JPH05178892A - Human epithelial cell growth factor mutant and its production - Google Patents

Abstract

PURPOSE:To obtain the subject new mutant useful for elucidating the canceration mechanism of cells or in the medical field due to a difference in receptor binding ability and cell growth promoting action on cultured cells as compared with those of the natural type growth factor. CONSTITUTION:The objective human epithelial growth factor (hEGF) mutant is a mutant obtained by substituting at least one amino acid residue at the 21-position (Met), 22-position (Tyr), 23-position (Ile), 24-position (Glu), 28-position (Lys), 29-position (Tyr) and 30-position (Ala) with other amino acid residues of the hEGF having a sequence expressed by the formula. The amino acid residue is substituted with Phe in the case of the 21-position, Ala or Phe in the case of the 22-position, Asp, Leu, Val, Trp or Phe in the case of the 23-position, Ala in the case of the 24-position, Glu, Phe or Met in the case of the 28-position, Phe, Gly or Pro in the case of the 29-position and Phe or Arg in the case of the 30-position. A recombinant microbial cell transformed with a recombinant plasmid containing a DNA region capable of coding the mutant is cultured to provide this mutant from the resultant culture.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a hEGF mutant having a different receptor-binding ability to cultured cells and / or a cell growth promoting activity as compared with natural hEGF, and a recombinant containing a DNA region encoding the mutant. Recombinant microbial cell transformed with plasmid and hE using the microbial cell
The present invention relates to a method for producing a GF variant.

[0002]

EGF was originally isolated and fixed from mouse submandibular gland as a substance having an early eye-cleavage action in mouse fetus by Cohen et al. (SN Cohen et.al. , Proc. Natl. Acad. Sc).
i.USA., 72 , 1317, '75), and then human EGF was found in urine (H. Gregory, Nature, 257 , 325, '75). Furthermore, the gene structure has been elucidated in detail today (GI Bell e
t.al .Nucleic Acids Res., 14 , 3427, '86).

[0003] EGF is known to have a cell growth promoting action and a gastric acid secretion inhibiting action. From this fact, it is expected to be applied to anti-ulcer, wound burn healing, corneal ulcer and the like in the medical field (SNCohen et.al. Ann.Rev.Biochem.,
48, 193, '79). From the viewpoint of elucidating the mechanism of cell proliferation or canceration, EGF is frequently used in a wide range as a research reagent.

Therefore, if an EGF mutant having a receptor binding ability and / or a cell growth promoting activity different from that of natural EGF can be obtained, this will make a great contribution in the medical field as well as in the field of basic research. Is expected.

[0005]

Accordingly, the present invention is intended to provide various novel EGF mutants having a different receptor binding ability and / or a cell growth promoting action as compared with natural EGF. ..

[0006]

[Means for Solving the Problems] As a result of intensive studies of the EGF mutants, the present inventors have found that the 19-32 positions having a beta sheet structure have a great influence on biological activity. When the amino acid in this region was changed, an interesting EGF mutant was obtained.

Accordingly, the present invention provides the following sequence: (H ₂ N) -Asn-Se
r-Asp-Ser-Glu-Cys-Pro-Leu-Ser-His-Asp-Gly-Tyr-Cys-
Leu-His-Asp-Gly-Val-Cys-Met-Tyr-Ile-Glu-Ala-Leu-As
p-Lys-Tyr-Ala-Cys-Asn-Cys-Val-Val-Gly-Tyr-Ile-Gly-
Glu-Arg-Cys-Gln-Tyr-Arg-Asp-Leu-Lys-Trp-Trp-Glu-Le
In human epidermal growth factor (hEGF) having u-Arg- (COOH), position 21 (Met), position 22 (Tyr), position 23 (Ile), position 24
(Glu), 28th (Lys), 29th (Tys) and 30th (Ala), at least one amino acid residue is replaced by another amino acid residue. By; Ala or Phe for position 22; Asp, Leu, Va for position 23
by l, Trp or Phe; by Ala in the 24th position; by Glu, Phe or Met in the 28th position; Phe, Gly in the 29th position
A recombinant human microbial cell transformed by a recombinant plasmid containing a human epidermal growth factor variant, and a DNA region encoding the variant, which is substituted by Phe or Arg at position 30 or Pro; The present invention provides a method for producing an hEGF variant, which comprises culturing, allowing the hEGF variant to be produced and accumulated in the culture, and separating the hEGF variant from the obtained culture.

In the present invention, amino acids and polypeptides will be abbreviated by the method adopted by the IUPAC-IUB Biochemistry Committee (CBN). The DNA sequence is abbreviated by the type of base contained in each deoxyribonucleotide constituting the DNA sequence, and for example, the following abbreviations are used. A deoxyadenylic acid G deoxyguanilic acid C deoxycytidylic acid T deoxythymidylic acid

Further, (H ₂ N) and (COOH) represent the amino terminal side and carboxy terminal side of the amino acid sequence, respectively.
5'and 3'indicate the 5'end side and the 3'end side of the DNA sequence, respectively.

