Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells.
We have purified recombinant human interleukin 4 (huIL-4), formerly named B-cell stimulatory factor-1, from supernatants of COS-7 monkey kidney and L-929 cells transfected with the cDNA for huIL-4. The purified protein exhibited a specific activity of 2.6 X 10(7) units/mg in a T-cell proliferation assay and consisted of multiple components on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibiting Mr values of 15,000, 18,000, and 19,000. All forms of huIL-4 eluted on gel filtration chromatography with an apparent Mr of 22,000. Gas-phase microsequencing identified 26 and 8 amino acid residues at the N and C termini, respectively, all of which were consistent with the cDNA sequence. The site of processing of the signal sequence was found to occur between Gly-24 and His-25. Incubation with N-glycanase converted the 18- and 19-kDa variants to a 15-kDa form. Treatment with endo-beta-N-acetylglucosaminidase H reduced the molecular mass of the 18-kDa variant to 15 kDa, but did not have any apparent effects on the mass of the 19-kDa species. The removal of oligosaccharide by any of these treatments did not affect bioactivity in the T-cell proliferation assay. Neither O-glycanase nor endo-beta-N-acetylglucosaminidase D affected the molecular weight of any of these species. These data suggest that differences in carbohydrate structure account, at least in part, for the observed microheterogeneity.
Le
H V
HV
Department of Biotechnology, Schering Corporation, Bloomfield, New Jersey 07003.
Ramanathan
L
L
Labdon
J E
JE
Mays-Ichinco
C A
CA
Syto
R
R
Arai
N
N
Hoy
P
P
Takebe
Y
Y
Nagabhushan
T L
TL
Trotta
P P
PP
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D006801:Humans; D015847:Interleukin-4; D007378:Interleukins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification*; D014162:Transfection
D007378:Interleukins; D011994:Recombinant Proteins; D015847:Interleukin-4; D004247:DNA