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 extracellular region of the membrane-bound human CD23 is processed into at least 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, certain autoimmune diseases, or chronic lymphocytic leukemia. Therefore, inhibition 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 proteins, the 33-kDa form has not been purified and characterized. To further investigate the multiple roles of sCD23 fragments and to devise assays to identify potent small-molecule inhibitors of CD23 processing, we have produced the 33-kDa form of sCD23 using Chinese hamster ovary (CHO) and Drosophila S2 cells. The CHO-expressed 33-kDa protein was found to undergo proteolytic degradation during cell growth and during storage of purified protein, resulting in accumulation of a 25-kDa form. The Drosophila system expressed the 33-kDa sCD23 in a stable form that was purified and demonstrated to be more active than the CHO-derived 25-kDa form in a monocyte TNFalpha release assay.
Khandekar
S S
SS
Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Mayer
R J
RJ
Cusimano
D M
DM
Katchur
S R
SR
Appelbaum
E R
ER
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004330:Drosophila / Q000166:cytology / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009000:Monocytes / Q000378:metabolism; D017455:Receptors, IgE / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D017455:Receptors, IgE; D014409:Tumor Necrosis Factor-alpha