Expression, Purification, Refolding, and Characterization of Recombinant Human Interleukin-13: Utilization of Intracellular Processing Elan Zohar Eisenmesser,* Rachel B. Kapust,† Joseph P. Nawrocki,‡ Marie J. Mazzulla,* ,1 Lewis K. Pannell,‡ David S. Waugh,† and R. Andrew Byrd* ,2 *Macromolecular NMR Section, Structural Biophysics Laboratory, and †Protein Engineering Section, Macromolecular Crystallography Laboratory, National Cancer Institute—FCRDC, Frederick, Maryland 21702; and ‡Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892 Received March 10, 2000, and in revised form May 30, 2000 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. So- lution studies of IL-13 and its soluble receptors may facilitate the design of antagonists/agonists which would require milligram quantities of specifically la- beled 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) pro- tease. Coexpression of the fusion protein and TEV pro- tease led to in vivo cleavage, resulting in high levels of hIL-13 production. hIL-13, localized to inclusion bod- ies, was purified and refolded to yield approximately 2 mg per liter of bacteria grown in minimal media. Sub- sequent biochemical and biophysical analysis of both the unlabeled and 15 N-labeled protein revealed a bio- active helical monomer. In addition, the two disulfide bonds were unambiguously demonstrated to be Cys29 –Cys57 and Cys45–Cys71 by a combined proteo- lytic digestion and mass spectrometric analysis. © 2000 Academic Press Key Words: fusion protein; interleukin-13; IL-13; mal- tose-binding protein; refolding; cytokine. IL-13 3 is a member of the short-four-helix-bundle cytokines, one of several classes of helical cytokines (1) that include IL-2, IL-3, IL-4, IL-5, and granulocyte- macrophage colony-stimulating factor (GM-CSF) (1– 4). IL-13 shares a 25% sequence identity with IL-4 and both genes are found on chromosome 5 along with those of IL-3, IL-5, IL-9, and GM-CSF (5). IL-13 is produced by T-cells, B-cells, mast cells, and basophils. Like other cytokines, IL-13 is pleiotropic, with both proinflammatory and anti-inflammatory immune re- sponses (5,6). Its proinflammatory effects include iso- type switching to IgE production in B-cells (7) and an increase in vascular cell adhesion molecule-1 produc- tion on endothelial cells (6). The latter effect has been implicated in allergenic asthma, and the resulting in- flammation may be therapeutically mediated by IL-13 (6). Two recent reports support a direct link between IL-13 treatment and airway hyperresponsiveness in murine models (8,9). Studies with anti-inflammatory cyclic nucleotide PDE inhibitors indicate that their effects are due to the downregulation of several cyto- kines, including IL-13 (10 –14). The anti-inflammatory effects of IL-13 include downregulation of proinflam- matory cytokines (i.e., IL-6, IL-12, and TNF-) and chemokines (i.e., MIP-1, MIP-1, and monocyte che- moattractant protein 3) and the upregulation of 12/15- lipoxygenases (5,6,15–18). Many functions of IL-13 overlap with those of IL-4, due to a shared receptor composed of IL-13R1 and IL-4R (gp140) that is present in synovial fibroblasts, endothelial cells, and hematopoietic cells (19 –22). IL-13 binding to the IL-13R1/IL-4R heterodimer leads to a number of signal transduction events that include Janus tyrosine kinases 1 and 2 (JAK1 and JAK2), tyrosine kinase 2 (TYK2), signal transducer 1 Current address: SmithKline Beecham Pharmaceuticals, 1250 S. Collegeville Road, Collegeville, PA 19426. 2 To whom correspondence should be addressed. Fax: (301) 846- 6231. E-mail: rabyrd@ncifcrf.gov. 3 Abbreviations used: NMR, nuclear magnetic resonance; CD, cir- cular dichroism; CIP, controlled intracellular processing; DTT, di- thiothreitol; IL, interleukin; IP, intracellular processing; MBP, mal- tose-binding protein; MIP, macrophage inflammatory protein; PBMC, peripheral blood monocytes; PDE, phosphodiesterase; TEV, tobacco etch virus; TNF-, tumor necrosis factor-. Protein Expression and Purification 20, 186 –195 (2000) doi:10.1006/prep.2000.1283, available online at http://www.idealibrary.com on 186 1046-5928/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.