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1.
Eur J Biochem. 1990 May 20;189(3):657-65.

Analysis of a soluble mutant des-methionine interleukin-2 receptor alpha chain (Tac protein) produced by transfected mammalian cells.

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1
Institut für Organische Chemie, Universität Frankfurt, Federal Republic of Germany.

Abstract

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.

PMID:
2190827
[Indexed for MEDLINE]
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2.
J Biol Chem. 1988 Jun 15;263(17):8359-65.

Expression and characterization of a des-methionine mutant interleukin-2 receptor (Tac protein) with interleukin-2 binding affinity.

Author information

1
Institut für Organische Chemie, Universität Frankfurt, Main, Federal Republic of Germany.

Abstract

A gene coding for the Tac protein (interleukin-2 receptor alpha-subunit, IL-2R alpha) of the interleukin-2 receptor was constructed by chemoenzymatic gene synthesis. The gene designed for mutagenesis codes for a receptor protein where all 10 methionines are substituted by alanine, valine, leucine, and isoleucine. In addition, aspartate at position 6 is substituted by glutamate. This desmethionine IL-2R alpha and the wild-type IL-2R alpha genes were integrated into a eukaryotic expression vector and transferred into different cell lines. The recipient cell lines express both wild-type and mutant receptor proteins on their cell surfaces which are recognized equally by different monoclonal antibodies. It was possible to establish cell lines with high level IL-2R alpha chain expression by fluorescence-activated cell sorting. The wild-type IL-2R alpha expressed in LTK- cells is a glycoprotein with an apparent molecular size of about 60 kDa and a typical low interleukin-2 binding affinity of KD = 12 nM. Despite the fact that 11 amino acids are altered, no significant difference in the mutant IL-2R alpha is observed, exhibiting the same molecular size and a low interleukin-2 binding affinity of KD = 26 nM.

PMID:
3131342
[Indexed for MEDLINE]
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