Purification and characterization of biologically active human recombinant 37 kDa soluble CD23 (sFc epsilon RII) expressed in insect cells.
Human recombinant soluble 37 kDa CD23 has been expressed in insect cells and secreted into the culture medium using the IL-2 leader sequence. The 37 kDa CD23 was purified 600-fold to homogeneity by monoclonal antibody affinity chromatography and gel filtration. The pure protein is monomeric, glycosylated, depleted of one N terminal amino acid and contains four disulphide bonds. It degrades into smaller fragments of 33, 29 and 25 kDa if purified in the absence of protease inhibitors. The same pattern of proteolytic fragments is observed when the pure preparation is incubated at room temperature for 3 weeks. Physical characterization of the 37 kDa CD23 by circular dichroism indicates that the protein contains mainly beta sheet and 20% of alpha helical structures. Specific binding of IgE to natural CD23 (low affinity IgE receptor) was inhibited by purified recombinant 37 kDa CD23. Moreover, purified recombinant 37kDa CD23 and interleukin-1 promoted the survival of germinal centre B cells.
Graber
P
P
Glaxo Institute for Molecular Biology S.A., Geneva, Switzerland.
Jansen
K
K
Pochon
S
S
Shields
J
J
Aubonney
N
N
Turcatti
G
G
Bonnefoy
J Y
JY
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000737:chemistry* / Q000302:isolation & purification* / Q000502:physiology; D001402:B-Lymphocytes / Q000502:physiology; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D007313:Insecta; D007375:Interleukin-1 / Q000494:pharmacology; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011961:Receptors, Fc / Q000737:chemistry* / Q000302:isolation & purification* / Q000502:physiology; D017455:Receptors, IgE; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D000944:Antigens, Differentiation, B-Lymphocyte; D007375:Interleukin-1; D011961:Receptors, Fc; D017455:Receptors, IgE; D011994:Recombinant Proteins; D007073:Immunoglobulin E