PMID: 2121481Oct 24, 1990Paper

Overexpression in Escherichia coli of a methionine-free designed interleukin-2 receptor (Tac protein) based on a chemically cleavable fusion protein

European Journal of Biochemistry
D HüskenJ W Engels

Abstract

Several fusion proteins of our previously chemically synthesized gene encoding the interleukin-2-receptor alpha subunit (IL-2R alpha or Tac protein) were constructed. They were designed in order to be cleavable by cyanogen bromide. Thus, the original internal methionines of the IL-2R alpha were replaced by either alanine, valine, leucine or isoleucine, based on secondary structure predictions. Additionally, aspartate at position 6 was substituted for glutamate in order to stabilize the acid-labile Asp-Pro bond. Direct C-terminal fusion of total beta-galactosidase and portions thereof did not result in substantial amounts of the expected construct. Ternary fusions consisting of beta-galactosidase domains N- and C-terminally fused to the mutant synthetic methionine-free interleukin-2 receptor alpha subunit (synIL-2R alpha) yielded inclusion bodies amounting to 4-7% of the total protein. This first overexpression of a type I membrane receptor can be rationalized by the known beta-galactosidase structure models. The fusion protein can be cleaved with cyanogen bromide, isolated and the resulting synIL-2R alpha detected by Western blot analysis.

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Citations

Mar 1, 1992·Trends in Pharmacological Sciences·A D Strosberg, S Marullo

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