The soluble form of E-selectin is an asymmetric monomer. Expression, purification, and characterization of the recombinant protein.
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 microM. 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 M(r) approximately 75,000 on nonreducing SDS-PAGE. sE-selectin eluted with M(r) approximately 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.
Hensley
P
P
Department of Macromolecular Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939.
McDevitt
P J
PJ
Brooks
I
I
Trill
J J
JJ
Feild
J A
JA
McNulty
D E
DE
Connor
J R
JR
Griswold
D E
DE
Kumar
N V
NV
Kopple
K D
KD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002465:Cell Movement; D002478:Cells, Cultured; D002850:Chromatography, Gel; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA; D019040:E-Selectin; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D007249:Inflammation / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014461:Ultracentrifugation
D015815:Cell Adhesion Molecules; D019040:E-Selectin; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D004247:DNA