ER retentional signal, which has been reported to have three specific motifs (Munro and Pelham, Cell, 48, 899-907, 1987). Vaux et al ., Nature, 345, 495-502, 1990; Koivunen et al ., EMBO J., 18, 65-74, 1999).

Currently, the primary amino acid structure of protein disulfide isomerase in various species, including vertebrates, has been revealed through full sequencing (Pihlajaniemi et al ., EMBO J., 6, 643-649, 1987; Edman et al ., Nature, 317, 267-270, 1985; Li Larkins, Mol. Biol., 30, 873-882, 1996; Veijola et al ., Biochem. J., 317, 721-729, 1996; LaMantia et al ., Proc. Natl. Acad. Sci. USA, 88, 4453-4457, 1991; Mazzarella et al ., J. Biol. Chem., 265, 1094-1101, 1990; Yamauchi et al ., Biochem. Biophys. Res. Commun., 146, 1485-1492, 1987; Fliegel et al ., J. Biol. Chem., 265, 15496-15502, 1990; Wilson et al ., Mol. Biochem. Parasitol., 68, 103-117 , 1994; Ngiam et al ., Curr. Genet., 31, 133-138, 1997; Chen Hayes, Plant Physiol., 106, 1705-1706, 1994).

However, only insects have reported the gene sequence and amino acid primary structure of protein disulfide isomerase only in Drosophila (McKay et al ., Mol. Biol., 25,647-654, 1995), but no other insects. It is true.

Thus, the present inventors conducted a study to find the nucleotide sequence and amino acid sequence of the gene encoding the protein disulfide isomerase from silkworms, and treated tunicamycin to Bm5 cultured cell line derived from silkworm ovary cells, The present invention was completed by inducing an abnormal structure and analyzing only the genes from which expression was induced, and discovering a new gene having a function similar to that of a protein disulfide isomerase previously known.

It is therefore an object of the present invention to provide a novel cDNA base sequence and its deduced amino acid sequence encoding the protein disulfide isomerase gene isolated from silkworm cultured cells.

Figure 1 shows a restriction map of the plasmid vector pGEMT-bPDI containing cDNA of protein disulfide isomerase isolated from silkworm culture cells.

Fig. 2 shows the results of RNA dot blotting of the expression of protein disulfide isomerase mRNA in each tissue of silkworms. (In Fig. 2, S, F, Ma, M and E are silk glands, respectively. , Fat body, epidermal organs, middle and epidermis.)

Figure 3 shows the results of the mRNA dot blotting method of the mRNA expression of protein disulfide isomerase when treated with a variety of stress inducing agents (in Figure 3, C, D, A, T, Ca, M and H represents cells treated with control, DTT, antimycin A, tunicamycin, Ca ²⁺ inophore A23187, monensin and H ₂ O ₂ , respectively.)

Figure 4 is a result of protein-specific analysis of the function of the protein disulfide isomerase gene through SDS-PAGE analysis and Western blot analysis (Fig. 4A is SDS-PAGE analysis, 4B is Western Blot analysis, where M, 1, 2 and 3 represent protein size marks, normal cell lines, cell lines transfected with wild-type virus and cell lines transfected with recombinant virus into which the protein disulfide isomerase gene was introduced.)

5 is a view showing the results of screening the cDNA clones by the differentiation screening method (in FIG. 5, C is a probe prepared from a normal cell line, T is a probe prepared from a tunicamycin-treated cell line Is used.)

Between protein disulfide isomerase genes of various species, they show relatively high homology at the deduced amino acid level, but little homology at the gene sequence level. For this reason, it is almost impossible to construct a probe by referring to a nucleotide sequence of a protein disulfide isomerase of a known species and to use it to obtain a cDNA encoding the entire protein disulfide isomerase according to a conventional method. did. In addition, since the function of protein disulfide isomerase revealed in Drosophila is not fully interpreted, and it is treated only as protein disulfide isomerase-like gene, it is not easy to identify the homologous sequence region between insects. It is very difficult to find the protein disulfide isomerase gene of silkworm from the protein disulfide isomerase gene.

