Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-10-31
2002-08-20
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...
C435S069100, C435S252300, C435S320100, C536S023100, C536S023400, C530S325000, C530S300000, C530S324000, C530S350000
Reexamination Certificate
active
06436674
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for secretory production of a human growth hormone having a molecular weight of about 20,000 using
Escherichia coli
. In particular, the present invention relates to a method for the production of growth hormone having a molecular weight of about 20,000, wherein
E. coli
is transformed by a recombinant plasmid carrying a DNA fragment in which the 5′ end of a DNA fragment coding for a human growth hormone having a molecular weight of about 20,000 is linked in series to the 3′ end of a DNA fragment coding for
E. coli
or Salmonella OppA secretion signal, or an amino acid modified form of
E. coli
OppA secretion signal, the resultant
E. coli
transformants are cultured, then the resultant culture fluid, cells or processed material thereof are used for the production.
Furthermore, the present invention relates to a method of producing a human growth hormone having a molecular weight of about 20,000, wherein
E. coli
is transformed with a recombinant plasmid carrying a DNA fragment coding for a human growth hormone having a molecular weight of about 20,000 and a DNA fragment coding for
E. coli
signal peptidase 1, the resulting
E. coli
transformants are cultured, then the resulting culture fluid, cells or processed materials thereof are used for the production.
Furthermore, the present invention relates to a method for increasing the production of a human growth hormone having a molecular weight of about 20,000 by altering the medium composition in culturing the transformants transformed by a recombinant plasmid with the abovementioned structure.
DESCRIPTION OF THE RELATED ART
There are two known types of human growth hormone (hGH) derived from the pituitary gland: one having a molecular weight of about 22,000 (hereinafter referred to as 22K hGH) and the other having a molecular weight of about 20,000 (hereinafter referred to as 20K hGH).
22K hGH is used for manufacturing pharmaceutical products, by means of recombinant DNA technology, for the treatment of pituitary dwarfism, pediatric chronic renal failure or the like. hGH has recently been found to have excellent activities such as immune promoting activity or lipolysis stimulating activity, as. well as growth promoting activity. Broader applications are greatly expected in the future.
On the other hand, recently, the risks of side effects in the clinical use of 22K hGH are widely reported and have become important issues in securing the safety of GH in clinical use and in broadening its applications. Risks arising from the use of 22K hGH reported to date include possibilities of inducing leukemia and causing diabetes. There is a need for a growth hormone with a lower risk of side effects if its clinical applications are to be broadened.
20K hGH has an amino acid sequence which corresponds to that of 22K hGH consisting of 191 amino acids except that 15 amino acid residues from the 32nd to the 46th inclusive from the N-terminal of 22K hGH are lacked. As compared to 22K hGH, this 20K hGH is reportedly low in leukemia cell proliferation activity and glucose intolerance, which is an index of diabetogenicity. Thus, the risks of side effects reported for 22K hGH are found to be less likely with 20K hGH. Also, according to results of in vitro experiments, 20K hGH has recently been found to be different from 22K hGH in its mode of binding to GH receptors. That is, M. Wada et al. revealed the difference between the two hGHs, showing that the binding activity of 22K hGH to a GH receptor decreases on the cell surface because 22K hGH binds to GH binding protein (GH receptor outer membrane protein) present in the serum at the level of physiological concentration, while a reduction in binding activity of 20K hGH to a GH receptor does not occur because 20K hGH hardly binds to a GH binding protein in physiological conditions, and that in a 1:2 complex (complex of 1 mole of growth hormone and 2 moles of GH receptor), which is necessary for the action of growth hormone via the GH receptor, 22K hGH forms a 1:1 complex (inactive type) while 20K hGH forms a 1:2 complex (active type) with the GH receptor (Mol. Endo 12, 1, 146-156, 19.97). From these results, it is expected that 20K hGH may have higher activity than 22K hGH. It has also been revealed that as to lipolysis stimulating activity, which to date was not clear, 20K hGH is as active as 22K hGH (Japanese Patent Laid-open (Kokai) No. 97/216832, or European Patent Publication No. 0753307).
As mentioned above, 20K hGH has been found to be a growth hormone which exhibits lower risks and higher activity than 22K hGH and has become a novel growth hormone expected to be useful as a pharmaceutical product.
Recent developments in recombinant DNA technology makes it possible to produce heterologous proteins using microorganisms as host organisms. Generally, intracellular expression methods and secretory production methods are used for the production of proteins using microorganisms. In intracellular expression methods, proteins carrying methionine at the N-terminal are accumulated in the cytoplasm. In order to obtain proteins without the methionine residue at the N-terminal, it is necessary to enzymatically cleave amino acid sequences containing the me thionine residue at the N-terminal, for example, by a peptidase. Further, proteins obtained by the intracellular expression methods are not active forms in their steric configurations and require refolding. Thus, intracellular expression methods are not necessarily recommended as efficient methods for protein production.
On the other hand, in secretory production methods using
E. coli
, target proteins are secreted and accumulated in the periplasm. Purification of proteins from the periplasm extract is easy because the level of impure proteins is lower than that in intracellular expression methods. Moreover, the secreted proteins have no methionine at the N-terminal and are naturally active forms in their steric configuration. Thus, secretory production methods are excellent.
However, secretory production methods using microorganisms require highly sophisticated techniques and thus rarely applied on an industrial scale. That is because precursor proteins synthesized in the cytoplasm pass through the cell membrane and because secretion signals have to be properly cleaved and removed by processing.
As to protein secretion in microorganisms, its mechanisms, relevant factors and their functions have been revealed. Based on these findings, various attempts have been made for efficient secretion methods.
Secretion signals are the first to be discussed. Secretion signals have an extremely important role in protein secretion to lead target proteins to the cytoplasmic membrane. It has been revealed that for the action of secretion signals, a positive charge in the positive charge region of their N terminals and hydrophobicity in the central hydrophobic area are essential (J. Biol. Chem., 267, 4882-4888, 1992). Based on this finding on structural characteristics of secretion signals, Udaka et al. made a modification in which a basic amino acid (Arg) was added to the N terminal positive charge region of a secretion signal of MWP (middle wall protein) derived from
Bacillus brevis
and a hydrophobic amino acid (Leu) was added to the central hydrophobic region in secretory production using
Bacillus brevis
as a host, and reported that the secretion efficiency of a sole fish growth hormone was improved to the level of 60 mg/L (Nippon Nogeikagaku Kaishi, 67(3), 372, 1993).
However, there are no theoretical guidelines for the modification of amino acid sequences of secretion signals and at present, preferable sequences have to be found by a try-and-error process. Further, there is no clear principle at present as to how to choose secretion signals derived from secretory proteins for target proteins for secretory production and thus, suitable secretion signals have to be found for the individual target proteins for secretory production.
The abovementioned problems are understood, for e
Honjo Masaru
Matsumoto Kazuya
Mochizuki Daisuke
Naitoh Naokazu
Uchida Hiroshi
Burns Doane Swecker & Mathis L.L.P.
Guzo David
Leffers, Jr. Gerald G.
Mitsui Chemicals Inc.
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