PMID- 7581857
OWN - NLM
STAT- MEDLINE
DCOM- 19951212
LR  - 20190913
IS  - 1572-6495 (Print)
IS  - 1572-6495 (Linking)
VI  - 668
IP  - 2
DP  - 1995 Jun 23
TI  - Biological and biophysical characterization of recombinant soluble human 
      E-selectin purified at large scale by reversed-phase high-performance liquid 
      chromatography.
PG  - 219-31
AB  - 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.
FAU - Burrows, S D
AU  - Burrows SD
AD  - Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of 
      Prussia, PA 19406-0939, USA.
FAU - Franklin, S G
AU  - Franklin SG
FAU - Brigham-Burke, M R
AU  - Brigham-Burke MR
FAU - Brooks, I S
AU  - Brooks IS
FAU - McNulty, D E
AU  - McNulty DE
FAU - Feild, J A
AU  - Feild JA
FAU - Anumula, K R
AU  - Anumula KR
FAU - O'Shannessy, D J
AU  - O'Shannessy DJ
LA  - eng
PT  - Journal Article
PL  - Netherlands
TA  - J Chromatogr B Biomed Appl
JT  - Journal of chromatography. B, Biomedical applications
JID - 9421796
RN  - 0 (Amino Acids)
RN  - 0 (Carbohydrates)
RN  - 0 (E-Selectin)
RN  - 0 (Recombinant Proteins)
SB  - IM
MH  - Amino Acids/analysis
MH  - Animals
MH  - CHO Cells
MH  - Carbohydrates/analysis
MH  - Cell Adhesion
MH  - Cell Line
MH  - Chromatography, High Pressure Liquid/*methods
MH  - Cricetinae
MH  - E-Selectin/*chemistry/isolation & purification/physiology
MH  - HL-60 Cells
MH  - Humans
MH  - Recombinant Proteins/chemistry/isolation & purification
MH  - Solubility
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
MH  - Spectrophotometry, Ultraviolet
EDAT- 1995/06/23 00:00
MHDA- 1995/06/23 00:01
CRDT- 1995/06/23 00:00
PHST- 1995/06/23 00:00 [pubmed]
PHST- 1995/06/23 00:01 [medline]
PHST- 1995/06/23 00:00 [entrez]
AID - 037843479500078W [pii]
AID - 10.1016/0378-4347(95)00078-w [doi]
PST - ppublish
SO  - J Chromatogr B Biomed Appl. 1995 Jun 23;668(2):219-31. doi: 
      10.1016/0378-4347(95)00078-w.