Therefore, in order to obtain a cDNA clone containing the protein disulfide isomerase gene of silkworms,

1) artificially treating tunicamycin with a Bm5 culture cell line derived from silkworm ovary cells used as a host cell of an insect baculovirus expression system to induce abnormal structure of the protein;

2) selecting the cDNAs from which expression is induced by differential screaning when the protein abnormal structure is induced in step 1);

3) analyzing partial nucleotide sequences of the selected genes, and comparing the analyzed nucleotide sequences with sequences of known protein disulfide isomerases to select cDNAs having similar functions; And,

4) determining the total nucleotide sequence of the cDNA selected in step 3), and deducing the amino acid sequence therefrom;

By the method comprising, characterized in that for cloning the protein disulfide isomerase gene of the silkworm.

By the above method, the nucleotide sequence of the protein disulfide isomerase cloned from silkworm was shown in SEQ ID NO: 1, and the amino acid sequence deduced therefrom is shown in SEQ ID NO: 2.

As shown in SEQ ID NO: 1, the protein disulfide isomerase gene of the present invention has a total size of 2,565 bp, and an open reading frame (hereinafter referred to as 'ORF') is one of the nucleotide sequences of SEQ ID NO: It was present at the positions of 117-1,601, the base sequence of the 3'-end includes a poly-A (poly-A) base, it was confirmed that the presence of two intermittent transcription termination signal 'AATAAA'. The 494 amino acids could be deduced from the ORF of the cDNA, and the estimated molecular weight was 55.6 kDa.

Further, the nucleotide sequence of the silkworm protein disulfide isomerase gene described in SEQ ID NO: 1 is known through the CLUSTAL W Program (https://www.clustalw.genome.ad.jp), which is a computer program for homology analysis. Comparing homology with the protein disulfide isomerases showed 55% homology with D. melanogaster, human ( H. sapians ), rat ( R. norvegicus ), corn ( Z. mays ), nematodes ( C. elegans ) and yeast ( S. cerevisiae ) showed low homology with 29%, 49%, 28%, 40% and 25%, respectively (see Table 1).

However, as shown in Table 2, 1) the N-terminal thioredoxin-like enzyme activity site (Region I), 2) the C-terminal, three specific active sites of the known protein disulfide isomerase in other species The thioredoxin-like enzyme activity site (region II) and 3) the ER (res) residual signal region (region III) were highly conserved.

It is reported that the sequence of the thioredoxin enzyme active site known as -Cys-Gly-Pro-Cys- is very similar to that of -Cys-Gly-His-Cys-, which is the enzyme active site sequence of protein-thiol oxidoreductase. Edman et al ., Nature, 317, 267-270, 1985; Darby et al ., Biochemistry 35, 10517-10528, 1996). In addition, according to the present studies, the protein disulfide isomerase contains these thioredoxin-like sequences in two places, N-terminus (Region I) and C-terminus (RegionII), and this site is protein disulfide isoform. It is the active center of Murrays.

It was confirmed that the protein disulfide isomerase of silkworm isolated in the present invention had an amino acid sequence of the tyroredoxin-like enzyme active site at the N-terminus and the C-terminus. That is, Region I is present in the sequences of 53 to 56 of SEQ ID NO: 2, and Region II is present in 395 to 398 of SEQ ID NO: 2, respectively.

In addition, region III is a vesicle residual signal motif involved in enzyme positioning in the endoplasmic reticulum, and its amino acid sequence is known as Lys-Asp-Glu-Leu (Munro Pelham, 1987; Vaux et al., 1990, Koivunen et al., 1999). In the deduced amino acid sequence of the protein disulfide isomerase of silkworm revealed in the present invention, it was confirmed that such a vesicle residual signal (SEQ ID NO: 461 to 464 of SEQ ID NO: 2) was also present.

