JPH0779780A - Production of recombinant human adf - Google Patents

Abstract

PURPOSE:To obtain a recomblnant human ADF having free radical elimination capability and useful e.g. for the treatment of inflammation, radiation injury, etc., in high efficiency by transforming an E.col1 with a plasmid containing a gene coding human ADF, culturing the transformed E.coli and collecting the product. CONSTITUTION:A gene coding a human ADF which is an interleukin-2 receptor inducing factor existing in the supernatant of ATL-2 cell is bonded with a promoter region, a ribosome bonding region, a translation initiation codon, a human ADF gene region, a translation termination codon and a terminator region in the order to construct a recombinant expression plasmid. The plasmid is introduced into a host such as E.coli and the obtained transformant is cultured in a medium to obtain the recombinant human ADF on an industrial scale in a mass. The recombinant human ADF is expected to be usable as an agent for the treatment of diseases accompanying the injury caused by the generation of free radicals such as inflammation and radiation injury.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for direct expression and production of human ADF polypeptide. Human ADF has a free radical scavenging ability and a refolding activity of a protein denatured and inactivated by free radicals. Therefore, it is useful as a therapeutic drug for inflammation, radiation injury, etc., which is accompanied by biological damage due to generation of free radicals It is a substance.

[0002]

Human ADF was reported by Tagaya et al. In 1985 as an IL-2 receptor-inducing factor present in the supernatant of ATL-2 cells (J. Immunol. Vo.
l. 134, 1623-1630, (1985), J. Am.
Mol. Cell. Immunol. Vol. 2, 17
-26 (1985)), its amino acid sequence and DNA sequence have already been determined, and production in E. coli has also been reported (JP-A-1-85097). Further, subsequent analysis revealed that human ADF has an amino acid sequence similar to thioredoxin, which is a redox protein widely existing from Escherichia coli to mammals, and has thioredoxin activity. Therefore, depending on the researcher, this substance may be called thioredoxin, but in the present invention, it will be unified under the name of human ADF as in the past. As described above, human ADF can eliminate active oxygen such as hydrogen peroxide and free radicals (hereinafter, collectively referred to as free radicals), and further, denatures and deactivates proteins denatured by free radicals. It can also be reactivated by folding. Therefore, inflammation accompanied by biological damage due to the generation of free radicals,
Utilization as a therapeutic agent for radiation disorders and the like is under consideration (Japanese Patent Laid-Open No. 3-204818).

Conventionally, in order to obtain the above-mentioned human ADF, normal human T cells isolated from human peripheral blood, etc. were stimulated with mitogen, or from a T cell line transformed with adult T leukemia virus (HTLV). A method for producing it has been adopted (J. Immunol. Vol. 140,
2614-2620 (1988)). However, in these methods, the amount of human ADF produced is low, and when mitogen is used, harmful mitogen is mixed and it is difficult to remove it, and in addition, serum components such as fetal bovine serum are added to the cell culture. Since it was necessary to add to the medium, these added proteins and human ADF could not be sufficiently separated, and there were many problems when used as a drug.

In order to overcome the above-mentioned problems, recently, recombinant human ADF using Escherichia coli by Yodoi et al.
Has been developed (see Japanese Patent Laid-Open No. 1-85097). In this method, in order to highly express human ADF in Escherichia coli, a gene encoding the N-terminal partial peptide of human interleukin-2 (IL-2) was first connected to the upstream of the gene encoding human ADF. A plasmid (pTfADF) that expresses a fusion protein of IL-2 partial peptide and human ADF (ΔIL-2-human ADF)
-2) was constructed. When E. coli transformed with pTfADF-2 was cultured, 2 mg of ΔIL-2-human ADF was expressed and accumulated per 100 mL of the culture solution.

