JPH03294293A - Modified human epithelial cell growth factor and production thereof - Google Patents

Abstract

NEW MATERIAL:A modified human epithelial cell growth factor expressed by the formula. USE:Antiulcer agents, curing agents for wounds and burns and remedies for corneal ulcer. PREPARATION:For example, a human epithelial cell growth factor (hEGF) is modified by using a genetic site mutational method to provide a DNA capable of coding a modified hEGF expressed by the formula in which (32)Ile is converted into (23)Phe. The resultant DNA is subsequently linked to a vector to form a recombinant plasmid containing a DNA region capable of coding the modified hEGF. The formed recombinant plasmid is then inserted into a microorganism such as Escherichia coli. transformation thereof is carried out to culture the transformed recombinant microbial cells. After culturing, the microbial cells are removed by centrifugation and ammonium sulfate is added to the supernatant. The resultant mixture is subjected to gel filtration to confirm a fraction containing the modified hEGF by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ammonium sulfate is subsequently added to the fraction to afford precipitates, which are then dissolved in water, further subjected to gel filtration and purified to provide the modified hEGF expressed by the formula.

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a modified human epidermal growth factor (hEGF) that has higher biological activity than natural epidermal growth factor (EGF), and a recombinant plasmid containing a DNA Atl region encoding the modified product. , relates to a recombinant microbial cell transformed with the plasmid and a method for producing a modified hEGF using the microbial cell. More specifically, hEGF variants having a cell proliferation-promoting effect, DN encoding the variants,
The present invention relates to a recombinant plasmid containing an ASI region, a recombinant microorganism cell transformed with the plasmid, and a method for producing bEGF variants using the microorganism cell. (Prior art) EGF was originally isolated and identified from the submandibular line of mice as a substance that has the effect of premature eye opening in mouse fetuses by Cboen et al. (S, N. Choen et al., Proc.
Natl. Acad, Sci, USA, +72.1317. '
75), human EGF was then discovered in urine (B,
Gregory, Nature. 257, 325. '75), and today the gene structure has also been elucidated in detail (G, 1.Be1l et al.
, al. Nucleic Acids Res, 1
4.3427. '86). EGF is known to have the effect of promoting cell proliferation and suppressing gastric acid secretion, and for this reason, it is expected to be applied in the medical field to anti-ulcer, wound/burn healing, corneal ulcer, etc. (S, N, Choen et, al. Ann. Rev. Biocbe+w, 48.193. '7
9) EGF is also frequently used as a research reagent in a wide range of fields from the perspective of elucidating the mechanisms of cell proliferation and canceration. [Problem to be solved by the invention] Therefore, EGF has higher biological activity than natural EGF.
If a derivative can be obtained, it is expected to make a significant contribution to both the medical field and the basic research field. Therefore, the present invention aims to provide a modified EGF that has higher biological activity than the natural type. Means for solving CrRB] The present inventors used a gene point mutation method (R.B. Wallace et, al., 5science, 2
09+ 1396+ '80), Hi) By modifying the EGF gene, (23) He was changed to (23) Phe
This is based on the fact that we have obtained a modified EGF that has been converted into ) f4sn-5et-Asp-5er-Glu-Cys-
Pro-Leu-5er-HisAsp-Gly-Ty
r-Cys-Leu-tlfs-Asp-Gly-Va
l-Cys-Met-Tyr-Phe-Glu-Ala
-Leu-Asp-Lys-Tyr-Ala-Cys-
Asn-Cys-Val-Val-Gly-Tyr41
e-Gly-Glu-Arg-Cys-Gln-Tyr
-Arg-^5p-Leu-Lys-Dingrp-Trp-
It is characterized by being represented by Glu-Leu-^rg (C-terminus) (I). The present invention further provides a DN encoding the hEGF variant described above.
A recombinant plasmid containing the A'l region; a microbial cell transformed with this plasmid; and hE characterized by culturing this microbial cell and isolating hEGF variants.
A method for producing a GF variant is provided. In addition, in the present invention, amino acids and polypeptides are
It shall be abbreviated according to the method adopted by the PAC-IUB Committee on Biochemistry (CBN). Furthermore, the DNA sequence is abbreviated by the type of base contained in each deoxyribonucleotide constituting it, and for example, the following abbreviations are used. A Deoxyazonylic acid G Deoxyguanylic acid C Deoxyazonic acid T Deoxyazonylic acid [Specific description] Polypeptide having the amino acid sequence represented by the above formula (I) (herein referred to as ("Phe") hEGF ) is, for example, a patent related to EGF secretion (
It can be obtained by converting the EGF gene shown in Japanese Patent Application Laid-Open No. 61-37099 by the known technique of point mutation (mentioned above) and then by the method described in the same patent. Of course, the structural gene for EGF derivatives can be newly synthesized (
The method of gene expression is not limited to the secretion method shown in the Examples of the present invention, but also the known direct method (T, Miyak
et,al,, Proc. Natl, Acad, Sci, USA,, 81.59
56. '85), fusion method (T, Miyake et,
al,, Proc, Natl, Acad, Sci, U
S.A. 79, 2475. '82) etc. can also achieve the objective. Direct methods and fusion methods are known as methods for expressing foreign genes, but in this case, extra amino acids or peptides involved in constructing the expression plasmid are placed on the N-terminal side of (i) or on the C-terminal side of (i).
