Expression, purification, refolding, and characterization of recombinant human interleukin-13: utilization of intracellular processing
| Autor: | Joseph P. Nawrocki, Rachel B. Kapust, Elan Z. Eisenmesser, R. Andrew Byrd, Marie J. Mazzulla, David S. Waugh, Lewis K. Pannell |
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| Rok vydání: | 2000 |
| Předmět: |
Protein Denaturation
Protein Folding Magnetic Resonance Spectroscopy Monosaccharide Transport Proteins medicine.medical_treatment Recombinant Fusion Proteins Protein Renaturation Inclusion bodies Maltose-Binding Proteins Mass Spectrometry law.invention Maltose-binding protein law Endopeptidases TEV protease medicine Escherichia coli Humans Disulfides Protease biology Tobacco etch virus Circular Dichroism Escherichia coli Proteins Proteolytic enzymes biology.organism_classification Fusion protein Biochemistry biology.protein Recombinant DNA ATP-Binding Cassette Transporters Electrophoresis Polyacrylamide Gel Interleukin-3 Carrier Proteins Protein Processing Post-Translational Biotechnology |
| Zdroj: | Protein expression and purification. 20(2) |
| ISSN: | 1046-5928 |
| Popis: | 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 (15)N-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. |
| Databáze: | OpenAIRE |
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