Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1988-04-07
1990-12-18
Weimar, Elizabeth C.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435212, 435219, 435815, C12N 964, C12N 948
Patent
active
049786202
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a novel process for selectively recovering single-chain tissue plasminogen activator (hereinafter called "tPA"), or single-chain tPA from an aqueous medium containing both single-chain tPA and double-chain tPA.
More specifically, it is concerned with a process for separating single-chain tPA and double-chain tPA from an aqueous medium, which contains both single-chain tPA and double-chain tPA, by using a carrier with the Erythrina trypsin inhibitor borne thereon.
Namely, this invention relates to a process for isolating and purifying single-chain tPA at a high purity from an aqueous medium containing both single-chain tPA and double-chain tPA and separating both tPAs from each other by using the Erythrina trypsin inhibitor (hereinafter abbreviated as "ETI") "which occurs in seeds of Erythrina latissima and other Erythrina plants and is an immobilized Kunitz inhibitor that serves as an inhibitor for trypsin, plasmin and tPA but does not act on urokinase" [F. J. Joubert et al., Hoppe Seyler's Z. Physiol. Chem. 362, 531-538 (1981)].
BACKGROUND ART
The following process has been known as one example of the use of ETI for the purification of tPA. Namely, the purification process comprises providing human melanoma cells, causing the cells to produce tPA in a serum-free medium, harvesting the culture crop, charging it into an ETI column to adsorb tPA, and then eluting ETI-adsorbed tPA with a 1-3M aqueous solution of potassium thiocyanate (Japanese Patent Laid-Open No. 118717/1984). This purification process is however a process for purifying tPA but does not relate to the separation and purification of single-chain tPA and double-chain tPA.
The present inventors have already developed inter alia a process for separating and removing tPA which occurs in a culture medium containing fetal calf serum, reacts to anti-human tPA antibody and has a molecular weight of 110,000.+-.20,000 daltons as well as a process for culturing cells, in which tPA gene has been integrated by using recombinant DNA technology, and then separating tPA derived from host cells and tPA originated from human cells (Japanese Patent Laid-Open No. 168601/1985).
Substances having the above tPA activity include both single-chain and double-chain ones. It has been found that both of them have a molecular weight of about 70,000 daltons but the single-chain tPA and double-chain tPA are different in plasminogen activating ability and affinity to fibrins. Namely, it has been uncovered that double-chain tPA has plasminogen activating ability as high as about ten times compared with single-chain tPA (Japanese Patent Laid-Open No. 118717/1984) and on the other hand, single-chain tPA has greater affinity to fibrins compared with double-chain tPA and upon adsorption on fibrins, is converted very quickly into double-chain tPA. Single-chain tPA is therefore preferable for obtaining the desired activity to the maximum extent at the site of clots [D. C. Rijken, et al., J. Biol. Chem., 257, 2920-2925 (1982)].
As a method known for obtaining single-chain tPA, it has been known to add a proteolytic enzyme inhibitor upon culture of cells so as to inhibit the conversion from single-chain tPA into double-chain tPA (D. C. Rijken, et al., J. Biol. Chem., 256, 7035-7041 (1981)]. This method is however very difficult to suppress the conversion into double-chain tPA completely under widely-varying conditions such as the kind of cells, the manner of cell cultivation and the cycle of cultivation. Namely, the kind and concentration of a proteolytic enzyme contained in a culture medium for cells generally differ from one medium to another and considerable difficulties are involved in controlling the action of such proteolytic enzymes. On the other hand, there is a wide variety of proteolytic enzyme inhibitors, including those giving adverse influence to the multiplication of cells. A limitation is therefore imposed on the kind of proteolytic enzyme inhibitors which are usable in the cultivation of cells. Furthermore, some kinds of p
REFERENCES:
J. Biological Chemistry, vol. 257, pp. 2920 to 2925 in Mar. 25, 1982, by D. C. Rijken et al.
J. Biological Chemistry, vol. 256, pp. 7035 to 7041 in Jul. 10, 1981, by D. C. Rijken et al.
Methods in Enzymology, vol. 36, Part B, pp. 59 to 72 and 77 to 102, edited by W. B. Jakoky and Aleir Wilchele.
Kawashima Nobuhiro
Mori Kunizo
Morii Mitsuyoshi
Morii Noriko
Ohoka Masaharu
Mitsui Toatsu Chemicals Incorporated
Patterson Jr. Charles L.
Weimar Elizabeth C.
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