Meanwhile, the silk gland (S), fat body (F), epidermal organ (Ma), middle intestine (M) and epidermal tissue (E) are separated from silkworms, and mRNA is extracted therefrom, followed by the protein disulfide isomerray of the present invention. As a result of RNA dot blotting using the cDNA of the probe as a probe, as shown in FIG. 2, it was expressed in all tissues except the middle intestine. . Since fat tissue in silkworms is the equivalent of mammalian liver, these results indicate that protein disulfide isomerase is most expressed in hepatocytes in mammals (Terada et al ., J. Biol. Chem. , 270, 20410-20416,1995; Mills et al ., Biochem. J., 213, 245-248, 1983; Nieto et al ., Biochem. J., 267, 317-323, 1990). Can be.

In addition, when silkworm cultured cells Bm5 were treated with various stress inducing agents of DTT, antimycin A, tunicamycin, Ca ²⁺ inophore A23187, monensin and H ₂ O ₂ , the amount of transcription of protein disulfide isomerase was increased. Increasing stress inducers were tunicamycin and Ca ²⁺ inophores A23187, as shown in FIG. 3. These results are consistent with previous reports that tunicamycin and Ca ²⁺ Inophore A23187 increase the amount of transcription of protein disulfide isomerase even when treated with various stress inducing agents in HeLa cells (Odani). et al ., Biochem. Biophys. Res. Commun., 220, 264-268, 1996).

Therefore, the protein disulfide isomerase disclosed in the present invention is the same as the conventional protein disulfide isomerase and the tissue expression form and the reaction form for the stress inducing agent, cDNA having a sequence of SEQ ID NO: 1 selected in the present invention is a protein It was confirmed that the disulfide isomerase was coded.

In addition, SDS-PAGE and Western blotting analysis was performed to confirm that the recombinant protein expressed as the protein disulfide isomerase gene isolated from silkworm cultured cells is exactly the same as the protein disulfide isomerase that has been previously identified. Western blot analysis was performed. As a result, protein bands not observed in normal cell lines or cell lines infected with wild-type virus were clearly observed on the SDS-PAGE (position of about 55.6 kDa), which was assumed to be protein disulfide isomerase protein.

In addition, Western blotting was performed using a protein disulfide isomerase antibody of rat to support this. As in the SDS-PAGE results, protein disulfide was detected in normal cell lines or wild-type virus-infected cell lines. Although expression of feed isomerase could not be confirmed, it was confirmed that protein disulfide isomerase was strongly expressed in the cell line infected with the synthetic virus to which the protein disulfide isomerase gene was introduced. In addition, since these results are a result of using a polyclonal antibody against rat protein disulfide isomerase, the silkworm protein disulfide isomerase isolated in the present invention has a protein disulfide isomerase in common. It can be seen that it has a specific epitope. Therefore, it was confirmed once again that the protein disulfide isomerase gene disclosed in the present invention has the same function as the conventional protein disulfide isomerase.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

In the following examples, the procedure for cloning the protein disulfide isomerase gene from silkworms (Example 1), and the protein disulfide isomerase in which the protein disulfide isomerase gene newly discovered in the present invention was previously identified In order to confirm that the gene has a function similar to that of the expression tissue assay (Example 2) and the expression regulation according to the stress inducer treatment was analyzed at the transcription level (Example 3). In addition, SDS-PAGE analysis and Western blot analysis (Example 4) were performed to confirm the function of the protein disulfide isomerase gene found in the present invention at the protein level.

