Synthesis and Purification of Active Human Tissue Plasminogen Activator From Escherichia coli

Sarmientos, Paolo; Duchesne, Marc; Denèfle, Patrice; Boiziau, Janine; Fromage, Nadine; Delporte, Nadine; Parker, Fabienne; Lelièvre, Yves; Mayaux, Jean‐François; Cartwright, Terence

doi:10.1038/nbt0589-495

Cited by 48 publications

(24 citation statements)

References 29 publications

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“…However, for a more efficient production of protein the method of fusion protein expression is necessary. Generally good refolding yields are obtained by using inclusion body fractions of proteins produced by direct expression without carrier protein [36,371, and the formation of heterogeneous cleavage fragments and the attendant solubility problems can be avoided. Unfortunately direct expression of IL-2Ra in E. coli [22] has not previously been achieved efficiently.…”

Section: Discussionmentioning

confidence: 99%

Isolation and refolding of a mutant methionine‐free interleukin‐2‐receptor α chain synthesized as a fusion protein in Escherichia coli

Seipelt

Engels

1994

European Journal of Biochemistry

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A soluble domain of the interleukin(IL)-2 receptor, the a chain synthesized in Escherichia coli, was employed to study expression and refolding of the protein. The results showed that it is possible to obtain biologically active synthetic methionine-free IL-2 receptor a chain (synIL-2Ra) after BrCN cleavage and renaturation of the crude cleavage material, although the a chain is expressed as a deglycosylated, methionine-free protein. The soluble receptor comprises amino acids 1 -219 and forms 5 disulfide bonds in its biologically active state. Biological activity has been analysed by affinity chromatography and ELISA with mutant [Ala125]IL-2 and monoclonal antibodies as ligands.Renaturation yield is limited mainly by the high aggregation rate of incorrectly folded protein. Aggregation could be limited by varying the oxidation conditions.The deletion of a non-bridging cysteine at position 192 in the synIL-2Ra did not affect the renaturation yield of the receptor protein. Additionally a cysteine-free and methionine-free /I-galactosidase derivative was fused to the soluble synIL-2Ra derivatives to prevent reoxidation of incorrect disulfide bonds in the crude BrCN-cleavage material. It is suggested that cysteine impurities from cyanogen-bromide-cleaved peptides might interfere seriously with the refolding process of the synthetic IL-2 receptor a-subunit.The interleukin-2-receptor (IL-2R)a-chain is part of the IL-2R occurring on the cell surface of lymphoid cells and has been analyzed in various complete and truncated glycosylated forms [l -41. The IL-2R is composed of at least three subunits of which the a chain (CD25, p55) was first characterized and cloned [5-81. The second domain, the p chain (p75), was cloned by Taniguchi and co-workers [9] in 1989. Several authors [lo, 111 identified a third subunit which was proposed to initiate internalisation of the biologically active a/p-complex bound to its ligand IL-2 [12, 131. This p64-protein, termed y chain, was cloned by Sugamura and coworkers [14]. The y chain is essential for internalisation of the high-affinity ligandheceptor complex.All three subunits are independently able to bind IL-2 with different affinities (IL-2/a-subunit, Kd = 10 nM; IL-2Ip-subunit, Kd = 100 nM). A stable interaction of IL-2 and the y-subunit was described recently [15] but the Kd is not known yet. The biologically active complex contains all three receptor subunits and binds IL-2 with high affinity (Kd = 10 pM). The existence of a kinase mechanism for receptor internalisation is currently under investigation.The / 3 and the y chains belong to the hematopoietin superfamily. They promote signal transduction for proliferation Abbreviations. GSH, glutathione (reduced form) ; GSSG, glutathione (oxidized form) ; IL-2, interleukin-2 ; IL-2R interleukin-2 receptor; IL-2Ra, interleukin-2 receptor a chain; synIL-2Ra synthetic methionine-free IL-2Ra chain.and clonal growth of resting T-cells after stimulation by antigen or ligand. The a subunit is mainly involved in ligand binding.The molecular clo...

