Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Patent
1999-02-08
2000-07-04
Weber, Jon P.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
530427, 435 694, 435 712, C07K 14475, C07K 106, A61K 3818, C12N 1509, C12P 2104
Patent
active
060840765
DESCRIPTION:
BRIEF SUMMARY
The following is the English translation of the Annexes to the International Preliminary Examination Report, Amended Sheets 26 and 27.
FIELD OF THE INVENTION
The present invention relates to a method of refolding denatured human activin A into natural-type human activin A having a biological activity. Natural-type human activin is a useful protein expected to be applied to pharmaceutical preparations etc.
BACKGROUND OF THE INVENTION
Human activin A is a homodimer protein consisting of 2 polypeptide chains composed of 116 amino acids, isolated from a culture supernatant of human leukemic cell strain THP-1 (IFO 50147). Its molecular weight is about 25,000 dalton, each polypeptide contains 9 Cys residues (that is, 18 Cys residues in the dimer) and there are 9 intramolecular and intermolecular disulfide bonds (Biochemical and Biophysical Research Communications, 142, 1095-1103, 1987).
Human activin A is purified and produced by repeatedly subjecting, to multi-stage chromatography, precipitation and concentration operation, a culture supernatant obtained after stimulation of human myelocytic leukemic cell THP-1 (IFO 50147) with phorbol ester ("Saibo Kogaku" (Cell Engineering), Separate Volume 4, pp. 48-58, 1988) or a culture supernatant of recombinant CHO cells highly producing human activin A, obtained by introduction of an expression vector having human activin A cDNA (Biochemical and Biophysical Research Communications, 151, 230-235, 1988). However, there are many problems where the production process involving purification of culture supernatants as the starting material derived from animal cells is used as an industrial production process.
That is, (1) because impurities which were secreted by animal cells as the producing host or derived from fetal bovine serum etc. previously added as medium ingredients should be removed at high degrees, the yield of purified human activin A remains extremely low; (2) as compared with the case where recombinant microorganisms are used as the producing host, productivity is extremely low, and highly productive animal cells or culture apparatuses are necessary to sufficiently supply the starting material to be purified; and (3) to culture the producing host, a high concentration of fetal bovine serum should be added or a serum-free medium containing growth factors etc. should be used. However, these are extremely expensive, and there is a problem with stable availability of constant quality essential for production. As described above, there are problems with low productivity etc. in the case where human activin A is produced using animal cells, and it is necessary to solve these problems in order to establish an industrial production process.
Conventionally, various attempts have been made to solve these problems. If a microorganism such as recombinant E. coli etc. is used as the producing host, its protein productivity is generally improved 100-fold or more as compared with that of animal cells, so it can be said that replacement of animal cells by microorganisms as the producing host is an effective means of improving productivity. However, the production of human activin A by E. coli results often in denatured human activin A having different intramolecular and/or intermolecular disulfide bonds to those of the natural-type (European Patent Application Publication (EP0222491), Japanese Patent Appln. Laid-Open Publication No. 119679/88).
As used herein, denatured human activin A refers to molecules with the polypeptide chain of human activin A but having lost both its tertiary structure and biological activity, such as those with intramolecular and/or intermolecular disulfide bonds cleaved to loose the tertiary structure thus forming a monomer structure, those with the disulfide bonds transferred to form a structure different from the natural type, or those polymerized via additional intermolecular disulfide bonds.
Because such denatured human activin A has no biological activity, the molecule should be reconstituted (refolded) so as to have the same tertiary struc
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Ejima Daisuke
Eto Yuzuru
Ono Kunio
Sasaki Michiro
Tsuchiya Shigekatsu
Ajinomoto Co. Inc.
Weber Jon P.
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