[0010]

The EGF mutant according to the present invention has an activity different from that of the natural type in the EGF receptor assay (JP-A-60-28994) and the cell growth promoting action. This is a strong expectation for new possibilities different from natural EGF. It also contributes greatly to the elucidation of cell proliferation or canceration mechanism, which will provide information that is indispensable for the development of anticancer agents.

[0011]

DETAILED DESCRIPTION OF THE INVENTION Preparation of Derivatives For the polypeptide having the amino acid sequence represented by the above formula (1), for example, the EGF gene disclosed in the EGF secretion patent (Japanese Patent Laid-Open No. 61-37099) is a known technique. After conversion by the point mutation method (RB Wallace et al., Science, 209 , 1
396 1980), and the method described in the patent. The codes corresponding to the converted amino acids are shown in Table 1 below, but are not limited to these.

[0012]

[Table 1]

Naturally, the structural gene of the EGF mutant may be newly synthesized, and the method of gene expression is not limited to the secretory method shown in the examples of the present invention, and the known direct method (T. Miyake et.al. , Proc.Natl.Acad.Sci.USA ., 81 , 5
956, '85), fusion method (T.Miyake et.al. , Proc.Natl.Acad.Sc
i.USA., 79 , 2475, '82) etc. can also achieve the purpose.

Direct methods, fusion methods, etc. are known as methods for expressing foreign genes. In this case, extra amino acids or peptides involved in the construction of expression plasmids should be added to the N-terminal side or C-terminal side of (1). There is.

For example, when expressed by the direct method, a Met residue due to ATG which is a translation initiation signal may be added to the N-terminal side (TMRoberts et.al. , Proc.Natl.Acad.Sc ) .
i.USA., 76 , 5596, '79), which is well understood by those skilled in the art. Therefore, the peptide (1) to which such amino acid or peptide is added is included in the scope of the present invention. Further, the mutant of the present invention can also be obtained by chemically synthesizing it without resorting to the method of gene recombination.

[0016]

EXAMPLES Next, the present invention will be described more specifically by way of examples. Example 1. Preparation of Expression Plasmid pTA152-21F 1 As an example, a method for producing a modified hEGF product in which the 21st position was converted to Phe will be shown. pTA1522-Eco (T.Oka et al , Agric.Biol.Che
m., 51 , 1099, '87) (JP-A-61-37099), phoA signal p
The EcoRI-Sal I fragment containing the eptide-hEGF gene was added to M13mp19.
Was inserted into the EcoRI-Sal I site of the multiple cloning site, and the Met at position 21 of hEGF was replaced with Phe using the method of oligo-nucleotide directed-mutagenesis (codon replacement was ATG → TTC).

The used oligo-nucleotide (GGCGTTTGCTTCT
ATATTGAA) was synthesized by NS-1 fully automatic DNA synthesizer (Shimadzu), and Muta-Gene ^™ manufactured by BioRad was used for modification.
The nucleotide sequence of the variant was confirmed by the Maxam-Gilbert method (Methods in Enzymology, 65 , 499-560, Academic Pr
ess). The modified PheA signal peptide-21Peh hEGF gene was excised as an EcoRI-Sal I fragment, and the modified gene and pTA1522-Eco phoA signal peptide-hEGF were excised.
21Phe hEGF expression vector pTA152
-21F was prepared.

Example 2. Production of 21Phe hEGF E. coli YK537 transformed with pTA152-21F (JP-A-
152297) was planted in 5 ml of medium and cultured overnight at 37 ° C. This E. coli solution was mixed with 3 L of medium (composition: 20 g / l tryptone, 10
g / lyeast extract, 30 g / l glucose, 1 g / l MgSO ₄ )
And cultured at 37 ° C. for 24 hours. AC-D3 (Iwashiya) was used for the culture, and the rotation speed during the culture was changed as follows. O-4hr / 500rpm, 4-6hr / 1000rpm, 6-10hr / 1200rpm, 10-2
4hr / 300rpm.

After culturing, ammonium sulfate was added to the medium from which cells were removed by centrifugation (7,000 rpm, 4 ° C., 10 minutes) so that the concentration was 130 g / l. Butyl-toyopearl 650M (Tosoh) column equilibrated with 130 g / l ammonium sulfate aqueous solution (φ3 cm x
(15 cm, flow rate: 1.6 ml / min), the adsorbed product was eluted with water. Fractions containing 21Phe hEGF are SDS-PAGE
Confirmed by.

Fractions containing 21 Phe hEGF were collected and ammonium sulfate
Ammonium sulfate precipitation was performed in addition to 0 g / l.
This precipitate was dissolved in 50 ml of water and applied to a column (φ2.6 cm × 90 cm, flow rate: 1 ml / min) of Sephadex G-50 (Pharmacia) equilibrated with ammonium acetate buffer (pH 5.8) to obtain the same buffer solution. The fraction containing 21Phe hEGF was collected and the fractions containing 21Phe hEGF were collected.