Example 1 cDNA Cloning and Gene Sequencing of Protein Disulfide Isomerase Isolated from Silkworms

1. Creating a cDNA Gene Bank

5m of tunicamycin per ml of cultured cells were treated for 5 hours at 25 ° C in silkworm cultured cells Bm5, and the expression levels of the genes increased when glycosylation and disulfide bonds were inhibited. Expression was induced. Then, silkworm cultured cells Bm5 was recovered, washed twice with PBS (phosphate buffered saline), and total RNA was separated therefrom. Then, only poly (A) + RNA was purified from the total RNA, according to a conventional method in the art. Then, single-chain cDNA and double-chain cDNA were synthesized in sequence using purely isolated poly (A) + RNA, and EcoR I and Xho I restriction enzyme recognition sequences were assigned to the 5'- and 3'-ends, respectively. It was. The cDNA thus prepared was directionally inserted into a Lambda ZAP II vector containing pBluescript SK-Plasmid, followed by in vitro packaging, and a cDNA gene bank was prepared. The pfu (plaque forming unit) of the constructed cDNA gene bank was 1.2 × 10 ⁶ .

Meanwhile, from the cDNA gene bank, randomly selected phages were converted into pBluescript SK- by in vivo excision in order to confirm the size of cDNA inserted into the vector and the presence or absence of insertion fragments. The converted plasmids were digested with EcoR I and Xho I restriction enzymes and found to have a fragment size of at least 500 bp.

Therefore, the fragment size of the clones involved in cDNA production efficiency, the pfu of the gene bank and the insertion rate of the fragment (Sung et al., 1998) were judged to be suitable for genetic analysis of the cDNA gene bank.

2. Selection of clones with increased expression by differentiation screening

From the cDNA gene bank prepared above, when screened with tunicamycin, cDNA clones with increased expression compared to normal cultured cells were screened differently (Hg et al ., Nucleic Acids Res., 19, 6123-6127). , 1991). In other words, the selection of clones with increased gene expression through differentiation screening was performed as follows.

768 cDNA phage clones randomly selected from the cDNA gene bank prepared above were converted to plasmid pBluescript SK- using an in vivo excision method. Then, using a hybri-dot manifold system (BRL Co., USA), the same blot was applied to two nylons, and one nylon membrane was synthesized using poly (A) + RNA isolated from normal cell lines. Hybridization was performed using a single chain cDNA as a probe and the other as a probe using a single chain cDNA synthesized using poly (A) + RNA isolated from cultured cells treated with tunicamycin. As a result, 40 cDNA clones were selected that showed a stronger signal in the nylon membrane for the tunicamycin treated cell line compared to the nylon membrane for the normal cell line (see FIG. 5).

3. Sequencing of Selected Clones in Differentiation

The sequencing of 40 cDNA clones selected by the differentiation screening method was performed using an automatic sequencing device (Perkin Elmer Co., ABI 377), and then the NCBI gene bank database search for the analyzed sequencing. CDNA clones were selected that showed high homology with protein disulfide isomerase.

4. Complete base sequence analysis of selected clones

The entire base sequence was analyzed for the cDNA estimated to be the protein disulfide isomerase gene selected above. The base sequence of the cDNA clone was determined using SP6, T7 and the following 10 synthetic primers.

SEQ ID NO: 1 5'-GTTGAATTCTATGCTCCATG-3 ' SEQ ID NO: 2 5'-GGCTGAAGAAGAAGACTGGC-3 ' SEQ ID NO: 3 5'-GGAGGCTGAAGATGAAGATG-3 ' SEQ ID NO: 4 5'-CGATTTCGAGAAATACCTTG-3 ' SEQ ID NO: 5 5'-CGGCACCCTGAAGCAGCATC-3 ' SEQ ID NO: 6 5'-GCCAACGAACTGGAACACAC-3 ' SEQ ID NO: 7 5'-GGAAGATGTGCCTGCCAAAG-3 ' SEQ ID NO: 8 5'-AATGCGAATCTTATTTTATG-3 ' SEQ ID NO: 9 5'-AGCTGTATTTATAATTTCTG-3 ' SEQ ID NO: 10 5'-CTTGTGTATCTCAAATTGGA-3 '

DNA for total sequence determination was isolated using Wizard Plus SV minipreps DNA purification system (Promega Co.). 250-500 ng of the isolated DNA and 3.2 pmole of primer were mixed with Termination Reaction Mix (Perkin Elmer Co.), followed by PCR reaction. Then, the nucleotide sequence was determined by developing on a 4.5% sequencing gel, and the nucleotide sequence was analyzed by DNA Sequencing Analysis Software (Perkin Elmer Co.).

