Process for preparing recombinant proteins using highly...

Details Process for preparing recombinant proteins using highly... Process for preparing recombinant proteins using highly...

1999-11-24

2002-05-21

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069400, C435S069500, C435S320100, C435S254210, C536S023100, C536S024100

Reexamination Certificate

active

06391585

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to a process for preparing recombinant proteins from yeast by using recombinant DNA technology. More particularly, the present invention relates to a process for preparing recombinant proteins by using yeast expression vectors which comprise hybrid promoter consisting of two kinds of yeast inducible promoters and secretory signal consisting of yeast killer toxin and the amino terminus of mature interleukin 1&bgr;(IL-1&bgr;).
In addition, the present invention relates to a process for preparing recombinant proteins such as hGCSF and hGH from yeast by using the yeast expression vector which comprises promoter and secretory signal of yeast heat shock protein 150.
In addition, the present invention relates to a process for preparing recombinant proteins by using the yeast expression vector with XbaI cleavage site inserted in order to facilitate the insertion of the recombinant protein genes.
By using the present expression vector, the recombinant proteins such as human granulocyte colony-stimulating factor(hGCSF) and human growth hormone(hGH) can be produced with high secretion efficiency. An experiment of bone marrow differentiation and proliferation has disclosed that the colony of neutrophilic granulocyte or monocytic macrophage is formed, and thereafter it has been known that colony stimulation factors exist in the living body [J. Cell. Comp. Physiol. 66: 319 (1965); Aust. J. Exp. Biol. Med. Sci. 44: 287 (1966)].
The factors called as colony stimulating factor (hereinafter it refers to “CSF”)are classified by their characteristics in biological activity as follows:
(i) GM-CSF (granulocyte-macrophage CSF) proliferates and differentiates stem cell of granulocytic leucocyte and monocytic macrophage, and finally forms colonies,
(ii) M-CSF (macrophage CSF) forms colony of monocytic macrophages, iii) multi-CSF (multi-lineage CSF) stimulates undifferentiated pluripotent stem cells and finally forms colony of pluripotent cells, (iv) G-CSF (granulocyte CSF) forms colony of granulocyte leucocytes [J. B. C. 252: 1998-2033 (1977), J. B. C. 252: 4045-4052 (1977), Biochem. J. 185: 341-343 (1980), J. B. C. 258: 9017-9021 (1983)].
GCSF, about 20 kDa glycoprotein, is derived from monocyte, monocytic macrophage, epithelial cell, fibroblast etc. And human GCSF (hereinafter it refers to “hGCSF”) gene exists on 17th chromosome. It is known that GCSF stimulates production of neutrophilia colony in vitro and of colonies of blast cell, macrophage in cooperation with IL-3, and get some myeloid leukemic cell line matured. GCSF increases the number of neutrophil and monocyte in vitro.
The clinical applications of hGCSF are as follows:
First, hGCSF increases the number of neutrophil dosage-dependently in treating a neutropenic patients with advanced solid and hematologic malignances.
Second, hGCSF recovers patients rapidly from neutropenia by chemotherapy for malign lymphatic tumor, lung cancer, testis cancer, urethra epithelioma and acute leukemia etc.
Third, hGCSF increases the number of neutrophil upon bone marrow transplantation for acute nonlymphocytic leukemia and chronic bone marrow leukemia patient.
Fourth, hGCSF recovers patients rapidly from neutropenia due to bone marrow dysplasia syndrome.
Fifth, hGCSF recovers patients rapidly from neutropenia due to aplastic anemia.
Sixth, hGCSF is useful for hereditary and idiopathic neutropenia.
Seventh, hGCSF prevents or reduces the incidence of mucositis and febrile neutropenia due to anti-tumor chemical treatment (Drug Evaluations Annual 1993, American Medical Associations p2232-2333).
Human growth hormone (hereinafter it refers to “hGH”) is nonglycosylated protein which is made up of 191 amino acids, and it is secreted from pituitary anterior lobe. The hGH containing 2 intramolecular disulfide bonds has 22,000 dalton of molecular weight. It is initially synthesized as a precursor and is secreted from the cell after processing. The hGH is produced in large quantities before adulthood, and is produced during a whole human life.
The hGH is necessary for normal growth and development, but several types of dwarfism are caused by the abnormal low-level production of hGH and the over production of hGH can be accompanied by acromegaly or gigantism.
The hGH shows various biological activities and reacts to various tissues, directly or indirectly. It has an effect on linear bone growth rate and lactation, and shows diabetogenic insulin-like activity. In addition, it promotes protein synthesis, and has an effect on metabolism of lipid and carbohydrate.
The followings are clinical applications of hGH:
It is known that abnormal growth can be recovered if the hGH is administrated at the childhood in the case of dwarfism caused by deficiency of hGH [Raben, M. S., J. Clin. Endocr. 18 901-904 (1958)]. It is known that hGH is also used for treatment of obesity, and effective on treatment of various ailments such as bone fracture, skin burn, bleeding ulcer etc [Proc. of NIAMDD Symp. Publ. No. 74-612 (ed. Raiti, S.) (Baltimore, Md., 1973)].
Base sequence of hGH DNA is known by cDNA cloning of this gene, and the expression of hGH DNA in
E. coli
has been reported [Martial et al., Science 205: 602-605 (1979)].
Many genetic engineering methods have been attempted for the overproduction of recombinant proteins.
First, a method of expressing protein in
E. coli
after cloning the target gene has been developed [Science 232: 61-64 (1986)]. But there are some disadvantages in the method using
E. Coli
as a host as described in the followings.
In a human body, protein is synthesized as precursor first and then is processed to mature form by proteolysis.
But when the protein is expressed in
E. coli,
the N-terminal methionine of the synthesized protein is not so effectively removed by the aminopeptidase as in the human body and hence the proteins with and without the methionine can coexist in the cytoplasm of
E. coli.
Then it is very difficult to separate the protein without methionine from the protein with methionine.
In many cases, protein is expressed in inactive, or insoluble form and then it should be converted to biologically active protein through a renaturation(refolding) process where the recovery yield of protein is sometimes significantly reduced.
And there is a problem of contamination by bacterial endotoxin in the purification process.
In addition, the post-translational modification of protein (e.g. glycosylation of hGCSF) is not possible in
E. coli.
Secondly, the cloned target gene has been expressed in animal cell such as CHU-2 (human GCSF-producing tumor cell line) or Chinese hamster ovary cell.
But the method using animal cell as a host has such disadvantages that culture condition is complicated with expensive serum media and recovery yield is generally very low since small amount of recombinant protein is usually purified from large volume of culture media [EMBO J. 5: 871-876 (1980)], (KR 91-5624).
As a plausible solution to the above problems, the expression system using yeast as a host has been developed. The method that can obtain target polypeptides or proteins in large amounts from recombinant yeast has been reported by Loison and others [Bio/Technol. 4:433-437 (1986); Burrow, “Baker's yeast, p349-420, in The Yeast, vol. 3, Rose and Harrison, eds. Academic Oress, London (1970)]. The expression system of recombinant yeast has significant advantages compared to the other expression systems employing animal cell or
E. coli
as a host.
The present inventors have studied a process of preparing hGCSF by using yeast. U. S. FDA noticed that yeast is not pathogenic to human body and most of regulation principles of gene expression in yeast are disclosed [Strathern et al., The Molecular Biology of the Yeast Saccharomyces, Metabolism and Gene Expression, Cold Spring Harbor Laboratory, New York (1982)].
Using yeast as host cell has advantages that it is generally regarded as safe organism to human body, and

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