Enzymatic Deglutathionylation to Generate Interleukin-4 Cysteine Muteins with Free Thiol

Interleukin-4 (IL-4) is a prototypical regulator protein of the immune system that is crucial for the pathogenesis and maintenance of asthma and other atopic diseases. It, together with IL-13, uses the IL-4 receptor α chain (IL-4Rα) to signal into immune and other cells. An IL-4 mutein acting as a dual IL-4/IL-13 receptor antagonist is in clinical development. Here, it is described how IL-4 muteins containing a single engineered cysteine with a free thiol can be prepared and used for site-specific chemical modification. The muteins were initially expressed in E. coli, refolded, and purified, but not in a fully reduced nonconjugated form. Attempts to reduce the cysteine chemically failed because the native disulfide bonds of IL-4 were also reduced under similar conditions. Therefore, an enzymatic procedure was developed to reduce glutathionylated IL-4 cysteine muteins employing glutaredoxin and reduced glutathione. Cysteine muteins engineered at four different positions around the IL-4Rα binding site were enzymatically reduced at different rates. All muteins were prepared with free thiol in reasonable yield and were modified by <i>N</i>-ethylmaleimide (NEM) or maleimido-PEG. The effect on IL-4Rα binding of cysteine substitution and of the site-specific modification by glutathione, <i>N</i>-ethylmaleimide (NEM), or a branched 2.36 kDa poly­(ethylene glycol) (PEG) will be discussed