HIGH-LEVEL EXPRESSION AND REFOLDING OF MOUSE INTERLEUKIN-4 SYNTHESIZED IN ESCHERICHIA-COLI

Mouse Interleukin 4 is a 20-kDa glycoprotein, synthesized by activated T lymphocytes and mast cells, which regulates the growth and/or diffe rentiation of a broad spectrum of target cells of the immune system, i ncluding B and T lymphocytes, macrophages, and hematopoietic progenito r cells. Using an inducible recA promoter and the g10-L ribosome bindi ng site, recombinant non-glycosylated interleukin 4 (IL-4) was express ed as 17% of total cellular protein in Escherichia coil inclusion bodi es, as a reduced, inactive 14.5-kDa polypeptide. The protein was refol ded and aggregates dissociated when three disulfide bonds were reforme d by slowly decreasing the concentration of guanidine hydrochloride an d cysteine. The oxidized monomer was purified to homogeneity by sequen tial ion-exchange and size exclusion chromatography. When compared wit h native IL-4, E. coil-derived IL-4 displayed an identical specific ac tivity of 4-7 x 10(7) units/mg. This recombinant IL-4 contained a thre e-amino-acid NH2-terminal extension, which did not affect its biologic al activity. Purified biologically active protein consisted of three i soforms as shown by two-dimensional gel electrophoresis, with a pI gre ater than 9.0. These data suggest that neither glycosylation nor the N H2 terminus of mouse IL-4 play a critical role in contributing to its in vitro biological activity.