It may be added to the end. For example, when expressed by the direct method, a Met residue resulting from ATG, which is a translation initiation signal, may be added to the N-terminus (T, M, Roberts et al., Proc.
, Natl., Acad. Sci, tlSA, 76, 5596. '79),
This is well understood by those skilled in the art. Therefore, peptide (I) to which such an amino acid or peptide is added is included in the present invention. Moreover, the derivatives of the present invention can also be obtained by chemical synthesis without resorting to genetic recombination techniques. [Effect] The modified EGF according to the present invention has stronger activity than the natural type in the EGF receptor assay (Japanese Patent Laid-Open No. 60-28994), and this means that it is more effective than the conventional natural EGF in terms of medicinalization. This gives us strong hope that it will be effective. Furthermore, it will greatly contribute as a reagent to the elucidation of cell proliferation and canceration mechanisms, and this will provide essential information for the development of anticancer drugs. Next, the present invention will be explained in more detail with reference to Examples. 1, 7A152-23F pTA1522-Eco (T, Oka et al.
Agric, Biol, Chew. 51, 1099. '87) (This plasmid is 12C600 (pYK283
), JP-A No. 60-30687, No. 61-37
It can be produced by treating pTA1522 induced according to the method described in 099 and 61-149087 by the point mutation method (see Figure 1)]
Eco containing the phoA signal peptide-hEGF gene
The RI-5al I fragment was inserted into the EcoRI-5al I site of the multiple cloning site of M13wp19, and Tie at position 23 of hEGF was replaced with Phe using oligomethreotide-directed mutagenesis (the codon replacement was ^TT-TTT). The oligomethreotide used (TTGCATGTATTTTG^^GCTCT
) was synthesized using an N5-1 fully automatic DNA synthesizer (Takashi), and modified using BioRad's Muta-Gene"
was used. The base sequence of the variant was confirmed by the Maxam-Gilbert method (Methods in Enzy++
ology, 65+ 499-560. Acades
+ic Press). Modified phoA signal peptide = (23Phe
) The hEGF gene was excised as an EcoRI-5al I fragment, and this modified gene and pTA1522-
Eco's phoA signal peptide-hEGF gene was recombined to create ("Phe)hEGP expression vector pTA.
152-23F was produced (Fig. 1). This expression vector pTA152-23P has the same structure as pTA1522-Eco except that the (23) rue codon of hEGF is changed to a Phe codon. 2. E. coli YK transformed with pTA152-23F of “Phe hEGF”
537 (JP-A-152297) was planted in a 5M1 medium and cultured at 37°C overnight. This E. coli solution was added to 3L of medium (
Composition: 20g/j! ) Ribton, 10g/l yeast extract, 30g/'! Glyose, 1 g/f! Mg5
O4) and cultured at 37°C for 24 hours. A for culture
C-03 (Iwashiya) was used, and the rotation speed during culture was changed as follows. 00-4hr1500rp, 4-6hr/100
0rps, 6-10hr/1200rpm
, and 10 12hr150rpm. After culturing, cells were removed by centrifugation (7000 rpm, 4°C, 10 minutes), and ammonium sulfate was added to the medium at 130 g/j! I added it so that it becomes . 130g/f of this solution! Butyl-toyopearl 650M equilibrated with an aqueous ammonium sulfate solution of
(Toyo Soda) column (φ3 cm x 15 cm, flow rate: 21.6 m/gein), and the adsorbed material was eluted with water. ("Phe) The fraction containing hEGF is 5O5-PAG
Confirmed by E. ("Phe) Both fractions containing hEGF were collected, and ammonium sulfate was added to 390 g/Il to perform ammonium sulfate precipitation. This precipitate was dissolved in 501 d of water and added with ammonium acetate buffer (pH 5, 8). Equilibrated 5ephadexG-50 (Pharsasia)
column (φ2.6 CIX90C1, flow rate: 1 d/
5 in), eluted with the same buffer, ("Phe)
The percentage containing bEGF was collected. 5ep-Pak (I
After freeze-drying the pretreated product using Aa ters), it was dissolved in 20% acetonitrile-0.1% trifluoroacetic acid, and 0DS-1207 (Toyo Soda; φ4.6
Reversed phase high performance liquid chromatography () IPLC) using GradyEnd 220% acetonitrile - 0.1% trifluoroacetic acid →
40% acetonitrile-0.1% trifluoroacetic acid/2
0 minutes), and the main beak eluted at the same position as wild-type hEGF is collected as ("Phe) hEGF. After lyophilization, the fraction is dissolved in 100 mM ammonium acetate buffer (pH 6,0). And DEAR-5P (
7.5m x 7.5 CI, flow rate = 0.5 d/s
in, gradient end: 100mM→400wM
The final purified product was obtained by subjecting it to ion-exchange HPLC using ammonium acetate buffer, pH 6.0, for 20 minutes, and fractionating the main peak eluting at the same position as wild-type hEGF (Figure 2). As a result of amino acid analysis, it was confirmed that the product contained phenylalanine (
T. Oka et al, Proc, Natl, Aca
d, Sci, USA, 82°7212, '85)
. (a) RRA 4xios KB cells (Dainippon Pharmaceutical) per tube were injected with human EGF (Cosmo Bio) or EGF.
Modified i, Rahelized E G F (' ”I-+wEG
When the radioactivity of the cells was measured (Japanese Patent Application Laid-open No. 6O-28994) when F. Amajam) was added, the efficacy ratio of the modified EGF to human EGF was 0.57. (b) Cell proliferation activity 96 Microplate with 2-fold serial dilutions of human EGF or EGF variants and 3 x 10' BALB/F
Cells CB, E, Weissyxan et al. C811132+599, '83), 37℃,
The cells were cultured for 5 days under humid conditions of 5% CO□ and 95% air. After culturing, it was fixed with 25% geltaraldehyde and 0.
The cells were stained with 0.5% methylene blue, and the absorbance of each person at 67 on- was measured (Figure 3). h) The efficacy ratio of the EGF variant to EGF was 1.67. . 3,