However, the entire base sequence of the analyzed cDNA contained the entire ORF, but part of the 3'-terminal untranslated region was cleaved. That is, the transcription termination codon (TAA) could be confirmed, but the transcriptional termination signal AATAAA sequence and the poly-A tail region could not be identified.

5. Securing the 3'-terminal sequence by RACE-PCR method

Therefore, 3'-terminal untranslated sites cut by the RACE-PCR method (rapid amplication of cDNA ends) were secured. Then, the PCR product was inserted at the Eco RI / Spe I restriction site of pGEM-T easy vector (Promega Co., USA) to prepare pGEMT-bPDI (see FIG. 1). Then, the entire nucleotide sequence of cDNA having the 3'-terminal untranslated site was obtained. The base sequence of the cDNA encoding the protein disulfide isomerase obtained as mentioned above and isolated from silkworm is shown in SEQ ID NO: 1.

6. Interpretation of the nucleotide sequence of cDNA encoding protein disulfide isomerase isolated from silkworm

As shown in SEQ ID NO: 1, the protein disulfide isomerase gene has a total size of 2,565 bp, the ORF was 117-1,601 in the nucleotide sequence of SEQ ID NO: 1, and the poly- Including the A (poly-A) base, it was confirmed that there are two potential transcription termination signals 'AATAAA'. The 494 amino acids could be deduced from the ORF of the cDNA, and the estimated molecular weight was 55.6 kDa. The amino acid sequence deduced above is shown in SEQ ID NO: 2.

On the other hand, as shown in SEQ ID NO: 2, the N-terminus of the amino acid sequence of the protein disulfide isomerase of the present invention had -CGHC-, an active site of a generally known protein-thiol oxidoreductase (SEQ ID NO: 2). At positions 53 to 56 and 395 to 398), and at the C-terminus thereof, a -KDEL motif (positions 461 to 464 of SEQ ID NO: 2), which is an ER retention signal, was also identified.

Therefore, it can be confirmed that the nucleotide sequence and the amino acid sequence described in SEQ ID NO: 1 and SEQ ID NO: 2 encode a protein disulfide isomerase separated from silkworms.

Example 2 Expression Tissue Assay of Protein Disulfide Isomerase Isolated from Silkworm Cultured Cells

For the assay of protein disulfide isomerase-expressing tissues in silkworms, silk gland, fat body, epidermal organs, mid- and epidermis were isolated from 5 larvae silkworms, and then SV Total RNA Isolation System (Promega Co.) Total RNA was isolated using. Then, the whole RNA isolated was warmed and denatured at 65 ° C. for 10 minutes, and then 5 μg of RNA isolated from each tissue was blotted onto the nylon membrane. Then, using Prime-It II Random Primer Labeling Kit (Stratagene Co.), cDNA of protein disulfide isomerase of silkworms labeled with [α- ³² P] dATP (ie, selected from the differentiation screening process) , Clones with untranslated sites) and 18S rRNA cDNA were prepared. Then, RNA dot blotting was performed using this as a probe. The results are shown in FIG.

As shown in FIG. 2, protein disulfide isomerase isolated from silkworm cultured cells was expressed in silk glands (M), fat body (F), epidermis (E) and epidermal organ (Ma), but in the middle intestine (M). Hardly expressed. In addition, it was confirmed that the protein disulfide isomerase was expressed the most amount, especially in fat body.

