Expression, purification, refolding, and characterization of recombinant human interleukin-13: utilization of intracellular processing.
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.
Eisenmesser
E Z
EZ
Macromolecular NMR Section, National Cancer Institute-FCRDC, Frederick, Maryland 21702, USA.
Kapust
R B
RB
Nawrocki
J P
JP
Mazzulla
M J
MJ
Pannell
L K
LK
Waugh
D S
DS
Byrd
R A
RA
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006801:Humans; D007377:Interleukin-3 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D058619:Maltose-Binding Proteins; D013058:Mass Spectrometry; D009004:Monosaccharide Transport Proteins; D011489:Protein Denaturation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D007377:Interleukin-3; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D010450:Endopeptidases; C099488:TEV protease