Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli

Pennica, Diane; Holmes, William E.; Kohr, William J.; Harkins, Richard N.; Vehar, Gordon A.; Ward, Carole; Bennett, William F.; Yelverton, Elizabeth; Seeburg, Peter H.; Heyneker, Herbert L.; Goeddel, David V.; Collen, Désiré

doi:10.1038/301214a0

Cited by 1,246 publications

(553 citation statements)

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“…The incorporation of diisopropylfluorophosphate into an active form of t-PA [4,5] and the primary structure of t-PA predicted from its c-DNA sequence [6] revealed that this protein is a typical serine protease. t-PA isolated from tissues [5,7] or cell culture [4] is an active enzyme that is Abbreviations: t-PA, tissue plasminogen activator; -p-NA, -p-nitro-anilide; SBTI, soybean trypsin inhibitor; LBTI, lima bean trypsin inhibitor; HMW-kininogen, high molecular mass kininogen; TBS, 50 mM Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl.…”

Section: Introductionmentioning

confidence: 99%

Proteolytic activation of tissue plasminogen activator by plasma and tissue enzymes

1984

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Tissue kallikrein and factor Xa were found to activate tissue plasminogen activator (t-PA) at a rate comparable with that of plasmin. During the activation reaction, the single-chain molecule was converted into a two-chain form. A slight t-PA activating activity was also found in plasma kallikrein. Other activated coagulation factors, factor XIIa, factor XIa, factor IXa, factor VIIa, thrombin and activated protein C had no effect on t-PA activation. t-PA was also activated by a tissue kallikrein-like enzyme that was isolated from the culture medium of melanoma cells. These results indicate that tissue kallikrein and factor Xa may participate in the extrinsic pathway of human fibrinolysis. Fibrinolysis t-PA Tissue kallikrein Coagulation factor

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Section: Introductionmentioning

confidence: 99%

Proteolytic activation of tissue plasminogen activator by plasma and tissue enzymes

1984

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“…The amino acid seqeunce of human urokinase was from Steffens et al (23) and Giinzler et al (8), and that deduced from the nucleotide sequence of human urokinase was from Heyneker et al (9) and Verde et al (28). The complete amino acid sequence of human tissue-type plasminogen activator deduced from the nucleotide sequence was the work of Pennica et al (21). From these data, twelve seqeunces constituting kringles were collected and subjected to computer analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The nucleotide sequence of urokinase was determined by Heyneker et al (9), who also showed the existence of a complete kringle arrangement in the A-chain. Pennica et al (21) recently deduced the amino acid sequence of a human tissue-type plasminogen activator from the nucleotide sequence. Two kringle structures are apparent in the heavy-chain region before the potential cleavage 16,20,26,27).…”

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confidence: 99%

Homology of kringle structures in urokinase and tissue-type plasminogen activator: The phylogeny with the related serine proteases.

Takahashi

Gojobori

Naora

1985

Cell Struct. Funct.

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ABSTRACT. Twelve amino acid sequences of kringle-forming polypeptides were compiled from the known sequences of urokinase A-chain (human), a tissue-type plasminogen activator (human), prothrombin (human and bovine), and plasminogen (human). Their sequence homologies with maximum match were examined by a computer program. A homology alignment and graphic matrix analyses did show that they had a great degree of homology. All the cystein residues responsible for the kringle structures of urokinase and the tissue-type plasminogen activator were confidently preserved as well as other proteins.

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“…64 The same cell line was used to clone the t-PA cDNA (2,530 bp) for the first time and it was expressed under the control of the trp promoter in E. coli. 67 An efficient, properly processed and glycosylated form of recombinant t-PA (rt-PA) was produced using mammalian cells with properties similar to the Bowes cell line t-PA. 68 Majidzadeh-A and co-workers (2010) expressed the gene encoding full-length human tPA under the control of the AOX1 promoter and S. cerevisiae a-mating factor signal sequence in P. pastoris GS115 with an amidolytic activity of 1,650 IU/ml. 69 Human t-PA (located on chromosome 8) was first isolated as a 70 kDa (527 amino acids) single chain polypeptide that is converted to the active 2-chain form via plasmin-mediated cleavage across the Arg 275 -Ile 276 bond.…”

Section: Second Generation Plasminogen Activatorsmentioning

confidence: 99%

The evolution of recombinant thrombolytics: Current status and future directions

Adivitiya

Khasa

2016

Bioengineered

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Cardiovascular disorders are on the rise worldwide due to alcohol abuse, obesity, hypertension, raised blood lipids, diabetes and age-related risks. The use of classical antiplatelet and anticoagulant therapies combined with surgical intervention helped to clear blood clots during the inceptive years. However, the discovery of streptokinase and urokinase ushered the way of using these enzymes as thrombolytic agents to degrade the fibrin network with an issue of systemic hemorrhage. The development of second generation plasminogen activators like anistreplase and tissue plasminogen activator partially controlled this problem. The third generation molecules, majorly t-PA variants, showed desirable properties of improved stability, safety and efficacy with enhanced fibrin specificity. Plasmin variants are produced as direct fibrinolytic agents as a futuristic approach with targeted delivery of these drugs using liposome technlogy. The novel molecules from microbial, plant and animal origin present the future of direct thrombolytics due to their safety and ease of administration.

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Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli

Cited by 1,246 publications

References 52 publications

Proteolytic activation of tissue plasminogen activator by plasma and tissue enzymes

Proteolytic activation of tissue plasminogen activator by plasma and tissue enzymes

Homology of kringle structures in urokinase and tissue-type plasminogen activator: The phylogeny with the related serine proteases.

The evolution of recombinant thrombolytics: Current status and future directions

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