Thus, reports that vertebrate disulfide isomerases derived from vertebrates are mainly expressed in hepatocytes (Terada et al ., J. Biol. Chem., 270, 20410-20416,1995; Mills et al ., Biochem. J., 213, 245-248, 1983; Nieto et al ., Biochem. J., 267, 317-323, 1990) and the silkworm protein disulfide as described in SEQ ID NO: 1 in that the fat of silkworm corresponds to the mammalian liver. It can be seen that the transcript expression form of isomerase is the same as the expression form of mammalian protein disulfide isomerase. Thus, it can be seen that the nucleotide sequence of SEQ ID NO: 1 of the present invention is protein disulfide isomerase.

Example 3 Expression Increase of Protein Disulfide Isomerase by Stress Inducer Treatment

Lee, Curr. According to Opinion in Cell Biol., 4, 267-273, 1992, genes were expressed to form protein peptides with normal sequences, but peptides in which protein folding was not normally performed due to the influence of the external environment When present in the endoplasmic reticulum of cells, it is said to increase the amount of transcription of protein disulfide isomerase to normalize the structure of these peptides.

Therefore, in order to confirm whether the silkworm protein disulfide isomerase of the present invention exhibits functions as an ER foldase and a molecular chaperone when abnormal structuring of the protein is caused by external stress. After treatment with various stress inducing agents to cells Bm5, the expression level of protein disulfide isomerase was examined.

The stress inducing agent used here is DTT (Dithioreitol), a reducing agent, Antimycin A, an inhibitor of ATP biosynthesis, Tunikamycin, an inhibitor of glycosylation and disulfide bond formation, and Ca ^{2+, an} inhibitor of homeostasis of calcium ions. Inophore A23187 (monensin) and H ₂ O ₂ , inhibitors of protein vesicle-to-Golgi shift, were used.

In silkworm cultured cells Bm5, DTT, antimycin A, tunicamycin, Ca ²⁺ inophore A23187, monensin and H ₂ O ₂ were added at concentrations of 3 mM, 8 μM, 5 μg / ml, 10 μM, 100 μM and 100 μM, respectively. After treatment for a period of time, the total RNA was isolated from the treated cells, and the mRNA expression level of protein disulfide isomerase in each of the treated cells was determined in the same manner as in the RNA dot blotting method of Example 2. Analyzed. As a control, a normal cell line without any substance was used. The results are shown in FIG.

As shown in FIG. 3, the protein disulfide isomerase gene of silkworm was strongly expressed in cells treated with tunisimacin or Ca ²⁺ inophore A23187, compared to other stress inducing agents. These results indicate that 1996 Odani et al. Treated HeLacell with heat shock, stress inducer A23187, tunicamycin, cyclohexiimide, DTT and Δ12-prostaglandin J2. When the mRNA expression level of protein disulfide isomerase in HeLa cells was increased (Odani et al ., Biochem. Biophys. Res. Commun., 220, 264-268, 1996).

In addition, through these results, the protein disulfide isomerase of the present invention also functions as an ER foldase and a molecular chaperone similarly to the protein disulfide isomerase previously known (LaMantia Lennarz, Proc). Natl.Acad.Sci. USA, 88, 4453-4457, 1991; Wang, Biochemistry, 63, 407-412, 1997; Cai et al ., J. Biol. Chem., 269, 24550-24552, 1994), It can be estimated that one of the proteins that bind to the calcium binding of the endoplasmic reticulum (Macer Koch, J. Cell Sci., 91, 61-70, 1988).

Therefore, it can be seen that the protein disulfide isomerase gene of the present invention has the same function as the previously known protein disulfide isomerase gene.

Example 4 Intracellular Introduction and Expression Confirmation of Recombinant Virus Incorporated with Protein Disulfide Isomerase Gene

In the present invention, SDS-PAGE and Western blot analysis were performed to confirm whether protein disulfide isomerase isolated from silkworm cultured cells had the same function as protein disulfide isomerase previously known at the protein level.

