Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Separation or purification
Patent
1991-11-29
1997-03-18
Davenport, Avis M.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Separation or purification
530324, 530325, 530326, 530300, A61K 3800, A61K 3802, C07K 100, C07K 500
Patent
active
056124546
DESCRIPTION:
BRIEF SUMMARY
[TECHNICAL FIELD]
The present invention relates to an improved process for purifying a polypeptide, more specifically, to a purification process carried out by subjecting an objective substance containing a polypeptide to a pretreatment, and then treating the resulting crude polypeptide aqueous solution with a packing material for reversed phase high performance liquid chromatography.
[BACKGROUND ART]
A very complicated proceudure is required to purify polypeptides produced by microorganisms, animal cells, and plant cells, while maintaining their physiological activities to high degree. Consequently, the present procedures require some to be improvement. For example, the purification of a human growth hormone releasing factor produced by transformed microorganisms involves a ten stage procedure, resulting in a large amount of production but at a yield too low for carrying out a bioassay (Vincent Geli et al., Gene, 80, 129-136 (1989)). For the purification of human calcitonin, it has been reported that an eight stage purification is carried out, using 6 types of columns, to isolate human calcitonin (J. P. Gilligan et al., Biochromatography, 2 (1), 20-27 (1987)).
These purification steps, however, are very complicated, and thus it may be considered that they lead to the decomposition of polypeptides, and to the disappearance of physiological activities of polypeptides during the purification.
The object of the present invention is, therefore, to provide a process which can isolate polypeptides in a stable form and isolate and purify polypeptides at a high yield by carrying out a simple procedure, in order to thus solve these problems.
[DISCLOSURE OF THE INVENTION]
Over the past several years, various physiologically active polypeptides, represented by the human growth hormone and human calcitonin, have been increasing produced with the aid of various cells manufactured by a genetic procedure. Of these, in addition to naturally found types of physiologically active polypeptides per se, there are many polypeptides produced as fused polypeptides (also referred to as "chimera proteins") to which other protein moieties are fused. Although these can be purified by using a conventional separation/purification process, there has been a particularly desire for the development of a process for efficiently recovering objective physiologically active polypeptides without any deactivation after cleaving fused polypeptides into physiologically active moieties and other protein moieties fused thereto. The present inventors found that, when the cleaved substances of the above-mentioned fused polypeptides are treated under a specific pH level, and the treated liquid thus obtained is treated with a packing material for reversed phase high performance liquid chromatography, objective physiologically active polypeptides can be efficiently obtained, and that this process also can be advantageously used for purifying samples containing physiologically active polypeptides per se, to thereby accomplished the present invention.
The above-mentioned object can be achieved by providing a process for purifying a polypeptide of the present invention, which process involves the following stages. That is, the present invention concerns a process which comprises
(a) a stage for regulating the pH range of an aqueous solution containing a crude polypeptide to 1-4 to cause impurities to precipitate, followed by removing these impurites, and
(b) adsorbing the supernatant obtained in the above-mentioned stage (a) on a packing material for reversed phase high performance liquid chromatography, followed by eluting an objective polypeptide.
[BRIEF DESCRIPTION OF THE DRAWINGS]
FIGS. 1 (a)-(e) show HPLC elution patterns of human calcitonin precursor solutions purified according to the process of the present invention accoding to stage order;
FIG. 2 shows an HPLC elution pattern of the specimen of FIG. 1 (e) after having been freeze-dried;
FIG. 3 shows an HPLC elution pattern of the highly pure purified human calcitonin precursor obtained by
REFERENCES:
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Raikher et al., Chemical Abstracts, vol. 102 No. 5, p. 411, Ab No: 43971j, Feb. 1985.
Analytical Biochemistry, (vol. 148) No. 1, Jul. 1985 pp. 93-100; Hans Peter Nick et al.
Birnbaum et al, J. Biol Chem., vol. 258, No. 9, May 10, 1983, pp. 5463-5466.
Wasserman et al., J. of Chromatography, vol. 411, pp. 345-354, 1987.
R. S. Birnbaum eta l., "Purification and Amino Acid Sequence of a Noncalcitonin Secretory Peptide Derived from Preprocalcitonin", J. Biol. Chem., vol. 258, No. 9, pp. 5463-5466, (1983).
G. Folena-Wasserman et al., "Assay, Purification and Characterization of a Recombinant Malaria Circumsporozoite Fusion Protein by High-Performance Liquid Chromtatography", J. Chromatogr., vol. 411 . . . .
M. Ohmori et al., "Genetic Construction and High-Level Gene Expression in Escherichia coli of a Precursor of Salmon Calcitonin I", Agric. Biol. Chem., vol. 52, No. 11, pp. 2823-2830, (1988).
J. S. Soldin et al., "Rapid Micromethod for Measuring Anticonvulsant Drugs in Serum by High-Performance Liquid Chromatography", Clin. Chem., vol. 22, No. 6, pp. 856-859, (1976).
Iida Toshii
Kaminuma Toshihiko
Tajima Masahiro
Davenport Avis M.
Shiseido Company Ltd.
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