However, since ΔIL-2-human ADF contains a partial peptide of human IL-2, human IL-2 is digested with proteolytic enzymes such as clostripain and kallikrein.
It was necessary to cleave the connection between the -2 partial peptide and human ADF. Purification was performed by anion chromatography and reverse phase chromatography to remove contaminating proteins such as the cleaved human IL-2 partial peptide, and the purified human ADF obtained was about 100 μg per 100 mL of E. coli culture solution. This is the ΔIL accumulated in E. coli.
-2-Only 5% of human ADF. Furthermore, this method requires a proteolytic enzyme equivalent to 10% (w / w) of the fusion protein, and the enzyme used is often expensive and difficult to obtain in large quantities. Therefore, many problems remain in the industrial mass production of recombinant human ADF by this method, such as yield, economic efficiency, and supply of materials.
In order to overcome this problem, a method for producing efficiently by a direct expression system, not as a fusion protein with a human IL-2 partial peptide, has been required.

[0006]

The object of the present invention is a method for producing human ADF, which is a substance useful as a therapeutic agent for inflammation, radiation injury, etc., which is associated with biological damage caused by the generation of free radicals, and is industrially mass-produced. To provide a method that can be adapted to. Specifically, it is a provision of a direct expression production method of recombinant human ADF.

[0007]

As a result of intensive studies to solve the above problems, the present inventor has found that the target recombinant human ADF can be efficiently produced in a large amount by the following method. I was able to complete the invention. That is, the present invention is a recombinant human being characterized by producing a desired human ADF polypeptide by culturing Escherichia coli transformed with a plasmid having a gene encoding human ADF, and obtaining the polypeptide. It is a direct expression production method of ADF polypeptide. Hereinafter, the present invention will be described in detail.

[0008] The human ADF of the present invention usually refers to one having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, but is not limited thereto. That is, it may be a polypeptide in which a methionine residue is added to the N-terminus (polypeptide described in SEQ ID NO: 2 in the sequence listing). Furthermore, a polypeptide in which the amino acid sequence has been replaced by a chemical modification or base substitution method, a polypeptide in which a part of the amino acid sequence is defective, a polypeptide in which an amino acid residue is inserted, or a sugar chain or the like is added to the side chain Human ADF even in the case of
As long as it has activity, it is included in the human ADF of the present invention. However, preferably N shown in SEQ ID NO: 1 of the sequence listing is used.
A polypeptide consisting of 104 amino acids starting from the terminal valine or a polypeptide consisting of 105 amino acids with methionine shown in SEQ ID NO: 2 in the sequence listing added to the N-terminus is preferable.

The human ADF gene usually refers to the gene described in SEQ ID NO: 3 or 4 in the sequence listing, but is not limited thereto. That is, it may be a gene having base substitution, base insertion, or base deletion. However, 104 shown in SEQ ID NO: 3 or 4 of the sequence listing is preferable.
Gene encoding a polypeptide consisting of 10 or 105 amino acids is preferred. The gene described in SEQ ID NO: 4 in the sequence listing is a gene having a translation initiation codon at the 5'end of the gene described in SEQ ID NO: 3 in the sequence listing. As described above, in order to industrially produce a eukaryotic protein industrially using Escherichia coli, gene expression that accumulates a large amount of the target polypeptide only in E. coli, not as a fusion protein, as described above. Systems and gene expression vectors need to be constructed. That is, first, from the 5 ′ upstream, a promoter region, a ribosome binding region, a translation initiation codon,
An expression vector containing a gene region encoding the desired human ADF, a translation stop codon, and finally a terminator region is constructed.

In the present invention, the origin of the promoter does not matter. For example, E. coli trp promoter, tac promoter, trc promoter, la
The c promoter, and further, λPL promoter and λPR promoter of λ phage can be used.

The ribosome binding region is, for example, t of E. coli.
ribosome binding region of rpL, trpE, lacZ,
The ribosome binding region of the CII protein of lambda phage can be used. Alternatively, a chemically synthesized DNA sequence can be used. In the vector of the present invention, two or more ribosome binding regions may be linked, and these ribosome binding regions may have the same sequence or a combination of different sequences. There are no particular restrictions on the ligation mode of the ribosome binding region, but the distance between the two ribosome binding regions is preferably about 5 to 20 bases.
Adenine (A), thymine (T) between the ribosome binding region and the translation initiation codon in order to increase the gene expression efficiency
Rich in nucleotide sequence may be inserted, and human ADF may be added as long as the amino acid sequence of the produced polypeptide is not changed.
The first half of the gene may be changed to a nucleotide sequence rich in adenine (A) and thymine (T).

