The Soluble Form of E-selectin Is an Asymmetric Monomer: Expression, Purification, and Characterization of the Recombinant Protein

Preston Hensley, Patrick J. McDevitt, Ian S Brooks, John J. Trill, John A. Feild, Dean E. McNulty, Janice R. Connor, Don E. Griswold, N. Vasant Kumar, Kenneth D. Kopple, Steven A. Carr, Barbara J. Dalton, Kyung Johanson

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Abstract

The gene coding for a soluble form of human E-selectin (sE-selectin) has been expressed in Chinese hamster ovary (CHO) cells. Cells seeded into a hollow fiber reactor secreted protein at a level of 160 mg/liter. The protein was purified to >95% pure and low endotoxin (<2 ng/mg), using physiological pH and buffers. The amino acid composition and N-terminal sequence were as predicted from the cDNA sequence. HL-60 cells bound to sE-selectin-coated plates in a dose-dependent manner, and this binding could be blocked up to 100% by pretreatment of HL60 cells with sE-selectin. The concentration of sE-selectin required for 50% inhibition was 1 μM. This value puts an upper limit for the affinity of E-selectin for its natural receptor. sE-selectin also inhibited inflammatory migration of neutrophils in a selective fashion. Purified sE-selectin exhibited a broad band of Mr ∼ 75,000 on nonreducing SDS-PAGE. sE-selectin eluted with Mr ∼ 310,000 from size exclusion chromatography at physiological pH and buffers, suggesting an oligomeric state. Matrix-assisted laser-desorption MS gave a molecular weight of 80,000, while the minimum monomer molecular weight from the gene sequence should be 58,571, demonstrating that the monomeric molecule thus expressed had 27% carbohydrate. Equilibrium analytical ultracentrifugation gave an average solution molecular weight of 81,600 (± 4,500). Velocity ultracentrifugation gave a sedimentation coefficient of 4.3 S and, from this, an apparent axial ratio of 10.5:1, assuming a prolate ellipsoid of revolution. An analysis of the NMR NOESY spectra of sE-selectin, sialyl-Lewis X, and sE-selectin with sialyl-Lewis X demonstrates that the recombinant protein binds sialyl-Lewis X productively. Hence, in solution, sE-selectin is a functional elongated monomer.

Original languageEnglish (US)
Pages (from-to)23949-23958
Number of pages10
JournalJournal of Biological Chemistry
Volume269
Issue number39
StatePublished - Sep 30 1994
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Hensley, P., McDevitt, P. J., Brooks, I. S., Trill, J. J., Feild, J. A., McNulty, D. E., Connor, J. R., Griswold, D. E., Kumar, N. V., Kopple, K. D., Carr, S. A., Dalton, B. J., & Johanson, K. (1994). The Soluble Form of E-selectin Is an Asymmetric Monomer: Expression, Purification, and Characterization of the Recombinant Protein. Journal of Biological Chemistry, 269(39), 23949-23958.