KR19980075710A - Proteins showing human thrombopoietin (hTPO) activity and methods for preparing the - Google Patents

Abstract

The present invention relates to a novel protein comprising its amino terminal region which exhibits the activity of human thrombopoietin (abbreviated as hTPO) and a method for producing the same. Specifically, the present invention includes a 153 amino acid sequence in which amino acid 1 is substituted with valine at the amino terminal site, which is important for the activity of hTPO, a hematopoietic protein that promotes the process of platelet production and differentiation, and at the amino terminal, Added new protein (hereinafter abbreviated as [Met- ¹ , Val ¹ ] hTPO _1-153 ), its gene, E. coli expression vector expressing it from the P _L promoter of lambda phage and [Met- ¹ , Val ¹ ] hTPO _{1 -153} relates to a method for producing a large amount of protein.

Description

Proteins showing human thrombopoietin (hTPO) activity and methods for preparing the

The present invention relates to a novel protein comprising its amino terminal region which exhibits the activity of human thrombopoietin (abbreviated as hTPO) and a method for producing the same.

More specifically, the present invention comprises a 153 amino acid sequence in which amino acid 1 is substituted with valine at the amino terminal site, which is important for the activity of hTPO, a hematopoietic protein that promotes the process of platelet production and differentiation, and at its amino terminal, methionine This added new protein (hereinafter abbreviated as [Met- ¹ , Val ¹ ] hTPO _1-153 ), its gene, E. coli expression vector expressing it from the P _L promoter of lambda phage and [Met- ¹ , Val ¹ ] hTPO _1-153 A method for producing a large amount of protein.

Human thrombopoietin (hTPO) is a glycoprotein composed of 332 amino acids that is produced and secreted by tissues such as the human liver or kidneys to act on the bone marrow, resulting in an increase in the number of platelets, hematopoietic cells in the bone marrow. It is a hematopoietic protein that stimulates and promotes differentiation.

Since Odel, et al., Proc. Soc. Exp. Biol. Med., 108, 428, 1961 , for the first time in 1961, Odel et al. Reported the presence of factors that regulate the number of platelets in rat blood . In 1994, the presence of hTPO was revealed, and its structure and action were studied (Sauvage, et al., Nature, 369 , 533-538, 1994; Bartley, et al., Cell, 77 , 1117-). 1124, 1994).

hTPO is a glycoprotein that acts on megakaryocyte colony forming progenitor cells, which are platelet precursor cells present in bone marrow cells, to proliferate platelet progenitor cells and ultimately increase the number of platelets and promote their differentiation. It is losing promising protein. In particular, indications that can be treated with hTPO are diseases caused by reduced platelet count (Thrombocytopenia) or deterioration of its function, especially for patients with thrombocytopenia and bone marrow transplantation caused by excessive administration of anticancer drugs or drugs. It can be usefully used for treatment.

To date, thrombocytopenia has been the only method for treating such thrombocytopenia, but the side effects due to bleeding, infection by blood-borne infections such as AIDS virus or hepatitis virus, and antigenicity of platelets (Lok, Stem Cells, 199 , 12, 586-598, 1994). Therefore, hTPO protein that can minimize or eliminate these side effects may be useful for treating various diseases related to platelets.

Human thrombopoietin (hTPO) is expressed as a protein consisting of 353 amino acids, including 21 signal sequences, which produces a hTPO consisting of 332 amino acids that are active through the maturation process. Specifically, hTPO consisting of 332 amino acids is divided into two regions, the amino terminal region (comprising about 153 amino acid residues) and the carboxy terminal region (comprising about 179 amino acid residues). Specifically, the amino terminus site shows about 23% amino acid sequence identity with erythropoietin (EPO) and includes four α-helix sites commonly found in hematopoietic proteins, mainly involved in the activity of the protein. Known. In fact, only the hTPO protein moiety (hereinafter abbreviated as hTPO ₁₅₃ protein) consisting of 153 amino acid residues, the site corresponding to the amino terminus of the mature TPO protein, has been reported to exhibit activity in vitro and in vivo . It is. In addition, it is known that the molecular weight of thrombopoietin extracted and purified directly from dog or pig plasma is about 18-25 kD, and the size corresponds to the amino terminus of the mature hTPO protein (hTPO ₃₃₂ ) consisting of 332 amino acids. About hTPO ₁₅₃ -hTPO ₁₉₈ ), it can be seen that the hTPO protein site alone exhibits biological activity.

On the other hand, the carboxy terminal region has six N-glycosylation sites, which are believed to be involved in the stability of the protein, but are not known in detail (Lok, S. and Foster, D., Stem cells, 12, 586-598, 1994).

Until now, in order to produce a large amount of hTPO, studies have been conducted to express hTPO using E. coli and mammalian cells, CHO cells and 293 cells. Savage et al. (Sauvage, WO95 / 18858) produced an expression vector pMP210-1 expressing the hTPO ₁₅₃ ([Met- ¹ ] hTPO ₁₅₃ ) gene from the tryptophan promoter containing the methionine codon at its amino terminus to express hTPO protein in E. coli. Reported. However, the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis are not specifically presented, so it is difficult to compare and predict direct expression results such as the expression amount and yield, but the expression amount is not high. It is expected. And Bartley, et al., EP Application No. 0 675 201 A1, et al. Lactose the hTPO ₁₆₃ ([Met- ² , Lys- ¹ ] hTPO ₁₆₃ ) gene, which includes a methionine codon and the first amino acid is substituted with lysine. An expression vector pAMG11-r-HuMGDF, which was expressed from a promoter induced by expression, was prepared to try to express hTPO protein, but the result of the expression amount was also not specifically mentioned.

In addition, the inventors have also prepared various types of expression vectors and studied their expression for a long time to mass produce hTPO in an amount sufficient for industrial use. Specifically, genes encoding hTPO ₃₃₂ and hTPO ₁₅₃ proteins were inserted next to the leader sequences of the Omp A (Outer membrane protein A) protein of Escherichia coli according to the protein translation sequence and expressed from the P _tac promoter in E. coli. Insignificant levels of less than about 1 mg / L. The hTPO ₁₅₃ protein was expressed by fusion with maltose binding protein and thioredoxin, etc., but this method was also weak in expression and expressed in E. coli cells. It is easily degraded, making it difficult to mass produce hTPO protein.

Therefore, expression of hTPO protein in Escherichia coli minimizes the degradation of hTPO by the action of proteolytic enzymes, and the development of expression vectors in which the production of hTPO protein is more strongly regulated by the inducer than the previously used promoter. This is urgent. Specifically, when there is no expression inducer, the expression is rarely expressed, but when there is an expression inducer, an expression vector capable of maximizing expression while minimizing degradation of hTPO protein can be expressed in a large amount. Is suitable.

In order to mass produce the hTPO protein having the amino acid sequence of its amino terminal region showing the activity of thrombopoietin, the present inventors included 153 amino acid sequences of the amino terminal region where serine 1 amino acid of hTPO was substituted with valine. And devised hTPO protein ([Met- ¹ , Val ¹ ] hTPO _1-153 protein) to which methionine was added at its amino terminus, and obtained its gene to prepare an expression vector capable of producing it from the P _L promoter of lambda phage. The present invention was completed by preparing a large amount of protein having hTPO activity in E. coli.

The present invention relates to a hTPO protein comprising 153 amino acid sequences of an amino terminal region in which amino acid 1 of human thrombopoietin is substituted with valine and methionine added to the amino terminal (hereinafter [Met- ¹ , Val ¹ ] hTPO _{1 For} the purpose of providing a _-153 protein). [Met- ¹ , Val ¹ ] hTPO _1-153 protein of the present invention has the amino acid sequence of SEQ ID NO: 6.

The present invention also provides a gene having the nucleotide sequence of SEQ ID NO: 5 encoding the [Met- ¹ , Val ¹ ] hTPO _1-153 protein.

It is also an object of the present invention to provide an E. coli expression vector capable of expressing the hTPO protein from the P _L promoter of lambda phage.

Specifically, the present invention provides an E. coli expression vector pLex404n capable of producing the [Met- ¹ , Val ¹ ] hTPO _1-153 protein using an expression cassette consisting of a P _L promoter of lambda phage and an asp A transcription termination codon.

In addition, an object of the present invention is to provide a transformant E. coli transformed with the expression vector pLex404n. The present invention provides a transformant of Escherichia coli GI724 strain (Accession No .: KFCC-10958).

In addition, the present invention is a method for producing a protein having human thrombopoietin activity by culturing the E. coli transformant to induce the expression of [Met ^-1 , Val ¹ ] hTPO _1-153 protein and then disrupting E. coli The purpose is to provide.

Figure 1 shows the genes of the amino terminal protein of hTPO ([Met- ¹ , Val ¹ ] hTPO _1-153 protein) and its amino acid sequence inferred therefrom.

Figure 2 shows the preparation of the plasmid vector pBlue404 containing the entire gene of human thrombopoietin (hTPO).

Figure 3 shows the manufacturing process of E. coli expression vector pLex404n comprising the gene of the [Met- ¹ , Val ¹ ] hTPO _1-153 protein of the present invention.

Figure 4 shows the results confirmed by agarose gel electrophoresis of the expression of the Escherichia coli pLex404n by cutting the restriction enzyme hTPO gene insertion and the presence of a single restriction enzyme recognition site.

Lane M: 1 kb molecular weight marker; Lane 1: Bam HI; Lane 2: Afl III,

Lane 3: Pst I and Hind III; Lane 4: Pst I and Kpn I

Figure 5 shows the expression of the hTPO protein with tryptophan in the E. coli strain containing the expression vector pLex404n of the present invention and analyzed the time-expressed hTPO protein by SDS-PAGE (A) and Western blot (B) The results are shown.

Lane 1: molecular weight marker; Lanes 2-4: Cell disruption solution after 0, 2, 4 hours after expression induction

The present invention comprises the amino acid sequence of amino acids No. 1 to 153 which is the site involved in hTPO activity of its amino terminus in mature form hTPO ₃₃₂ from which the signal peptide sequence of human thrombopoietin has been removed, wherein amino acid serine 1 A new hTPO protein (hereinafter abbreviated as [Met- ¹ , Val ¹ ] hTPO _1-153 protein) introduced with amino acid No. 1 and substituted with this valine is provided and its gene (see FIG. 1).

First, in order to obtain a gene of the [Met- ¹ , Val ¹ ] hTPO _1-153 protein, the Korean patent application No. 96- filed on March 6, 1997, the entire hTPO gene from the cDNA library of human fetal hepatocytes The plasmid vector pBlue404 containing the hTPO gene was cloned by the method of Reference Example 1 and Reference Example 2 carried out in No. 7512 (Name of the Invention: Method for Producing hTPO Using Insect Cells). [Met- ¹ , Val ¹ ] hTPO Used to clone _1-153 genes (see FIG. 2).

REFERENCE EXAMPLE 1 cDNA Preparation of hTPO Whole Genes from cDNA Library of Human Fetal Hepatocytes

CDNA library (clontech, cat # HL1064b) in which cDNA prepared by extracting mRNA from 26 week-old human fetal liver tissue was inserted into λgtll phage DNA was used to clone hTPO gene.

(Step 1) Titer determination of λ phage

Escherichia coli Y1090r- was used as a strain for infecting λgtll phage. A single colony of Escherichia coli Y1090r ⁻ was inoculated using LB medium without MgSO ₄ (10 g of bactotriptone per liter of medium, 5 g of yeast extract, 10 g of NaCI) and incubated at 37 ° C. until the absorbance at 600 nm reached 2.0. The fiji was diluted to 1: 250000 with 1 × lambda dilution buffer, and the dilutions were added to 200 μ1 of the culture medium in which the E. coli was incubated for 15 hours at 2 μ1, 5 μ1, 10 μ1, and 0 μ1 (control). . This was incubated for 15 minutes with shaking incubator again at 37 ° C, and then mixed homogeneously by adding ₁₀ mM MgSO ₄ and LB softtop agar medium containing 0.7% agar to each of the four test tubes, and adding LB solid with ₁₀ mM MgSO _4. Plated to media. Next, the number of plaques generated by incubating at 37 ° C. for 7 hours was counted to determine titers of λgtll phage representing 4.58 × 10 ¹⁰ plaque forming units (PFU) per ml of stock solution.