As a translation termination codon, the ocher (TA
A), opal (TGA), amber (TAG), preferably ocher (TAA), opal (TGA) are used, and in addition, in order to ensure translational termination of the target gene, 2 of TAA and / or TGA is used. Heavy stop codons may be placed. The transcription termination site is, for example, t of E. coli.
rpA terminator, rrnB terminator, re
A cA terminator or the like can be used. Also,
Generally, the copy number of the expression plasmid is preferably high, and the pUC system is more preferable as the replication origin than the pBR replication origin.

The constructed expression plasmid may be transformed into Escherichia coli as a host and expressed by a usual method. In the present invention, Escherichia coli is used as the expression cell, but a prokaryote or eukaryote other than E. coli may be used as the host. For reference, examples of prokaryotes include Bacillus subtilis. Examples of eukaryotic cells include yeast and CHO cells.

As a method for incorporating the expression vector into these E. coli, a known method may be used. For example, by treating cells in the logarithmic growth phase with 50 mM calcium chloride in ice for about 30 minutes, the structure of the cell wall of E. coli is changed,
Subsequently, about 10 minutes after injection of plasmid DNA, 30 ° C to 4
After heat treatment at 2 ° C for 2 minutes, add the medium to 30 ° C ~
The expression plasmid DNA can be incorporated into an organism by culturing at 37 ° C. for about 60 minutes. Alternatively, the expression vector may be introduced into a host such as Escherichia coli by electroporation.

Human ADF can be accumulated in the E. coli body by culturing the E. coli carrying the expression vector. As the medium, a known medium capable of culturing Escherichia coli may be used, and the culture conditions may be known conditions. In addition, the recombinant human A accumulated in the E. coli body
The DF may be obtained by a known method. For example, salting out, ultrafiltration, chromatofocusing, hydroxyapatite chromatography, hydrophobic chromatography, reverse phase chromatography, anion exchange chromatography, cation exchange chromatography, various affinity chromatography, gel filtration chromatography, etc. It may be purified by a combination of known methods.

Next, purified recombinant human ADF
May be used after being dissolved in distilled water, physiological saline or an appropriate buffer solution. Alternatively, a freeze-dried product may be used. Concentration is usually 0.01-100 mg / mL
, But is not limited to this range. Further, excipients such as mannitol and cyclodextrin, and stabilizers may be contained in the purified recombinant human ADF for use. The content of the recombinant human ADF is usually the formulation containing the recombinant human ADF 10.
0.1 to 100 parts by weight, preferably 10 parts by weight
~ 80 parts by weight. Of course, it is not limited to the above range. Hereinafter, the present invention will be described in more detail based on examples. The present invention is not limited to the embodiments.

[0017]

【Example】

(Example 1) Measurement of thioredoxin activity In order to measure and quantify the activity of human ADF, an insulin reducing activity measuring method (Biochemistry, Vol. 21,
6628-6633 (1982)) was used. The measurement principle is shown in (Reaction 1). When human ADF reduces insulin, hydrogen ions flow in the direction of the arrow, and as a result, NADPH is converted to NADP. NADPH to N
Since the conversion rate to ADP is proportional to the concentration of human ADF, 340 nm accompanying the conversion from NADPH to NADP
The human ADF can be quantified by obtaining the decrease rate of the absorbance. Incidentally, TxR is an abbreviation for thioredoxin reductase. (Reaction 1) NADPH → TxR → ADF (thioredoxin) → insulin

