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Expression, purification, refolding, and characterization of recombinant human interleukin-13: Utilization of intracellular processing

  1. Author:
    Eisenmesser, E. Z.
    Kapust, R. B.
    Nawrocki, J. P.
    Mazzulla, M. J.
    Pannell, L. K.
    Waugh, D. S.
    Byrd, R. A.

  2. Author Address

    NCI, Frederick Canc Res & Dev Ctr, Macromol NMR Sect, Struct Biophys Lab, Frederick, MD 21702 USA. NCI, Frederick Canc Res & Dev Ctr, Macromol NMR Sect, Struct Biophys Lab, Frederick, MD 21702 USA. NCI, Frederick Canc Res & Dev Ctr, Prot Engn Sect, Macromol Crystallog Lab, Frederick, MD 21702 USA. NIDDKD, Bioorgan Chem Lab, Bethesda, MD 20892 USA.
    1. Year: 2000
  2. Journal: Protein Expression and Purification
    1. 20
    2. 2
    3. Pages: 186-195
  4. Type of Article: Article
  1. Abstract:

    Interleukin-13 (IL-13) is a pleiotropic cytokine that elicits both proinflammatory and anti-inflammatory immune responses. Recent studies underscore its role in several diseases, including asthma and cancer. Solution studies of IL-13 and its soluble receptors may facilitate the design of antagonists/agonists which would require milligram quantities of specifically labeled protein. A synthetic gene encoding human IL-13 (hIL-13) was inserted into the pMAL-c2 vector with a cleavage site for the tobacco etch virus (TEV) protease. Coexpression of the fusion protein and TEV protease led to in vivo cleavage, resulting in high levels of hIL-13 production. hIL-13, localized to inclusion bodies, was purified and refolded to yield approximately 2 mg per liter of bacteria grown in minimal media. Subsequent biochemical and biophysical analysis of both the unlabeled and N-15-labeled protein revealed a bioactive helical monomer. In addition, the two disulfide bonds were unambiguously demonstrated to be Cys29- Cys57 and Cys45-Cys71 by a combined proteolytic digestion and mass spectrometric analysis. (C) 2000 Academic Press.

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