Cloning and expression in murine erythroleukemia cells: the soluble forms of the type I and type II tumor necrosis factor receptors fused to an immunogenic affinity tag.
We have cloned, expressed, and purified the extracellular domains of types I and II human tumor necrosis factor receptors. Both proteins were expressed in and secreted by murine erythroleukemia cells under the control of the human beta-globin promoter placed down-stream from the human globin locus control region. Secretion of both proteins was directed by the respective tumor necrosis factor receptor signal sequence. Each tumor necrosis factor receptor extracellular domain was expressed as a chimeric protein, fused to a carboxy terminal flexible peptide linker and an antigenic affinity tag. Secretion of both proteins into the growth medium in a hollow fiber bioreactor was achieved. A monoclonal antibody generated against the affinity tag allowed the purification of both proteins. These were isolated as biologically active products in that they bound human tumor necrosis factor-alpha in a 125I-radioiodinated ligand binding assay. The two proteins also bound tumor necrosis factor-alpha at approximately equimolar ratios as demonstrated by BIAcore sensorgram analysis.
Newton
C R
CR
ZENECA Pharmaceuticals, Macclesfield, Cheshire, United Kingdom.
MacCallum
L J
LJ
McPheat
W
W
Carr
H
H
Foster
H R
HR
Abbott
W M
WM
Needham
M
M
Dinsmore
A
A
Copley
C G
CG
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D000939:Epitopes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D008969:Molecular Sequence Data; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012641:Selection, Genetic; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000345:Affinity Labels; D015703:Antigens, CD; D000939:Epitopes; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha