Plasminogen Activator (Urokinase) from Cultured Cells

Lj, Lewis

doi:10.1055/s-0038-1666938

Cited by 29 publications

(11 citation statements)

References 2 publications

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“…When the urokinase activity reached a particular threshold value, the cells were again induced to secrete urokinase into the medium leading to a temporary rise in the urokinase secretion profile. Park et al (1999) reported the production of plasminogen activators from oocytes and cumulus cells and according to their findings urokinase activity in the cultured broth increased at 12 h of culture without any further increase at 24 h. A minor decline in urokinase activity in the spent medium was observed after 144 h. Similar losses of urokinase activity on prolonged maintenance of the cultures have been reported previously by Lewis (1979). Further studies are needed to be performed on inhibitor secretion by this cell line in order to have a complete understanding of the urokinase secretion profile of the cell line.…”

Section: Production Of Urokinasesupporting

confidence: 76%

“…The time course of plasminogen activator production is controlled by negative feedback and stops when its level in the medium reaches a critical concentration (Lewis, 1979). However, on removal of the enzyme by continuously replacing the medium, the cells continue to produce enzyme at a constant rate for a period of several weeks.…”

Section: Integrated Production and Separation Of Urokinase From Ht108mentioning

confidence: 99%

“…5). Losses of urokinase activity on prolonged maintenance of the cultures might also play a role in the occasional decline in urokinase activity in the medium (Lewis, 1979). Hence, it can be concluded that there are different regulating mechanisms operating inside the system, which include urokinase, PAIs, and the (inactive , active) zymogen forms of urokinase.…”

Section: Integrated Production and Separation Of Urokinase From Ht108mentioning

confidence: 99%

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Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

Kumar

Bansal

Nandakumar

et al. 2006

Biotechnol. Bioeng.

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An integrated cell cultivation and protein product separation process was developed using a new type of supermacroporous polyacrylamide gel, called cryogel (pAAm-cryogel) support matrix. Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. The secreted protein was isolated from the circulating medium using a chromatographic capture column. A pAAm cryogel support with covalently coupled gelatin (gelatin-pAAm cryogel) was used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. The cells were attached to the matrix within 4-6 h of inoculation and grew as a tissue sheet inside the cryogel matrix. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor. No morphological changes were observed in the cells eluted from the gelatin-cryogel support and re-cultured in T-flask. The gelatin-pAAm cryogel bioreactor was further connected to a pAAm cryogel column carrying Cu(II)-iminodiacetic acid (Cu(II)-IDA)-ligands (Cu(II)-IDA-pAAm cryogel), which had been optimized for the capture of urokinase from the conditioned medium of the cell lines. Thus an automated system was built, which integrated the features of a hollow fiber reactor with a chromatographic protein separation system. The urokinase was continuously captured by the Cu(II)-IDA-pAAm cryogel column and periodically recovered through elution cycles. The urokinase activity increased from 250 PU/mg in the culture fluid to 2,310 PU/mg after recovery from the capture column which gave about ninefold purification of the enzyme. Increased productivity was achieved by operating integrated bioreactor system continuously for 32 days under product inhibition free conditions during which no backpressure or culture contamination was observed. A total 152,600 Plough units of urokinase activity was recovered from 500 mL culture medium using 38 capture columns over a period of 32 days.

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Section: Production Of Urokinasesupporting

confidence: 76%

Section: Integrated Production and Separation Of Urokinase From Ht108mentioning

confidence: 99%

Section: Integrated Production and Separation Of Urokinase From Ht108mentioning

confidence: 99%

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Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

Kumar

Bansal

Nandakumar

et al. 2006

Biotechnol. Bioeng.

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“…Because t-PA production is regulated by negative feedback control, the replacement of culture broth with fresh medium can release the control and increase productivity (Kadouri and Bohak 1983;Lewis 1979). We have investigated continuous t-PA production with a perfusion culture device using non-woven fabrics and ceramic pieces as cell matrices.…”

Section: Introductionmentioning

confidence: 99%

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth

Mitsuda

Kobayashi

Matsuda

et al. 1991

Appl Microbiol Biotechnol

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Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm3 per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed.

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“…Urokinase (EC 3.4.99.26), an enzyme synthesized in human kidney (Andrassy et al, 1976; Lewis, 1979) and released in urine (Williams, 1951;Sobel et al, 1952), functions as an activator of plasminogen. Active plasmin resulting from the proteolytic cleavage of its precursor, plasminogen, then exhibits fibrinolytic activity.…”

Section: Introductionmentioning

confidence: 99%

Molecular Cloning, Sequencing, and Expression inEscherichia coliof Human Preprourokinase cDNA

Jacobs¹,

Cravador²,

Loriau³

et al. 1985

DNA

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A cDNA library derived from human carcinoma cells was used to isolate a clone, pULB1000, coding for the preproenzyme form of human urokinase. This clone carries the full-length sequence coding for the signal peptide and for the A chain (157 amino acids) and B chain (253 amino acids) of urokinase in tandem. The sequence of the cDNA predicts the presence of a single lysine residue between the last amino acid of the mature A polypeptide (Phe-157) and the first amino acid of the mature B polypeptide (Ile-1). The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence data with three exceptions, the reported cysteine residue at position 131 in the A chain is a tryptophan, and glycine 366 and alanine 410 in the B chain are, respectively, a cysteine and a valine in our clone. A large Bgl I fragment (1482 bp), derived from the clone pULB1000 coding for most of the signal peptide and for the A and B chains, has been subcloned into the expression vector pCQV2. Heat induction of E. coli cells carrying the recombinant plasmid leads to the production of urokinase-like polypeptides having the expected molecular weights and being specifically recognized by antibodies raised against natural human urokinase.

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Plasminogen Activator (Urokinase) from Cultured Cells

Cited by 29 publications

References 2 publications

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth

Molecular Cloning, Sequencing, and Expression inEscherichia coliof Human Preprourokinase cDNA

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