[Brief explanation of drawings]

Figure 1 shows the plasmid pTA1 used in the present invention.
The structures of 522-Eco and pTA1522 are shown, and the open parts in the figure are the base parts where point mutations have occurred. FIG. 2 is a chart showing that ["Phe"] hEGF of the present invention is eluted at the same position as native hEGF in high performance fluid chromatography. FIG. “Phe
) is a graph comparing hEGF and natural hEGF.

Claims (1)

[Claims] 1. A human epidermal growth factor variant represented by the following amino acid sequence: (N-terminus) [There is an amino acid sequence] (C-terminus). 2. The following amino acid sequence: (N-terminus) [There is an amino acid sequence] (C-terminus) D encoding the human epidermal growth factor variant represented by
Recombinant plasmid containing the NA region. 3. The following amino acid sequence: (N-terminus) [There is an amino acid sequence] (C-terminus) D encoding the human epidermal growth factor variant represented by
A recombinant microbial cell transformed with a recombinant plasmid containing the NA region. 4. The following amino acid sequence: (N-terminus) [There is an amino acid sequence] (C-terminus) D encoding the human epidermal growth factor variant represented by
A method for producing a modified human epidermal growth factor, which comprises culturing a recombinant microbial cell transformed with a recombinant plasmid containing an NA region, and isolating the modified human epidermal growth factor.