JPH022390A - Novel stimulation factor for human granulocyte macrophage colony - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides human granulocyte-macrophage colony-stimulating factor (hereinafter referred to as human granulocyte-macrophage colony-stimulating factor GM-C3) which has a novel structure useful as a medicine.
(abbreviated as F).

(Conventional technology and means for solving problems) GM-
C3F stimulates bone function and promotes the differentiation and differentiation of white blood cells such as granulocytes and macrophages, which play an important role in infection defense and immunity.
It is a type of cytotoxic protein that promotes proliferation, and its secondary structure has already been reported. (Gordon G, W
ong et, a+,, 5science228+ 8
10-815. (1985); Antony W, B
Urges et, a+,.

Blood, 6943-501987) The present inventors isolated and recovered GM-C3F produced in the cells of Escherichia coli transformed by genetic recombination.
Since GMC3F is obtained as an inactive aggregate in E. coli, it can be solubilized and reduced under appropriate conditions to form linear GM-C.
In the process of isolating SF and oxidizing it to lead to active GM-C3F having two sets of disulfide bonds, a new GM-C3F different from those already reported was created.
It was discovered that 3F could be obtained, and the present invention was completed.

The GM-CSF of the present invention has N
There are seven amino acids from the N-terminus of GM-C3F preceded by a methionine residue (Met-Ala-Pr).

This is a novel GM-C3F identified by the amino acid sequence from the 8th to the 128th amino acid sequence of the amino acid sequence shown in FIG. 1, which has been removed (Ala-Arg-5er-Pro).

Below, in this specification, the amino acid sequence shown in FIG.
GM-C3F in which a methionine residue precedes the N-terminus specified by
GM-C3F of the present invention in which seven amino acids are removed from the N-terminus of M-C3F is designated as type B) GM-C3F type B of the present invention can be obtained by the method described below.

The GM-CSF gene encoding GM-C3F shown in FIG. 1 is isolated by a conventional method, and a recombinant E. coli expressing this gene is prepared by a conventional method.

Preferably human leukemia cell line 0937 (ATCC CRL
SG9 derived from 112 strains of Escherichia coli by a plasmid carrying the human GM-C3F gene derived from 1593).
36 strain was transformed, and the resulting E. coli was cultured in a suitable medium such as L broth medium according to a conventional method to produce GM-C3F (see PC application WO37102060). GM
-C3F accumulates in the host as insoluble aggregates.

The cultured bacterial cells are collected by centrifugation or the like, and the bacterial cells are disrupted using an appropriate means (eg, ultrasonication, French press treatment, etc.), and insoluble aggregates are removed by centrifugation. This aggregate is dissolved using an appropriate solubilizing agent (e.g., high concentration guanidine hydrochloride or urea) and a reducing agent (e.g., 2-mercaptoethanol) to cleave disulfide bonds that may exist within and between molecules. Solubilize. CM-C3F contained as a reduced product is primarily purified by means such as gel filtration chromatography. In the step of taking out the aggregate, host-derived proteins and nucleic acids contaminating 0MC3F, soluble components among cell membrane components, and contaminant components having a molecular weight different from that of CMC5F are removed by the above operation. At this time, it is desirable that the purity of the reduced GM-C3F is as low as not to interfere with the next regeneration step, and preferably, the purity is about 20 to 50%.

The thus obtained reduced form of GM-C3F is formed into a 2m disulfide bond in a solution containing an appropriate redox system (e.g., a mixture of oxidized and reduced forms of glutathione or cysteine) to form an active GM-C3F. Play the CSF. After regeneration, when the GM-C3F concentration is low, it is concentrated to a concentration of 0.1 mg/IR1 or higher, more preferably 0.5 mg/m or higher, using a method such as an ultrafilter. This regeneration solution contains impurities such as redox agents and chelating agents in addition to host-derived substances that could not be removed by the next bath.
The impurities are removed using gel filtration chromatography or ion exchange high performance liquid chromatography. - The thus obtained mixture of GM-C3FA type and B type is subjected to reverse phase high performance liquid chromatography and separated. By separating each GM-C3F active fraction, further purifying it, and assaying the structure of GM-C3F, the desired G
M-C3FB type can be obtained. GM-C3F type B obtained by the above operations was found to have higher biological activities than type A, such as neutrophil survival maintenance activity and hamster bone marrow cell colony forming activity.

The present invention will be explained in more detail with reference to Examples below.

Susichitsu1 Human leukemia cell line 0937 (A T CCCRL
SG936 strain derived from E. coli strain 112 (
ATCC39264) and the obtained E. coli (
DSM3474) (known strain described in PCT application No. 087102060) was cultured at 30'C for 14 hours in Pross medium containing ampicillin and kanamycin.
Next, the temperature was raised to 42°C, induction was applied, and the mixture was further roasted for 3 hours. After culturing, the bacterial cells are destroyed by ultrasonic disruption,
12. Pellet by centrifugation at OOOrpm for 30 minutes.

The obtained pellet was treated with O, 1M Tri-11 (:1(p
fl 7.5), 6M guanidine hydrochloride and I(
0.1M with 1mM 2-mercaptoethanol
Solubilized using Tri-11CI (pH 7,5) and then loaded onto a Sephacryl 5200 Superfine column (
Trademark: Manufactured by Pharmacia) (2.6cm x 94co)
Gel filtration chromatography was performed at 4°C over 500ad resin). ) The GM-C3F purity in the total protein content of Yoshikawa's CMC3F-containing solution was 47%.

