Expression and purification of stable 33-kDa soluble human CD23 using the Drosophila S2 expression system

CD23, a 45-kDa type II membrane glycoprotein present on B cells, monocytes, and other human immune cells, is a low-affinity receptor for IgE. The extr acellular region of the membrane-bound human CD23 is processed into at leas t four soluble (s) CD23 forms, with apparent molecular masses of 37, 33, 29 , and 25 kDa. High levels of sCD23 are found in patients with allergy, cert ain autoimmune diseases, or chronic lymphocytic leukemia. Therefore, inhibi tion of the processing of membrane-bound CD23 to control the cytokine-like effects of sCD23 offers a novel therapeutic opportunity. While the 37-, 29- , and 25-kDa forms of sCD23 have been expressed previously as recombinant p roteins, the 33-kDa form has not been purified and characterized. To furthe r investigate the multiple roles of sCD23 fragments and to devise assays to identify potent small-molecule inhibitors of CD23 processing, we have prod uced the 33-kDa form of sCD23 using Chinese hamster ovary (CHO) and Drosoph ila S2 cells. The CHO-expressed 33-kDa protein was found to undergo proteol ytic degradation during cell growth and during storage of purified protein, resulting in accumulation of a 25-kDa form. The Drosophila system expresse d the 33-kDa sCD23 in a stable form that was purified and demonstrated to b e more active than the CHO-derived 25-kDa form in a monocyte TNF alpha rele ase assay. (C) 2001 Academic Press.