Production of Leukemia Inhibitory Factor in Escherichia coli by a Novel Procedure and Its Use in Maintaining Embryonic Stem Cells in Culture

Gearing, David P.; Nicola, Nicos A.; Metcalf, Donald; Foote, Simon J.; Willson, Tracy A.; Gough, Nicholas M.; Williams, R.Lindsay

doi:10.1038/nbt1189-1157

Cited by 67 publications

(56 citation statements)

References 23 publications

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“…The glutathione Stransferase (GST) fusion proteins were expressed in Escherichia coli and purified by using glutatione Sepharose 4B beads (Amersham Pharmacia). The viral protein was cleaved from GST while still bound to gluthatione-agarose beads by thrombin, as described (24). The p17 protein was further purified (Ͼ98%) by reverse-phase FPLC, reaching a purity more than 98%.…”

Section: Methodsmentioning

confidence: 99%

HIV-1 matrix protein p17 increases the production of proinflammatory cytokines and counteracts IL-4 activity by binding to a cellular receptor

Francesco

Baronio

Fiorentini

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Purified recombinant HIV-1 p17 matrix protein significantly increased HIV-1 replication in preactivated peripheral blood mononuclear cell cultures obtained from healthy donors. Because HIV-1 infection and replication is related to cell activation and differentiation status, in the present study, we investigated the role played by p17 during the process of T cell stimulation. Using freshly isolated peripheral blood mononuclear cells, we demonstrate that p17 was able to enhance levels of tumor necrosis factor ␣ and IFN-␥ released from cells stimulated by IL-2. IL-4 was found to downregulate IFN-␥ and tumor necrosis factor ␣, and p17 restored the ability of cells to produce both cytokines. The property of p17 to increase production of proinflammatory cytokines could be a mechanism exploited by the virus to create a more suitable environment for HIV-1 infection and replication. Our data show that p17 exerts its biological activity after binding to a specific cellular receptor expressed on activated T lymphocytes. The functional p17 epitope involved in receptor binding was found to be located at the NH 2-terminal region of viral protein. Immunization of BALB͞c mice with a 14-aa synthetic peptide representative of the HIV-1 p17 functional region (SGGELDRWEKIRLR) resulted in the development of p17 neutralizing antibodies capable of blocking the interaction between p17 and its cellular receptor. Our results define a role for p17 in HIV-1 pathogenesis and contribute to our understanding of the molecular mechanism of HIV-1 infection and the development of additional antiviral therapeutic strategies.

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Section: Methodsmentioning

confidence: 99%

HIV-1 matrix protein p17 increases the production of proinflammatory cytokines and counteracts IL-4 activity by binding to a cellular receptor

Francesco

Baronio

Fiorentini

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…Transformants of E. coli were produced and selected by conventional procedures [11]. Induction of transformants, lysis of cells, and the recovery of rlTF was carried out as described by Gearing et al [12]. Purified samples were dialysed against 0.l M Tris-HC1, pH 7.4, 1 mM EDTA, l mM EGTA, and injected onto a Superose 12 gel filtration column (Pharmacia) equilibrated with the same buffer, to confirm the identity of the peptide.…”

Section: Expression and Pur(fication Of Recombinant Rltf In E Colimentioning

confidence: 99%

Characterisation of the single copy trefoil peptides intestinal trefoil factor and pS2 and their ability to form covalent dimers

Chinery

Bates

et al. 1995

FEBS Letters

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A bacterial recombinant expression system was established to produce biologically active rat Intestinal Trefoil Factor (rITF). Characterisation of purified rITF shows that both monomers and dimers can be observed under reducing and non-reducing conditions, respectively. Site-directed mutagenesis studies show that Cys57 is necessary for rITF dimer formation. Samples of human gastrointestinal tissue following biopsy also demonstrated the presence of reducible human pS2 and ITF covalent dimers. Three-dimensional models for pS2 and ITF support the hypothesis that both pS2 and ITF can exist as disulphide-linked dimers in vivo and that any proposed function for these peptides must take dimer formation into account.

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“…Gearing et al [12] expressed murine and human LIF in a glutathione-Stransferase based fusion construct while Samal et al [13] reported the expression of mature human LIF as insoluble inclusion bodies with subsequent refolding procedures. In this work we describe the development of a novel production and purification process for recombinant human LIF (hLIF).…”

mentioning

confidence: 99%

Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner

Tomala¹,

Lavrentieva²,

Moretti³

et al. 2010

Protein Expression and Purification

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CitationPreparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner. This is a pre-or post-print of an article published in Tomala, M., Lavrentieva, A., Moretti, P., Rinas, U., Kasper, C., Stahl, F., Schambach, A., Warlich, E., Martin, U., Cantz, T., Scheper, T. Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner regard to large scale cultures of these cells, LIF is needed in high quality and quantity and represents the major costs (90%) of the culture media. In this report, we describe a novel production and purification process for human LIF (hLIF) from recombinant E. coli cultures. hLIF was cloned into pET32b-trx and expressed as soluble protein in fusion with thioredoxin. After purification based on membrane adsorber technology, the fusion protein underwent cleavage by specific TEV protease. Released, soluble hLIF was subsequently purified by cation exchange chromatography and successfully tested for its biological activity using suspension cultures of murine embryonic and induced pluripotent stem cells. Our novel protocol for the production of recombinant hLIF is very suitable and effective for the production of low soluble proteins through expression in fusion with the solubilizing partner thioredoxin.

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Production of Leukemia Inhibitory Factor in Escherichia coli by a Novel Procedure and Its Use in Maintaining Embryonic Stem Cells in Culture

Cited by 67 publications

References 23 publications

HIV-1 matrix protein p17 increases the production of proinflammatory cytokines and counteracts IL-4 activity by binding to a cellular receptor

HIV-1 matrix protein p17 increases the production of proinflammatory cytokines and counteracts IL-4 activity by binding to a cellular receptor

Characterisation of the single copy trefoil peptides intestinal trefoil factor and pS2 and their ability to form covalent dimers

Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner

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