This fraction was further treated with 0.1M Tri-HCI (ptl 7.5) containing 0.4mM cystine.
Dialysis was performed for 12 hours at 5°C, and further dialysis was performed for 11 nights at 4"C against a fresh buffer solution to obtain a regenerated solution containing oxidized GM-C3F. The GM-C2F content in this solution was 0.05 mg/ml. Therefore, the regeneration solution was
After centrifugation at 2.000 rpm for 30 minutes, the supernatant was concentrated to 0.4 mg 7 ml by ultrafiltration. The obtained concentrated solution was subjected to gel filtration chromatography using Sephadex G-100 (trademark; manufactured by Pharmacia) (2.6cn+X94cm, 500m resin), and the eluted GM
-C3F-containing solution was filtered again using an ultrafilter at 1.0 m
g/ml.

1.88 as GM-C3F thus obtained
A solution containing mg was purified using HPLC under the following conditions.

Column: Sumipax ODS A-213 (10m
m x 250m) (manufactured by Sumitomo Chemical) Mobile phase: A solution 0.
12% trifluoroacetic acid aqueous solution B liquid under certain water elution conditions (liquid A 43%, liquid B 57%)? Let's roll 8.

Flow rate: 3 mfl/min Column temperature: Room temperature Detector: Ultraviolet absorption meter (measurement wavelength 230 nm) Retention time j (1, M-C3F Type A 17 minutes C, M-C3F
Type B Each component of GM-C3F type A and type B was fractionated for 21 minutes.
dford et al.'s method (Bradford, M. et al.
,al,, Anal.

As a result of measuring the protein amount using Biochem, Yu2248 (1976)), GM-C3F type A was 0.54.
mg, and 0.4 hg of GM-C3FC type C were obtained.

The second high-performance liquid chromatogram of the crude product of GM-C3F
As shown in the figure.

The N-terminal amino acid sequence of GM-C3F type B obtained in Example 1 was analyzed using a gas phase amino acid sequencer. As a result, GM-C3F type B has seven N-terminal amino acids of type A (Met-Ala-Pr.

It was found that the protein had been deleted (Ala-Arg-5er-Pro).

J. Hamster Bone Marrow Cell Colony Forming Activity Test Method Hamster femurs were removed aseptically and cultured in a-MEM culture medium containing 2% tallow serum (Stanners+C, P, e
t.

al., Nature New Biology, 23
0:52 (1972) (manufactured by Flow)),
Bone marrow cells were washed out. The cells are broken up by pipetting and allowed to stand for 3 minutes. After taking the cell suspension and washing it with the same culture medium, add α-MEM containing 40% tallow serum.
Adjust the cell concentration to 1X10/ml with culture medium. This bone marrow cell suspension and human GM-C3F serially diluted with α-MEM culture medium containing 2% tallow serum were added to wells for 10
0 Il each and cultured at 37°C under 5% carbon oxide conditions. After 42 hours of culture, incubate with α-MEM culture solution for 40 hours.
μci/xi! Tritium-labeled thymidine prepared in
After adding 5 μl to each well and culturing for a further 6 hours, the radioactivity incorporated into the cells is measured.

The specific activity of CM-C3FO was determined by defining the amount of GM-C3F that induces the incorporation of tritium-labeled thymidine in CCV/CC)/2 as 50 units/rd. However, Cv represents the maximum amount of tritium-labeled thymidine incorporated; CC represents the amount of tritium-labeled thymidine incorporated in the absence of GM-C3F.

Test results Table 1 From the results in Table 1, GM-C3F type B is GM-C3F
Like type A, it was found to have hamster bone marrow cell colony forming activity.

[Brief explanation of the drawing]

FIG. 1 shows the amino acid sequence of GM-C3F derived from recombinant E. coli. However, A: alanine, C nigistein, D = aspartic acid, E: glutamic acid, F: phenylalanine, G nigericin, H: histidine, 1: isoleucine, K: lysine, L: leucine, M: methionine, N: asparagine, P Nibroline, Q: Glutamine, R: Arginine,
S: serine, T: threonine, v: valine, w nitributophan, Y dityrosine, respectively. FIG. 2 shows a high performance liquid chromatogram of the crude product GM-C3F used in the examples. Figure 1 M A P A R S P S P
S T Q P W E Hl 2
3 4 5 6 7 8 9] 011 12
131415+6V N A r Q E'
A RRL L N L S R17
181920212223242526272g 2
9 30 31DTAAEMNETVEV I
5E32 33 34 35 36 37 38 39
40 4+ 42 43 44 45 46MFD
LQEPTCLQTRLE 47 48 49 EO5152535455565
758596061 YKQGLRGSLTKLK
G PLTMIASHYKQHCPP TPETSCATQ I ITFES92 93
94 9:l 96 97 98 9910010
1102103104105106FKENLKDFL
LV I PFD1071081091101
1+ 112113114115116117118
119120121-SS-bond 55th and 97th
Seventh! Sting 89th and 1225th Sting

Claims (1)

[Claims] Human granulocyte-macrophage colony-stimulating factor polypeptide specified by the amino acid sequence from the 8th to the 128th amino acid sequence shown in Figure 1