High-level expression of the truncated alpha chain of human high-affinity receptor for IgE as a soluble form by baculovirus-infected insect cells. Biochemical characterization of the recombinant product.
The binding subunit of human high-affinity receptor for IgE (Fc epsilon RI alpha) was efficiently expressed as a truncated form in insect cells. The soluble (s)Fc epsilon RI alpha purified from culture medium by affinity chromatography with an anti-(alpha chain) mAb was nearly homogeneous and had an IgE-binding activity. The amino acid composition and the revealed N-terminal amino acid sequence of sFc epsilon RI alpha suggested that it was properly processed in insect cells. The apparent molecular mass (35 kDa) of purified sFc epsilon RI alpha was smaller than that of sFc epsilon RI alpha produced by CHO transfectants. The reduction of the apparent molecular mass after N-glycanase treatment showed the recombinant product was N-glycosylated. Peptide mapping of native and deglycosylated sFc epsilon RI alpha indicated that three Asn residues (Asn21, Asn42 and Asn166) should be almost fully glycosylated, and that two Asn residues (Asn74 and Asn135) were partially glycosylated.
Yagi
S
S
Corporate Research and Development Laboratory, Tonen Corporation, Iruma-gun, Japan.
Yanagida
M
M
Tanida
I
I
Hasegawa
A
A
Okumura
K
K
Ra
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D016367:Baculoviridae; D015153:Blotting, Western; D002460:Cell Line; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D017455:Receptors, IgE / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D017455:Receptors, IgE; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D001216:Asparagine