(Step 2) DNA Separation from λgtll Phage

A single colony of Escherichia coli Y1090r- was inoculated in 3 ml LB medium containing ₁₀ mM MgSO ₄ and 0.2% maltose and incubated at 37 ° C. for 18 hours. , Medium containing ₂ g of MgCl ₂ ) and cultured at 37 ° C. until the absorbance at 600 nm of the culture became 0.6. 30 μl of λgtll phage stock solution was inoculated into the culture solution, and cultured again at 37 ° C. for 6 to 10 hours until the culture became clear.

2 ml of chloroform was added thereto, followed by shaking culture for 15 minutes to obtain a phage pulverized product in solution. The pulverized product was centrifuged (Sovall SS34) for 10 minutes at 7000 rpm at 4 ° C. to obtain a supernatant, and added to an equal volume of 20% polyethyleneglycol 8000 / 2.0M NaCl for 1 hour on ice. This was again centrifuged at 6000 rpm for 10 minutes at 4 ° C. to obtain a precipitate of λgtll phage, which was dissolved in 6 ml of TM buffer (20 mM Tris-Cl (pH8.0), 20 mM MgCl ₂ ) and added with the same amount of chloroform. Mix well, centrifuge at 5,000 rpm at 4 ° C for 10 minutes, take supernatant, load into DEAE-cellulose column (1.5 X 5 cm, Bio-Rad.) Fully equilibrated with 3 ml TM buffer and buffer The phage solution was eluted with the solution. 3 ml of the initial eluate was removed, and 2 ml of 5M NaCl and 10 ml of ice-cold 100% ispropanol were added to 13 ml of the collected eluate, followed by standing at −20 ° C. for 15 minutes. It was centrifuged at 5,000 rpm for 10 minutes at 4 ° C. (Sorvall SS34) to obtain a precipitate thereof, which was completely dissolved in 0.8 ml TE buffer (pH 8.0) once with the same amount of phenol, phenol: chloroform: isoamyl alcohol ( 25: 24: 1) twice, and finally extracted once with chloroform, 1/10 volume of 3M sodium acetate (pH 6.0) and 2 volumes of ice-cold 100% ethanol were added. DNA pellets thus obtained were washed with 70% ethanol, air dried and then dissolved in 100 µl TE buffer (pH 8.0) and stored at 4 ° C.

(Step 3) Screening of hTPO gene using polymerase chain reaction (PCR)

Carboxylic transcription transcription frame of the oligonucleotide of SEQ ID NO: 1 having the base sequence comprising the leader sequence and amino terminal sequence of the TPO gene and the DNA of the phage library isolated by the above method as a template (open A polymerase chain reaction (PCR) was performed using oligonucleotide of SEQ ID NO: 2 having a base sequence including a reading frame (ORF) region and a stop codon as a primer. Specifically, each primer was dissolved by 10 pmol per μl, adjusted to the range of 100 pmol-150 pmol, and 1 unit of Taq polymerase was added to proceed the PCR reaction to the final 20 μl volume. PCR reactions include primary denaturation; 94 degreeC 5 minutes, primer conjugation; 58 degreeC 2 minutes, enzyme extension reaction; 72 ° C. 1 minute 20 seconds, denatured; The process was repeated 30 times at 94 ° C. for 1 minute, and then the final enzyme reaction was performed at 72 ° C. for 5 minutes to terminate the reaction, and 1% agarose gel electrophoresis was performed. In addition, the DNA band corresponding to the size including the cDNA sequence of the TPO gene was found, and then the band corresponding to 1079 bp including the cDNA sequence of the hTPO gene was amplified again. Specifically, the PCR was performed under the following reaction conditions by adding primers to the primary PCR reaction product under the same conditions as described above. PCR reactions include primary denaturation; 94 ° C., 5 minutes, primer conjugation; 58 ° C., 2 minutes, enzyme extension reaction; 72 ° C., 80 seconds, denaturation; The process of 94 ° C. 1 minute was repeated 30 times, and the final enzyme reaction was completed by proceeding to 72 ° C. 5 minutes.