The measuring method is as follows. In a measurement cuvette of the spectrophotometer, add 0.1M Tris-HCl buffer, pH
7.5 (450 μl), 5 units / mL TxR (50
μl) and a solution containing human ADF (50 μl) are sequentially added, and finally 10 mg / mL insulin solution (50 μl)
Was added to start the reaction. Immediately after adding insulin, set the cuvette in the spectrophotometer and repeat every 3 minutes for 3 minutes.
Absorbance at 40 nm (A340) was recorded. The maximum value of change in absorbance per minute (ΔA340 / min) was defined as thioredoxin activity. Further, the specific activity was calculated using (Equation 1). (Equation 1) Specific activity (ΔA340 / min · mg) = ΔA
340 / min x 0.6 x (1 / 0.05) x (1 / sample concentration)

Table 1 below shows purified recombinant human A
It is the thioredoxin activity of DF. For quantification of recombinant human ADF expressed in cells or recombinant human ADF in the purification process, a calibration curve prepared based on this table was used. In addition, TxR is a known method from human placenta (Biochemistry, Vol. 21, 662).
8-6633 (1982)) was used.

[0020]

Table 1 Example of thioredoxin activity measurement of purified recombinant human ADF Amount of added ADF (μg) Thioredoxin activity (ΔA340 / min) 0 0.0049 0.5 0.1102 1.0 1.0 0.2134 2.0 0.0. 3743

(Example 2) Construction of human ADF direct expression plasmid DNA (pADFDE) In order to express human ADF in a large amount in Escherichia coli, a human ADF expression plasmid DNA was constructed by the following method (FIG. 1, FIG. 1). 2). First, from the transcription start point to human ADF
The DNA sequence extending to the N-terminus of was designed as shown in FIG. In addition, the following points were considered in order to increase the translation efficiency of human ADF mRNA. That is, optimization of the Shine-Dalgarno sequence (SD sequence) that is a ribosomal RNA binding site, A-Trich conversion of the nucleotide sequence between the SD sequence and the start codon (ATG), and A of the codon encoding the N-terminus of human ADF. -For Trich formation, and for the region away from the N-terminal, consider the frequency of codon usage in E. coli.
The A sequence was designed.

1 and 2 show a method for preparing a plasmid DNA for direct expression of human ADF (pADFDE). First, as shown in FIG. 1, an oligonucleotide (adapter 1, 2; adapter 1, 2,
3, 4) were synthesized. Next, SEQ ID NOS: 3 and 4 in the sequence listing
After cleaving the ΔIL2-human ADF fusion protein expression plasmid pTfADF-2 having the described human ADF cDNA sequence with the restriction enzymes ClaI and PstI, the above-mentioned mixture of adapters 1, 2, 3, 4 was added and ligated with T4 DNA ligase. To allow the plasmid pADFD
E (pBR) was constructed. The plasmid pTfADF
Escherichia coli having -2 has been deposited at the Institute of Microtechnology (currently Life Research Institute). The deposit number is BP-1885.

Furthermore, in order to amplify the ADF gene, the drive unit is a high copy number plasmid DNA p
We constructed pADFDE by replacing it with the UC system (Fig. 2).
After the construction, it was confirmed that pADFDE had the correct nucleotide sequence encoding human ADF using the DNA nucleotide sequencing method by the Sanger method.

The plasmid pAD thus obtained
Human ADF expression plasmid D, which FDE certainly aims for
Whether or not it was NA was confirmed as follows. First, E. coli strain H transformed with the plasmid pADFDE
After culturing B101 in 1 mL of LB medium at 31 ° C. overnight, the cells were ultrasonically disrupted and separated into a soluble fraction and an insoluble fraction by centrifugation. The insoluble fraction was resuspended in the same amount of distilled water. 1 μL of each fraction was separated by SDS-polyacrylamide gel electrophoresis, the protein band was stained by silver staining, and then the band was quantified by a densitometer. In addition, thioredoxin activity was measured using 5 μL of each fraction to quantify the recombinant human ADF accumulated in the cells. As shown in Table 2, the soluble fraction of the cells was 12 kD, which corresponds to the molecular weight of human ADF.
The expression of the protein a was recognized, and the thioredoxin activity was also confirmed depending on the expression level of the 12 kDa protein. Immunoblotting using an anti-human ADF antibody revealed that the 12 kDa protein was recognized by the anti-human ADF antibody. From the above,
By transforming E. coli with pADFDE, it was confirmed that recombinant human ADF was expressed in the cells. The expression level of recombinant human ADF reached about 30% of the soluble protein. The transformed Escherichia coli pADFDE / HB101 is Microorganism Research Institute No. 12606 (FERM P-12606).

