Preparation and characterization of mouse IL-22 and its four single-amino-acid muteins that act as IL-22 receptor-1 antagonists.

Recombinant mouse interleukin 22 (mIL-22) and its variants encoding four muteins (Y51A, N54A, R55A and E117A) were expressed in Escherichia coli, refolded and purified to homogeneity as monomeric proteins by one-step ion-exchange chromatography. The binding of IL-22 and its four muteins to immobilized mIL-22 receptor α1 extracellular domain (mIL-22 Rα1-ECD) exhibited similar affinity, indicating that the single-amino-acid mutations do not affect its binding properties. Similarly, no differences were found in binding to IL-22 binding protein expressed on the surface of yeast cells, although the affinity of all five proteins to the binding protein was higher than that to IL-22 Rα1-ECD. In an in vitro bioassay, recombinant mIL-22 stimulated signal transducer and activator of transcription-3 phosphorylation in HepG2 cells, whereas the four muteins were completely (Y51A) or almost completely (N54A, R55A and E117A) devoid of this agonistic activity. Furthermore, the agonistic activity of mIL-22 could be inhibited in a dose-dependent manner by the four muteins with almost identical efficiency. mIL-22 and its Y51A mutein were pegylated by methoxy polyethylene glycol-propionylaldehyde-20 kDa, yielding a mixture of mono (75-80%) and double (20-25%) pegylated proteins. The pegylated proteins showed lower affinity (50 and 25%) toward immobilized mIL-22 Rα1-ECD than their non-pegylated analogs. Wild-type pegylated IL-22 exhibited 5- to 10-fold lower activity in the HepG2 bioassay than its non-pegylated counterpart. Preparation of recombinant mIL-22 antagonists provides new tools for the study of IL-22 activity and of eventual therapeutic means for attenuating its negative effects.