BIOLOGICAL AND BIOPHYSICAL CHARACTERIZATION OF RECOMBINANT SOLUBLE HUMAN E-SELECTIN PURIFIED AT LARGE-SCALE BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A first step in the development of a high-throughput screening assay f or antagonists of human E-selectin is the purification and characteriz ation of the selectin. In the present paper we describe a single-step, rapid, reversed-phase HPLC purification protocol for the recombinant, soluble form of human E-selectin (rshE-selectin) produced in Chinese hamster ovary cells. The procedure resulted in high protein yields wit h recoveries of greater than 98%. Characterization of the reversed-pha se purified rshE-selectin showed this product to be analogous to rshE- selectin purified using conventional chromatographic techniques with r espect to biological activity and molecular shape. However,;the carboh ydrate composition of reversed-phase purified rshE-selectin, which had been variable with conventionally purified material, was found to be constant across several isolations. The protocol described herein elim inated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for addi tional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are disti nct advantages over those previously published. It is expected that ot her mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.