[0025]

[Table 2] Table 2 Content of 12kDa protein in pADFDE / HB101 cells and thioredoxin activity 12kDa protein Thioredoxin activity (band area (relative value)) (ΔA340 / min) HB101 (control) soluble fraction 0.21 0.0315 Insoluble fraction 0.14 0.0082 pADFDE / HB101 soluble fraction 31.31 0.3526 insoluble fraction 0.18 0.0103

Example 3 Preparation of Recombinant Human ADF In order to examine the biochemical properties and physical properties of the recombinant human ADF, purified recombinant human ADF is required. Therefore, purified recombinant human ADF was obtained by the following method. First, E. coli HB101 (FERM transformed with the plasmid pADFDE obtained in Example 2)
P-12606) was added to 100 mL of LB medium,
The culture was carried out at 100 ° C. with shaking at 100 rpm overnight. Next, 100 mL of this E. coli culture solution was added to 2 L of LB medium,
After further shaking culture at 31 ° C. for 16 hours at 100 rpm, cells were collected by centrifugation (1000 × g, 15 minutes). The cells were suspended in 200 mL of 20 mM piperazine hydrochloric acid buffer (pH 6.0) and then ultrasonically disrupted (100
W, 30 minutes) to disrupt and centrifuge (10000
Xg, 15 minutes) to remove insoluble components was used as a crude Escherichia coli extract. The expression level of recombinant human ADF determined by measuring thioredoxin activity was 1 L of culture solution.
It was 500 mg per unit.

Purification of recombinant human ADF from E. coli crude extract was by anion exchange chromatography. That is, the E. coli crude extract was preliminarily equilibrated with 20 mM piperazine hydrochloric acid buffer (pH 6.0) on a Q sepharose column (diameter 10 cm × 40 cm, Pharmaci.
non-adsorbed components were eluted with the same buffer solution. Next, the components adsorbed on the column were eluted with a linear gradient of NaCl concentration of 0 to 100 mM. The flow rate is 5
It was set to 0 mL / min and the volume of the gradient was 5 L. Recombinant human ADF was collected in a fraction of about 50 mM NaCl.

The recovered recombinant human ADF was 100 mg per liter of the culture broth, and the yield was 100 times that of the conventional fusion protein method with IL-2 (JP-A-1-85097). Moreover, the recovery rate was 20%, which was improved to 4 times that of the conventional method. Next, when SDS polyacrylamide gel electrophoresis was performed, the purified recombinant human ADF showed an almost uniform band with a molecular weight of 12 kDa, and when quantified using a densitometer, the purity was determined to be 95% or more. It was Further, when the N-terminal amino acid sequence was determined using a peptide sequencer, it had the sequences described in SEQ ID NOs: 1 and 2 in the sequence listing. The recombinant human ADF thus obtained has two types of N-terminal structures because a part of the polypeptide (methionine type) described in SEQ ID NO: 2 in the sequence listing originally created in E. coli It is considered that the N-terminal methionine was cleaved by aminopeptidase in E. coli and converted into the polypeptide (valine type) described in SEQ ID NO: 1 in the sequence listing. The thioredoxin activity of the recombinant human ADF thus obtained was 80Δ340 / min per mg. Furthermore, the refolding activity of denatured proteins (Biochem. J., V
ol. 275, 349-353 (1991)) is 0.0000017 when the recombinant human ADF is 3 μM.
It was μmol / min. Depending on the culture conditions, the ratio of N-terminal structure of recombinant human ADF was methionine type:
The methionine-type and valine-type specific activities are considered to be the same, since there was no difference in the thioredoxin activity and the refolding activity although the valine-type varied between 1: 9 and 9: 1. .