First, the protein disulfide isomerase gene isolated in Example 1 was inserted into a pBAC-1 insect baculovirus transfer vector to prepare a plasmid recombinant transfer vector pBAC1-bPDI. Then, the recombinant transfer vector pBAC1-bPDI and wild-type insect baculovirus were cotransfected into Sf-9 cell line derived from ovary tissue of pupa of Spodopetera frugiperda (fall armyworm) as a host cell. Homologous recombination led to the integration of the recombinant transfer vector pBAC1-bPDI onto the wild-type virus genome. As used herein, "homologous recombination" means a conventional DNA recombination method using sequence homology of a border sequence.

Insect baculovirus incorporating the recombinant transfer vector pBAC1-bPDI was selected by the following method. In other words, after confirming the co-infected Sf-9 cells on the microscope, 1 to 2 cells per well were plated by using a limiting dilution method in the 96-well plate. . The cell cultures incubated for 4 days were then transferred to a new 96-well plate and stained with X-gal to select unstained white plaques, thereby incorporating the combinatorial transition vector pBAC1-bPDI. Insect baculovirus were selected and named for this recombinant virus vAc-bPDI.

Recombinant virus vAc-bPDI, into which the protein disulfide isomerase gene was isolated from silkworm cultured cells, was transfected into sf-9 insect cells to express the protein disulfide isomerase gene. To confirm expression, cells transfected with the recombinant virus were incubated for 72 hours, and then proteins were separated therefrom and subjected to SDS-PAGE. The results are shown in Figure 4a. As shown in FIG. 4A, protein bands not observed in the normal cell line (lane 1) or the cell line infected with the wild type virus (lane 2) were confirmed in the cell line (lane 3) transfected with the recombinant virus vAc-bPDI. The size of this band was confirmed to be consistent with the estimated size of protein disulfide isomerase (about 55.6 kDa) through the genetic analysis. Thus, the expressed protein was confirmed to be protein disulfide isomerase protein.

In addition, to confirm the above results, Western blot analysis was performed using a polyclonal antibody against rat protein disulfide isomerase (prepared from Peter Arvan, Harvard Medical School). Western blots were performed using routine methods in the art and the results are shown in FIG. 4B.

As shown in FIG. 4B, expression of protein disulfide isomerase was not detected in normal cell line (lane 1) or wild-type virus-infected cell line (lane 2), but the protein disulfide isomerase gene was introduced. In the cell line transfected with virus (lane 3), it was confirmed that protein disulfide isomerase was strongly expressed. In addition, since the result of Figure 4b is a result of Western blot using a polyclonal antibody against rat protein disulfide isomerase, the silkworm protein disulfide isomerase isolated from the present invention is a protein disulfide isomerase. It has a specific epitope which it has in common. Therefore, it was confirmed again that the protein disulfide isomerase gene disclosed in the present invention has the same function as the conventional protein disulfide isomerase.

The protein disulfide isomerase nucleotide sequence and amino acid sequence of the present invention can normalize the recombinant protein of abnormal structure that occurs excessively when the recombinant protein is produced through a baculovirus expression system that hosts insect organisms and cultured cells. Therefore, the expression level of the useful protein in which the original activity is maintained can be improved.

In addition, when the protein disulfide isomerase is prepared using the protein disulfide isomerase sequence of the present invention, protein disulfide isomerase, which is currently separated from the liver of vertebrates, can be replaced by an expensive product. In addition, insects or cultured cells transformed with the protein disulfide isomerase gene of the present invention may be prepared to produce more stable useful materials, and by directly making recombinant proteins and adding them to the cell culture medium, the useful proteins may be cheaper. It can also be mass produced.

Claims (2)

A base sequence of a protein disulfide isomerase isolated from silkworms and isolated from silkworms having the nucleotide sequence shown in SEQ ID NO: 1. An amino acid sequence of a protein disulfide isomerase gene isolated from the silkworm having the amino acid sequence of SEQ ID NO: 2 deduced from the nucleotide sequence of claim 1.