Reference Example 2 Preparation of the plasmid vector pBlue404 containing the hTPO gene

In order to clone the hTPO gene amplified by the above method, 250 µl was taken from the test tube containing it, extracted twice with phenol, and an equal amount of 100% ethanol was added, followed by precipitation. This was completely dissolved in 100 μl secondary distilled water, and 33 μl of this was subjected to 1% agarose gel electrophoresis. Next, a DNA band of a 1079 bp hTPO gene was cut with a razor and eluted using a JinClean II elution kit, which was finally dissolved in secondary distilled water to 50 μl. 30 μl of this was taken and the hTPO gene was completely cleaved by reaction with restriction enzymes Xba I and Hind III at 37 ° C. for 5 hours.

In addition, the plasmid vector pBluescriptII SK (+) (Statagene) was digested with restriction enzymes Xba I and Hind III to separate 2979 bp DNA fragment by 1% agarose gel electrophoresis to separate only the corresponding band as described above, and then to the TPO. Together with the gene DNA fragment, it was dissolved in 35 μl of secondary distilled water. In order to link the cDNA PCR product of the TPO gene with the plasmid vector pBluecriptIISK (+), they were mixed in a molar ratio of 1: 1 and reacted at 16 ° C. for 10 hours using a T4 DNA ligase. E. coli XL1 blue (Stratagene) strain was transformed with this reaction solution to obtain E. coli transformants (Hanahan, et al., DNA Cloning, Vo1.1: A Practical Approach IRL Press, 1985, p109). 135). The plasmid vector containing the hTPO gene thus prepared was named pBlue404, which was subjected to single and double cleavage using restriction enzymes Xba I, Hind III, Bam HI, Kpn I, Pst I and Hind III, followed by 1% agar. Oz gel electrophoresis was performed to confirm that the hTPO gene was correctly inserted.

Finally, Sanger's didioxy nucleotide sequencing method was performed to confirm that the hTPO gene cloned in the present invention was an hTPO gene having the same base sequence as the known sequence.

The present invention uses the plasmid vector pBlue404 including the entire hTPO gene as a template for PCR amplification and obtains the gene encoding the [Met- ¹ , Val ¹ ] hTPO _1-153 protein, and the bases of SEQ ID NO: 3 and SEQ ID NO: 4 Polymerase chain reaction (PCR) is performed using oligonucleotides having sequences as primers. In this case, the primer of SEQ ID NO: 3 has the nucleotide sequence of the Kpn I restriction enzyme recognition site and the amino terminal region of hTPO, and the primer of SEQ ID NO: 4 has the nucleotide sequence of the carboxy terminal region of the [Met- ¹ , Val ¹ ] hTPO _1-153 protein. , Protein translation stop sequence and Nsi I restriction enzyme recognition site. As a result, a 470 bp DNA fragment which is a gene encoding the [Met- ¹ , Val ¹ ] hTPO _1-153 protein of the present invention is obtained.

The present invention also provides an expression vector capable of producing [Met- ¹ , Val ¹ ] hTPO _1-153 protein. Specifically, the present invention cuts the 470bp DNA fragment which is the gene encoding the [Met- ¹ , Val ¹ ] hTPO _1-153 protein with Kpn I and Nsi I restriction enzymes to separate the 462bp fragment, and then uses the P of lambda phage. E. coli expression vector pLex404n of the present invention was prepared by introducing into an existing plasmid vector pLex (Invitrogen) comprising an expression cassette consisting of an _L promoter and an asp A transcription termination codon (see FIGS. 3 and 4).

Gene base sequence was analyzed using the expression vector pLex404n (Sanger, PNAS 74, 5463, 1977) using a nearly dideoxy method to encode a complete [Met- ¹ , Val ¹ ] hTPO _1-153 gene sequence. Confirm that it exists.

The expression vector of the present invention uses a P _L promoter of lambda phage, which is more than 3-5 times higher in expression efficiency of protein expression and can be produced in large quantities than hTPO, compared with the pTac promoter or Pac promoter of the present invention. It is very useful for expressing the same unstable foreign protein.

