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 for antagonists of human E-selectin is the purification and characterization 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 with recoveries of greater than 98%. Characterization of the reversed-phase purified rshE-selectin showed this product to be analogous to rshE-selectin purified using conventional chromatographic techniques with respect to biological activity and molecular shape. However, the carbohydrate 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 eliminated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for additional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are distinct advantages over those previously published. It is expected that other mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.
Burrows
S D
SD
Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, USA.
Franklin
S G
SG
Brigham-Burke
M R
MR
Brooks
I S
IS
McNulty
D E
DE
Feild
J A
JA
Anumula
K R
KR
O'Shannessy
D J
DJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D002448:Cell Adhesion; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae; D019040:E-Selectin / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D018922:HL-60 Cells; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D002241:Carbohydrates; D019040:E-Selectin; D011994:Recombinant Proteins