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Section: Discussionmentioning

confidence: 99%

Isolation and refolding of a mutant methionine‐free interleukin‐2‐receptor α chain synthesized as a fusion protein in Escherichia coli

Seipelt

Engels

1994

European Journal of Biochemistry

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“…tPA was initially synthesised for therapeutic purposes using Bowes melanoma cells but the need for a reliable process and higher yield led to development of prokaryotic and eukaryotic cell to yield the recombinant tPA [28]. Escherichia coli due to its low production cost has been a favoured choice of organism for researchers all over [29] [30]. The first time human tPA was isolated was from E coli as a single chain serine protease consisting of 527 amino acids and Ser at the N-terminus [31].…”

Section: In Vitro Synthesis Of Rtpa: An Overview Of Available Methodsmentioning

confidence: 99%

A Review on Recombinant Tissue Plasminogen Activator: <i>In Vitro</i> Synthesis and Its Use in Treatment of Acute Ischemic Stroke

Sukumaran¹,

Poulse²

2014

OALib

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Tissue-type Plasminogen Activator (tPA) is a serine protease produced by endothelial cells. They target plasminogen, an inactive precursor of plasmin and convert the plasminogen into active plasmin, which degrades the fibrin component of thrombus to aid in dissolution of blood clots. Hence tPA is commonly used in treatment of acute Ischemic stroke in the form of recombinant tissue plasminogen activator (rtPA). rtPA is produced using recombinant technology from different types of vectors depending on the quantities required for the treatment of different diseases. Alteplase (intravenous rtPA) is the most common therapy drug used to treat ischemic strokes. This review gives an outline of the in vitro synthesis of rtPA and its usage in treatment of Acute Ischemic stroke.

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“…Both full-length enzymes had similar specific activities in fibrinolytic assays. ⌬FtPA and protease domain were generated as described below using the same plasmid as for the full-length protein (pXL130), provided by RhonePoulenc Rorer and expressed and renatured from E. coli inclusion bodies in the same way as full-length nonglycosylated tPA (14). Expression of tPA was induced by addition of 40 mg/ml naladixic acid (15) to cultures of E. coli strain N99cI ϩ (Pharmacia, Uppsala, Sweden) grown at 37°C with aeration up to an A 600 nm of approximately 1.0.…”

Section: Methodsmentioning

confidence: 99%

“…Expression of tPA was induced by addition of 40 mg/ml naladixic acid (15) to cultures of E. coli strain N99cI ϩ (Pharmacia, Uppsala, Sweden) grown at 37°C with aeration up to an A 600 nm of approximately 1.0. Cultures were grown at 37°C for a further 90 -120 min before harvesting and processing as described previously for the full-length enzyme (14) with the following modifications. After concentrating renatured protein using SP Trisacryl (IBF Biotechnics, Villeneuve-la-Garenne, France).…”

Section: Methodsmentioning

confidence: 99%

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Regulation of Tissue Plasminogen Activator Activity by Cells

Sinniger

Merton

Fàbregas

et al. 1999

Journal of Biological Chemistry

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A number of cell types have previously been shown to bind tissue plasminogen activator (tPA), which in some cases can remain active on the cell surface resulting in enhanced plasminogen activation kinetics. We have investigated several cultured cell lines, U937, THP1, K562, Molt4, and Nalm6 and shown that they bind both tPA and plasminogen and are able to act as promoters of plasminogen activation in kinetic assays. To understand what structural features of tPA are involved in cell surface interactions, we performed kinetic assays with a range of tPA domain deletion mutants consisting of fulllength glycosylated and nonglycosylated tPA (F-G-K1-K2-P), ⌬FtPA (G-K1-K2-P), K2-P tPA (BM 06.022 or Reteplase), and protease domain (P). Deletion variants were made in Escherichia coli and were nonglycosylated. Plasminogen activation rates were compared with and without cells, over a range of cell densities at physiological tPA concentrations, and produced maximum levels of stimulation up to 80-fold with full-length, glycosylated tPA. Stimulation for nonglycosylated full-length tPA dropped to 45-60% of this value. Loss of N-terminal domains as in ⌬FtPA and K2P resulted in a further loss of stimulation to 15-30% of the full-length glycosylated value. The protease domain alone was stimulated at very low levels of up to 2-fold. Thus, a number of different sites are involved in cell interactions especially within finger and kringle domains, which is similar to the regulation of tPA activity by fibrin. A model was developed to explain the mechanism of stimulation and compared with actual data collected with varying cell, plasminogen, or tPA concentrations and different tPA variants. Experimental data and model predictions were generally in good agreement and suggest that stimulation is well explained by the concentration of reactants by cells.

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Synthesis and Purification of Active Human Tissue Plasminogen Activator From Escherichia coli

Cited by 48 publications

References 29 publications

Isolation and refolding of a mutant methionine‐free interleukin‐2‐receptor α chain synthesized as a fusion protein in Escherichia coli

Isolation and refolding of a mutant methionine‐free interleukin‐2‐receptor α chain synthesized as a fusion protein in Escherichia coli

A Review on Recombinant Tissue Plasminogen Activator: <i>In Vitro</i> Synthesis and Its Use in Treatment of Acute Ischemic Stroke

Regulation of Tissue Plasminogen Activator Activity by Cells

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