The product pretreated with Sep-Pak (Waters) was freeze-dried and then dissolved in 20% acetonitrile-0.1% trifluoroacetic acid to obtain ODS-120T (Tosoh; φ4.6 mm × 15c).
reverse phase high performance liquid chromatography (HPLC)
(Flow rate: 1 ml / min), gradient, 20% acetonitrile-0.1% trifluoroacetic acid → 40% acetonitrile-
0.1% trifluoroacetic acid / 20 minutes), wild type hEGF
The main peak eluting at about the same position as 21Phe hEG
Sorted as F.

The collected fractions were lyophilized, dissolved in 100 mM ammonium acetate buffer (pH 6.0), and then DEAE-5PW (φ7.
5 mm x 7.5 cm, flow rate: 0.5 ml / min, gradient:
The final purified product was obtained by subjecting it to ion exchange HPLC using 125 mM → 500 mM ammonium acetate buffer, pH 6.0, for 20 minutes) and collecting the main peak eluting at almost the same position as wild type hEGF. The result of amino acid analysis
It was confirmed that Phe was included (T.Oka et.al. , Pro
c.Natl.Acad.Sci.USA., 82 , 7212, '85).

Example 3. Measurement of biological activity (a) RRA When 4 × 10 ₅ cells / tube of KB cells (Dainippon Pharmaceutical) were added with human EGF (Cosmobio) or EGF mutant, labeled EGF ( ¹²⁵ I-mEGF, Amersham) The radioactivity of the cells was measured.

(B) Cell proliferation activity In a 96-well microplate, a human EGF or EGF mutant solution of each 2-fold dilution series and 3 × 10 ³ BALB / MK cells (B.
E. Weissman et.al. , Cell, 32 , 599, '83), and added 37
Cultivation was carried out for 5 days under humid conditions of ° C, 5% CO ₂ and 95% air. After culturing, fix with 25% glutaraldehyde, 0.05
% Methylene blue, and the absorbance at 670 nm of each well was measured.

Example 4. Example 1 was repeated, except that the codons shown in Table 1 were used as amino acid substitution codons, various expression plasmids were made, various hEGF variants were made according to the method of Example 2, and Example 3 The biological activity was measured according to the method described in 1. The results are shown in Table 2.

In Table 2, the leftmost column (column "Sample") shows that the amino acids shown on the right side are present at the amino acid positions indicated by the numbers on the left side. AAA (μg / ml) is the concentration of the test peptide in the sample determined by amino acid analysis, and RRA (μg / ml) is the concentration of the peptide converted from RRA activity (converted based on the activity of natural hEGF). Shown and Bioassay (or Bio) (μg / m
l) is the peptide concentration (natural hEGF
Is calculated based on the cell proliferation activity of the above). Therefore, RRA / AAA (%) and Bio / AAA (%) respectively show the relative values of the specific activity of RRA and the specific activity of cell proliferation of each sample.

[0027]

[Table 2]

[Sequence listing] Sequence number: Sequence length: Sequence type: Topology: Linear Sequence type: Peptide Other information: Xaa at position 21 is Met or Phe; Xa at position 22
a is Tyr, Ala or Phe; Xaa at position 23 is Tyr, Asp,
Len, Val or Trp; Xaa at position 24 is Glu or Ala; Xaa at position 28 is Lys, Glu, Phe or Met; 29
Xaa at position is Tyr, Phe, Gly or Pro; and Xaa at position 30 is Ala, Phe or Arg. Array:

Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display location C12N 15/18 ZNA (C12P 21/02 C12R 1:19) C07K 99:00

Claims (2)

[Claims] 1. The following sequence: (H ₂ N) -Asn-Ser-Asp-Ser-Glu-
Cys-Pro-Leu-Ser-His-Asp-Gly-Tyr-Cys-Leu-His-Asp-Gl
y-Val-Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-
Cys-Asn-Cys-Val-Val-Gly-Tyr-Ile-Gly-Glu-Arg-Cys-Gl
In human epidermal growth factor (hEGF) having n-Tyr-Arg-Asp-Leu-Lys-Trp-Trp-Glu-Leu-Arg- (COOH), position 21 (Me
t), 22nd (Tyr), 23rd (Ile), 24th (Glu), 28th (Lys)
, At least one amino acid residue at positions 29 (Tys) and 30 (Ala) is replaced by another amino acid residue,
Here in case of 21st place by Phe; in case of 22nd place Ala or
By Phe; Asp, Leu, Val, Trp or Phe for position 23; By Ala for position 24; Glu, Phe for position 28
Or Met; a human epidermal growth factor variant substituted by Phe, Gly or Pro at position 29; and Phe or Arg at position 30. 2. A recombinant microbial cell transformed with a recombinant plasmid containing a DNA region encoding a human epidermal growth factor variant having the amino acid sequence of claim 1 is cultured, and the hEGF mutation is introduced into the culture. Let the body generate and accumulate,
A method for producing a hEGF mutant, which comprises isolating the hEGF mutant from the obtained culture.