Claims
- 1. A method of purifying crude human tissue plasminogen activator (tPA) containing, as an impurity, a protein which is reactive with an anti-human tPA antibody and has a molecular weight of 110,000 .+-. 20,000 daltons, existing in a culture medium containing serum, which method consists essentially of the steps of:
- (a) passing the crude tPA to be purified through a column of an affinity reagent containing an immobilized Kunitz inhibitor which is produced in seeds of Erythrina latissima and other Erythrina plants and acts as an inhibitor to trypsin, plasmin and tPA but not to urokinase, to thereby bind the tPA to the affinity reagent and separate unbound material
- (b) washing the column with a saline buffer solution containing salts at neutral or alkaline pH containing 0.5 to 3.0M NaCl; and thereafter
- (c) eluting the tPA-bound affinity reagent with a buffer of acidic pH.
- 2. The method as claimed in claim 1, wherein the crude tPA is produced by a transformant strain obtained by using non-human animal cells as host cells and integrating the human tPA gene therein, and the tPA-bound affinity reagent is eluted to produce tPA in a purified form free of protein having the molecular weight of 110,000 .+-. 20,000 daltons and tPA derived from the non-human cells.
- 3. The method as claimed in claim 1, wherein the tPA is secreted from culture cells derived form melanoma cells.
- 4. The method as claimed in claim 1, wherein said tPA is secreted from culture cells derived from normal human tissues.
- 5. The method as claimed in claim 1, wherein said tPA is secreted from culture cells obtained by integrating the human tPA gene using recombinant DNA technology.
- 6. The method as claimed in claim 5, wherein said human tPA gene is derived from melanoma cells.
- 7. The method as claimed in claim 5, wherein said human tPA gene is derived from normal human tissues.
- 8. The method as claimed in claim 5, wherein said tPA is produced by a transformant strain obtained using mammalian cells as host cells and integrating the human tPA gene therein.
- 9. The methods as claimed in claim 8, wherein said mammalian cells are mouse fibroblast (C127).
- 10. The method for preparing purified human tissue plasminogen activator (tPA) free of a protein which is reactive with an anti-human tPA antibody and has a molecular weight of 110,000 .+-. 20,000 daltons, said method consisting essentially of the steps of:
- (A) cultivating a tPA-yielding transformant strain non-human animal cell culture obtained by integrating the human tPA gene in the animal cells in a suitable culture medium containing fetal bovine serum;
- (B) allowing the tPA-yielding cells to secrete tPA in the presence of fetal bovine serum to produce tPA together with a protein which is reactive with an anti-human tPA antibody and has a molecular weight of 110,000 .+-. 20,000 daltons;
- (C) passing the tPA-containing mixture of step (B) through a column of an affinity reagent containing an immobilized Kunitz inhibitor which is produced in seeds of Erhthrina latissima and other Erythrina plants and acts as an inhibitor to trypsin, plasmin and tPA but not to urokinase, to thereby bind the tPA to the affinity reagent and separate unbound material,
- (D) washing the column with saline buffer solution containing salts at neutral or alkaline pH containing 0.5 to 3.0M NaCl;
- (E) eluting the tPA-bound affinity reagent with a buffer of acidic pH; and
- (F) collecting the tPA in substantially pure form essentially free from the 110,000 .+-. 20,000 daltons protein.
Priority Claims (1)
Number |
Date |
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60-168601 |
Aug 1985 |
JPX |
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Parent Case Info
This is a continuation of application Ser. No. 07/287,655, filed Dec. 21, 1988, now abandoned. This is a continuation of application Ser. No. 06/887,514, filed Jul. 21, 1986, now abandoned.
Foreign Referenced Citations (2)
Number |
Date |
Country |
0112122 |
Jun 1984 |
EPX |
0143081 |
May 1985 |
EPX |
Non-Patent Literature Citations (2)
Entry |
Chemical Abstracts, vol. 102, No. 1, Jan. 7, 1985, p. 238 Abstract No. 2474f, C. Heussen et al "Purification of Human Tissue . . . ". |
Kluft, C. et al., (1983) Advances in Biotechnological Processes, vol. 2, pp. 97-110, A. R. Liss, Inc, N.Y. (Mizrahi and van Wezel, editors). |
Continuations (2)
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Number |
Date |
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Parent |
287655 |
Dec 1988 |
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Parent |
887514 |
Jul 1986 |
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