In the present invention, as a result of attempting intracellular expression of hTPO protein using several kinds of promoters, the lambda phage P _L promoter and the asp A transcriptional sequence are included, and are composed of the ampicillin resistance gene and the ColE1 replication start site (ori), and the plasmid the small size and high expression efficiency of the expression vector pLex (Invitrogen, Inc.) are pTac control expression of the protein than the expression vector pKK223-3 (4.5 kb) of the promoter is significantly superior to the 2.9 kb of, [Met ^-1, Val ¹ ] found to be useful for producing hTPO _1-153 protein.

The present invention transforms E. coli with the expression vector pLex404n to obtain E. coli transformants. Specifically, the E. coli GI724 strain inhibits the expression of the cI inhibitor by adding tryptophan, which is useful for inducing the expression of the [Met- ¹ , Val ¹ ] hTPO _1-153 protein. The present invention is directed to the E. coli transformant GI724 / pLex404n. It was deposited with the Korean spawn association on March 12, 2017 (accession number: KFCC-10958).

In addition, the present invention is to culture the E. coli transformant to add tryptophan as an expression inducer (inducer) and then induce the expression of the protein and centrifuged by crushing it from E. coli crushing solution obtained from [Met ^-1 , Val ¹ ] Prepare _{hTPO 1-153} protein.

In addition, the present invention is analyzed by [Set- ¹ , Val ¹ ] hTPO _1-153 protein produced through the above process by performing SDS-polyacrylamide gel electrophoresis and Western blot (see Fig. 5). As a result, [Met- ¹ , Val ¹ ] hTPO _1-153 protein showed the highest expression level after 4 hours of induction of expression, density measuring device (Pharmacia) and Bradford protein quantitative analysis (Bio-Rad) As a result of quantifying the human thrombopoietin, it is confirmed that the expression in the amount of about 15mg / ℓ-25mg / ℓ.

Hereinafter, the present invention will be described in detail by way of examples.

The following examples illustrate the present invention in detail, and the content of the present invention is not limited by the examples.

Example 1 Preparation of Escherichia Coli Expression Vector pLex404n

To prepare an expression vector capable of producing hTPO ([Met- ¹ , Val ¹ ] hTPO _1-153 ) protein with thrombopoietin activity in which Escherichia coli is added methionine and amino acid number 1 is substituted with valine, Approximately 500 ng (2 μl) of plasmid vector pLex (Invitrogen) was added 2 μl of Nsi I (NEB, 10 units / μl) restriction enzyme, 23 μl of secondary distilled water, and 3 μl of 10X buffer solution (NEB). The reaction was completely cleaved for 3 hours at. Then, 1% agarose gel electrophoresis was performed to separate 2886 bp DNA fragments using the Geneclean II DNA elution kit (The Geneclean II, Bio 101) and to completely dissolve the expression vector DNA in 30 μl distilled water. . This was digested with Nsi I restriction enzyme and then mixed with 25 µl of the expression vector pLex DNA solution and 2 µl of Kpn I restriction enzyme (NEB, 20 units / µl) and 3 µl of 10X enzyme buffer solution (NEB) at 37 ° C. Reaction was carried out for 2 hours. Thus, 2803 bp expression vector pLex DNA fragments digested with Kpn I and Nsi I restriction enzymes were obtained.

In addition, in order to obtain a gene encoding the [Met- ¹ , Val ¹ ] hTPO _1-153 proteins, the oligonucleotide of SEQ ID NO: 3 and SEQ ID NO: 4 as a primer and the cloning vector pBlue404 of Reference Example 2 as a template The gene was amplified by a polymerase chain reaction (PCR). PCR amplification reaction was to dissolve each primer by 10 pmol per μl, adjust the volume in the range of 100 pmol-150 pmol, add 1 unit of Taq polymerase, add 1-3 μl of cloning vector pBlue404 template (1 to 3 ng) The final volume was 20 μl. Reaction conditions include primary denaturation; 94 ° C. 5 minutes, denaturation; 94 ° C. 1 min, primer conjugation; 58 ° C. 2 min, enzyme extension reaction; The reaction was terminated by repeating the above procedure 30 times at 72 ° C. for 1 minute 30 seconds and performing final enzymatic reaction at 72 ° C. for 5 minutes.

Next, 1% agarose gel electrophoresis was performed to separate the 470bp PCR product using the JinClean II DNA elution kit, and cleaved with Kpn I and Nsi I restriction enzymes under the same conditions as above to obtain 462bp DNA fragments. Thus, the PCR product of the gene encoding the [Met- ¹ , Val ¹ ] hTPO ₁₅₃ protein was cut into 462 bp DNA fragment digested with Kpn I and Nsi I restriction enzymes, and expressed with the same Kpn I and Nsi I restriction enzymes. The vector pLex404 was ligated at 16 ° C. for 18 hours by adding 1 μl of T4 DNA ligase, and then transformed into E. coli GI724 strain (Invitrogen) to obtain an E. coli transformant.

The genotype (Genotype) of the E. coli GI724 strain is F ⁻ , λ ⁻ , lacI ^q , lacP8, ampC :: P _trp cI, and when the tryptophan is added to the medium, the expression of the cI inhibitor is suppressed from the P _L promoter of expression vector pLex. Expression of hTPO protein is induced. Through the transformation, an expression vector having a gene encoding [Met- ¹ , Val ¹ ] hTPO _1-153 protein was inserted into the expression vector pLex, named pLex404n, and designated as Bam HI, Afl III, Bgl I, Hind III, It was confirmed that the gene was correctly inserted by cutting with Pst I restriction enzyme, and the base sequence of the [Met- ¹ , Val ¹ ] hTPO _1-153 gene was confirmed by the _{Sangdiger didioxy} sequencing method (Fig. 4).

Escherichia coli transformants obtained by transforming Escherichia coli GI724 strain with the expression vector pLex404n of the present invention were deposited on March 12, 1996 to the Korean spawn association, an international depository institution (Accession Number: KFCC-10958).

Example 2 Mass Expression and Analysis of Human Thrombopoietin in Escherichia Coli Containing an Expression Vector pLex404n

To mass produce hTPO protein with human thrombopoietin activity, the E. coli GI724 / pLex404n strain transformed with the expression vector pLex404n capable of producing hTPO was transferred to Na ₂ HPO ₄ (6 g / L), KH ₂ PO ₄ (3 g). / l), NaCl (5g / l), NH ₄ Cl (10g / l), MgCl _2. 6H ₂ O (0.233g / l), Glycerol (10ml), casaminoacid (20g / l), Ampicillin (100 mg / L), inoculated in 3 ml of the medium having a composition of pH 7.4 and incubated at 30 ℃ for 18 hours. The culture was washed with Na ₂ HPO ₄ (6 g / l), KH ₂ PO ₄ (3 g / l), NaCl (5 g / l), NH ₄ Cl (10 g / l), MgCl _2. 6H ₂ O (0.233 g / l). ), 100 ml of medium having a composition of glucose (5 g / L), casamino acid (2 g / L), ampicillin (100 mg / L) and pH7.4 was inoculated to have an initial E. coli concentration of 0.1 (OD). When the E. coli concentration reached 0.5 (OD) while shaking culture at 30 ° C. and 250 rpm, L-tryptophan was added to a final concentration of 100 mg / l and the culture temperature was raised to 37 ° C. to induce the expression of hTPO. After inducing the expression, 1 ml of sample was taken every hour, centrifuged at 15000 rpm for 3 minutes, and the supernatant was discarded to collect E. coli. The collected E. coli samples were resuspended by the addition of phosphate buffer solution (PBS, pH 7.4) so that the E. coli concentration was the same.

In order to extract the hTPO expressed in E. coli, E. coli was crushed by applying 10 times of ultrasonic waves to each sample for 20 seconds using an ultrasonic mill. The sample pulverized by ultrasound was centrifuged at 15000 rpm and 4 ° C. for 20 minutes, and then the supernatant was discarded and 100 μl of distilled water was added to prepare a sample for human thrombopoietin analysis.

