Analysis of a soluble mutant des-methionine interleukin-2 receptor alpha chain (Tac protein) produced by transfected mammalian cells.
By using recombinant DNA technology the cytoplasmic and trans-membrane domain of the human interleukin-2 receptor alpha chain (IL-2R alpha, Tac) and of a mutant protein lacking methionine-residues 18, 25, 44, 88, 92, 126, 149, 167, 205, and 209 (des-Met IL-2R alpha) encoded by a chemically and enzymatically synthesized gene, were deleted. This leads to secretory expression of soluble wild-type and des-Met mutant Tac protein of 42-45 kDa after transfection of BHK-21 cells. Transfectants secreted up to 1.6 micrograms soluble wild-type IL-2R alpha protein/10(6) cells in 24 h into the culture medium. LTK- cell lines, expressing a large number of wild-type and des-Met mutant low-affinity IL-2R alpha of 50-55 kDa on their surface, shed a truncated form of the Tac protein of about 40 kDa into the culture medium. In contrast to wild-type IL-2R alpha, shedding of mutant Tac protein is strongly reduced. This phenomenon might be the result of higher protein stability of the mutant receptor which may also explain the about 10 times higher surface expression of des-Met IL-2R alpha in LTK- cells. There are no significant differences in the biosynthesis and post-translational modification of mutant or wild-type Tac proteins either in transfected LTK- or BHK-21 cells as analysed by pulse/chase labeling experiments.
Institut für Organische Chemie, Universität Frankfurt, Federal Republic of Germany.
T
T
Beckers
H
H
Hauser
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D
Hüsken
J W
JW
Engels
D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D004247:DNA / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000096:biosynthesis; D008715:Methionine; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000032:analysis; D010957:Plasmids; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
D008565:Membrane Proteins; D010446:Peptide Fragments; D015375:Receptors, Interleukin-2; D004247:DNA; D008715:Methionine