(Example 4) Large-scale preparation of high-purity recombinant human ADF In order to carry out a drug efficacy test of human ADF using animals such as mice, rats, rabbits, dogs and monkeys, highly purified recombinant human ADF was used. You need to get a lot. Therefore, as a result of diligent studies by the present inventor, a method for obtaining a large amount of highly purified recombinant human ADF by combining high-density Escherichia coli culture and a multistep purification process was found. Table 3 shows a summary of the purification results.

[0030]

Table 3 Summary of large-scale preparation of high-purity recombinant human ADF Step yield Step yield Yield (cell crude extract) 400 g Butylspharose 133 33% 33% Sephadex G-25 117 88 88 29 CM Sepharose FF 93 79 23 CM Sepharose FF (concentrated) 83 89 21 Sepharose S-100HR 60 72 15

Each step and yield will be described below based on Table 3. First, E. coli HB101 (FERM P-12606) transformed with the plasmid pADFDE was treated with 1 L of L.
After adding to the B medium, the cells were cultured at 31 ° C. overnight with shaking at 100 rpm. Next, 1 L of this E. coli culture solution was mixed with 20 L of M-9 casamino acid medium (0.6% sodium hydrogen phosphate, 0.3% sodium dihydrogen phosphate, 0.05% sodium chloride, 0.1% chloride). Ammonium, 0.05% magnesium sulfate, 0.00147% calcium chloride,
0.2% glucose, 0.2% casamino acid, 0.02%
L-leucine, 0.02% L-proline, 0.0002
% Thiamine hydrochloride, 100 μg / ml ampicillin, 2
5 μg / ml streptomycin, pH 7.4), high-density culture was carried out at 31 ° C. for 16 hours using a 30 L fermentor, and then the entire amount of the culture solution was added to 180 L of M-9 casamino acid medium, High-density culture was performed using a 300 L fermentor at 31 ° C. for 16 hours. After culturing, the cells were collected using a continuous centrifuge and suspended in 50 L of 10 mM sodium phosphate buffer, pH 7.4. The cell suspension was disrupted using a continuous cell disruption device, and then insoluble components were removed using a continuous centrifuge to obtain a crude cell extract. The recombinant human ADF in the crude cell extract was quantified by the thioredoxin activity assay method to calculate the total amount of recombinant human ADF.
g, total 400 g. The expression level of recombinant human ADF per culture medium was 4 times that in the shaking culture.

Recombinant human ADF was purified from the crude cell extract using the following method. First, ammonium sulfate was added to the crude cell extract to make it 45% saturated, and then 50
% Saturated ammonium sulfate, 50 mM sodium acetate, p
It was added to and adsorbed on a butyl sepharose column (diameter 25.2 cm × 15 cm, 7.5 L) equilibrated with H5.5. After elution of non-adsorbed components with the same buffer, 50 to 0
Linear gradient of% saturated ammonium sulfate (gradient volume 60
When the adsorbed component was eluted with L at a flow rate of 0.5 L / min), recombinant human ADF was recovered in a fraction of ammonium sulfate of about 20% saturation. In consideration of the throughput of the column, the treatment amount per treatment was set to 1/6 of the total amount of the crude bacterial cell extract, and the same operation was repeated 6 times to treat the whole amount. As shown in Table 3, the yield at the end of this step was 133 g, and the yield was 33%.

Next, 20 mM sodium acetate, pH 4.
After adding 1/18 volume of the above recombinant human ADF fraction to a Sephadex G-25 column (diameter 25.2 cm × 25 cm, 12.5 L) equilibrated with 5.
The buffer solution was replaced by elution with the same buffer solution (flow rate 0.5 L / min). The same operation was repeated 18 times to treat the whole amount. As shown in Table 3, the yield at the end of this step was 117 g, and the yield was 29%.