10 μl of each sample and 10 μl of reducing buffer solution (2 ×) for SDS-PAGE were mixed and treated at 100 ° C. for 5 minutes, followed by SDS-PAGE analysis. Lane 1 in Fig. 5 (A) is the standard molecular weight marker of the protein (BioRad, SDS-PAGE Standard, Cat.No. 161-0318) from above 203,000, 118,000, 86,000, 51,600, 34,100, 29,000, 19,200, 7,500 Dalton size, lanes 2-4 are samples that induced expression for 0, 2 and 4 hours, respectively. 5 (b) is a Western blot result of the same sample as above. At this time, the primary antibody was used by diluting 1: 1000 goat primary antibody (RD Corporation Cat. No. AB-288-NA) prepared for hTPO ₃₃₂ with phosphate buffer solution, and the secondary antibody was alkaline phosphata A secondary antibody (Cig., Cat. No. A4187, Sigma Co., Ltd.) bound with an akaline (Alkaline phosphatase) was used.

As a result, a dark band (arrow) appears at a position corresponding to about 18,000 Daltons from 2 hours after the expression of the hTPO _1-153 protein was induced [Met ^-1 , Val ¹ ] hTPO _1-153 The protein was expressed (see Figure 5 (a)) and confirmed that the protein was hTPO by Western blot analysis (see Figure 5 (b)). [Met ^-1 , Val ¹ ] hTPO _1-153 protein showed the highest expression level after 4 hours of induction of expression. The human _thrombin was measured by the density meter (Pharmacia) and the Bradford protein quantitative assay (Bio-Rad). As a result of quantifying bopoietin, the expression level of about 15 mg / L-25 mg / L was confirmed.

As described above, the expression vector of the present invention can regulate the expression of proteins within a short time by using the P _L promoter of lambda phage, which is very useful for mass production of human thrombopoietin by genetic engineering methods. Producing human thrombopoietin by the method is expected to effectively treat thrombocytopenia and the like in cancer patients and bone marrow transplant patients.

(Sequence list)

SEQ ID NO: 1

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 1]

5'-GGTCTAGAAT GGAGCTGACT GAATTGC-3 '

(Sequence list)

SEQ ID NO: 2

Sequence length: 30

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 2]

5'-TTAAGCTTAT CACCCTTCCT GAGACAGATT-3 '

(Sequence list)

SEQ ID NO: 3

Sequence length: 24

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 3]

5'-GGTACCGGCTCCTCCTGCTTGTGA-3 '

(Sequence list)

SEQ ID NO: 4

Sequence length: 29

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 4]

5'-CAATGCATCACCTGACGCAGAGGGTGGAC-3 '

(Sequence list)

SEQ ID NO: 5

Sequence length: 537

Type of sequence: nucleic acid

Number of chains: 2 prints

Form: Straight

Type of sequence: DNA

[SEQ ID NO: 5]

AAGAAGGGCT TTATTTGCAT ACATTCAATC AATTGTTATC TAAGGAAATA CTTACATATG 60

GTACCGGCTC CTCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT 120

GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 180

CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 240

CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 300

CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 360

CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 420

CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 480

TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGGT GAATCTAGAG TCGACCT 537

(Sequence list)

SEQ ID NO: 6

Length of sequence: 154

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

[SEQ ID NO: 6]

Met Val Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys 15

Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys 30

Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val 45

Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys 60

Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val 75

Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu 90

Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu 105

Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr 120

Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu 135

Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr 150

Leu Cys Val Arg 154

Claims (6)

A hTPO protein having the amino acid sequence of SEQ ID NO: 6 containing the 153 amino acid sequence of the amino terminal region in which amino acid 1 of human thrombopoietin (hTPO) is substituted with valine, and methionine added to the amino terminal. An hTPO gene having the nucleotide sequence of SEQ ID NO: 5 encoding the hTPO protein of claim 1. An E. coli expression vector pLex404n capable of producing the hTPO protein of claim 1 using an expression cassette consisting of a lambda phage P _L promoter and an asp A transcription termination codon. E. coli transformants transformed with the expression vector pLex404n of claim 3. The transformant of claim 1, wherein the transformant of Escherichia coli GI724 strain (Accession No .: KFCC-10958). A method of preparing a protein having human thrombopoietin activity by culturing the E. coli transformant of claim 5 to induce the expression of the hTPO protein of claim 1 and then disrupting E. coli.