A 1/6 volume of the fraction from the previous step was previously set to 20 m.
C equilibrated with M sodium acetate buffer, pH 4.5
M Sepharose Fast Flow Column (Diameter 25.2
cm × 15 cm, 7.5 L), and then the non-adsorbed components were eluted with the same buffer solution. Subsequently, a linear gradient of 0 to 0.5 M sodium chloride (gradient volume 75 L, flow rate 0.5
L / min), the adsorbed components were eluted,
Recombinant human ADF was collected in a fraction of sodium chloride of about 0.2M. The whole amount was processed by repeating the same operation 6 times. As shown in Table 3, the yield at the end of this step was 93 g, and the yield was 23%.

Next, concentration was performed using a CM sepharose fast flow column (diameter 11.3 cm × 3 cm, 0.3 L). The method was as follows: 1/12 of the fraction of the previous step was added to a column equilibrated with 20 mM sodium acetate buffer, pH 4.5, and the non-adsorbed components were eluted with the same buffer, followed by 0.2 M acetic acid. The adsorbed component was concentrated and recovered with sodium, pH 6.0 (flow rate 0.3 L / min). The same operation was repeated 12 times in order to treat the whole amount. As shown in Table 3, the yield at the end of this step was 83 g, and the yield was 21%.

Finally, gel filtration chromatography was used to remove the buffer and trace impurities. That is, 0.
Sephacryl S-100HR column (diameter 10 cm x 64 c, equilibrated with 2 M ammonium acetate, pH 6.0).
m, 5 L), 1 / 24th of the fraction of the previous step was added, and elution was carried out with the same buffer (flow rate 20 ml / min). The same operation was repeated 24 times to treat the whole amount.
The yield of recombinant human ADF purified in this manner was 60 g, and the recovery rate was 15% (Table 3).

When the purified recombinant human ADF was analyzed by SDS polyacrylamide gel electrophoresis, it showed a uniform band with a molecular weight of 12 kDa, and a purity test with a densitometer confirmed that the purity was 99% or higher. Furthermore, the content of Escherichia coli-derived protein was determined by an ELISA method using an anti-Escherichia coli protein antibody.
It was below. In addition, the endotoxin content was determined using the Limulus assay kit and found to be 0.001 endotoxin unit / mg or less. Moreover, when the molecular weight was determined using an electrospray ionization mass spectrometer, it was in agreement with the molecular weight expected from the base sequence. That is, the molecular weights of the two types of polypeptides shown in SEQ ID NOS: 1 and 2 of the sequence listing were shown. When the N-terminal amino acid sequence was determined using a peptide sequencer, two types of sequences described in SEQ ID NOs: 1 and 2 in the sequence listing were confirmed. The thioredoxin activity was 80Δ34 per mg.
0 / min, the refolding activity of the denatured protein was 0.0000017μ when human ADFP was 3μM.
It was mol / min.

[0038]

EFFECTS OF THE INVENTION Recombinant humans are obtained by optimizing the SD sequence, converting the SD sequence to the N-terminal of the peptide by A-Trich, and establishing the direct expression production method of the present invention using pUC plasmid DNA. A dramatic increase in ADF expression efficiency and productivity have been achieved. Furthermore, it has become possible to obtain high-purity and large amounts of recombinant human ADF by combining high-density culture and multi-step purification process. Human ADF, which is a substance useful as a therapeutic agent for inflammation, radiation damage, etc., which is accompanied by biological damage due to the generation of free radicals, obtained by the present invention
It can be manufactured in large quantities and at low cost.

[0039]

[Sequence list]

SEQ ID NO: 1 Sequence length: 104 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Human Strain name: ATL-2 Sequence characteristics Characteristic symbol: active-site Location: 31..34 Method of characterizing: E 1 5 10 15 Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp Ala 16 20 25 30 Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly 32 35 40 45 Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys Tyr 48 50 55 60 Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp Val 64 65 70 75 80 Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe Lys 80 85 90 95 Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys Leu 96 100 Glu Ala Thr Ile Asn Glu Leu Val 104

SEQ ID NO: 2 Sequence length: 105 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Human Strain name: ATL-2 Sequence features Characteristic symbols: active-site Location: 32..35 Method of characterizing: E 1 5 10 15 Met Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp 16 20 25 30 Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys 32 35 40 45 Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys 48 50 55 60 Tyr Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp 64 65 70 75 80 Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe 80 85 90 95 Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys 96 100 105 Leu Glu Ala Thr Ile Asn Glu Leu Val 105

SEQ ID NO: 3 Sequence Length: 312 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: cDNA to mRNA Origin Organ Name: Human Strain Name: ATL-2 Sequence Feature Characteristic symbol: active-site Location: 91..102 Method of determining feature: E 1 5 10 15 GTG AAG CAG ATC GAG AGC AAG ACT GCT TTT CAG GAA GCC TTG GAC GCT 48 Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp Ala 20 25 30 GCA GGT GAT AAA CTT GTA GTA GTT GAC TTC TCA GCC ACG TGG TGT GGG 96 Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys Gly 35 40 45 CCT TGC AAA ATG ATC AAG CCT TTC TTT CAT TCC CTC TCT GAA AAG TAT 144 Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys Tyr 50 55 60 TCC AAC GTG ATA TTC CTT GAA GTA GAT GTG GAT GAC TGT CAG GAT GTT 192 Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp Val 65 70 75 80 GCT TCA GAG TGT GAA GTC AAA TGC ATG CCA ACA TTC CAG TTT TTT AAG 240 Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr Phe G ln Phe Phe Lys 85 90 95 AAG GGA CAA AAG GTG GGT GAA TTT TCT GGA GCC AAT AAG GAA AAG CTT 288 Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys Leu 100 105 GAA GCC ACC ATT AAT GAA TTA GTC 312 Glu Ala Thr Ile Asn Glu Leu Val

SEQ ID NO: 4 Sequence length: 315 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA Origin organism name: Human strain name: ATL-2 sequence Feature Symbol representing the feature: active-site Location: 94..105 Method by which the feature was determined: E 1 5 10 15 ATG GTG AAG CAG ATC GAG AGC AAG ACT GCT TTT CAG GAA GCC TTG GAC 48 Met Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala Leu Asp 20 25 30 GCT GCA GGT GAT AAA CTT GTA GTA GTT GAC TTC TCA GCC ACG TGG TGT 96 Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys 35 40 45 GGG CCT TGC AAA ATG ATC AAG CCT TTC TTT CAT TCC CTC TCT GAA AAG 144 Gly Pro Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Ser Glu Lys 50 55 60 TAT TCC AAC GTG ATA TTC CTT GAA GTA GAT GTG GAT GAC TGT CAG GAT 192 Tyr Ser Asn Val Ile Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp 65 70 75 80 GTT GCT TCA GAG TGT GAA GTC AAA TGC ATG CCA ACA TTC CAG TTT TTT 240 Val Ala Ser Glu Cys Glu Val Lys Cys Met Pro Thr P he Gln Phe Phe 85 90 95 AAG AAG GGA CAA AAG GTG GGT GAA TTT TCT GGA GCC AAT AAG GAA AAG 288 Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys 100 105 CTT GAA GCC ACC ATT AAT GAA TTA GTC 315 Leu Glu Ala Thr Ile Asn Glu Leu Val

[Brief description of drawings]

FIG. 1 shows the construction process of plasmid pADFDE (pBR).

FIG. 2 is the construction process of plasmid pADFDE.

[Fig. 3] Fig. 3 shows human AD from the designed transcription start point.
The DNA sequence extending to the N-terminus of F is shown.

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Claims (3)

[Claims] 1. A method for culturing Escherichia coli transformed with a plasmid having a gene encoding human ADF in a medium to produce the desired human ADF polypeptide and obtaining the polypeptide. A method for direct expression and production of recombinant human ADF polypeptide. 2. The direct expression production method according to claim 1, wherein the recombinant human ADF produced has the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing. 3. The direct expression production method according to claim 1, wherein the recombinant human ADF produced has the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing.