Open AccessOverexpression in Escherichia coli of a methionine‐free designed interleukin‐2 receptor (Tac protein) based on a chemically cleavable fusion protein
Author(s) -
HÜSKEN Dieter,
BECKERS Thomas,
ENGELS Joachim W.
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb19351.x
Subject(s) - cyanogen bromide , fusion protein , methionine , valine , biochemistry , alanine , protein subunit , microbiology and biotechnology , g alpha subunit , chemistry , protein a/g , ternary complex , interleukin 10 receptor, alpha subunit , leucine , amino acid , biology , peptide sequence , gene , recombinant dna , enzyme
Several fusion proteins of our previously chemically synthesized gene encoding the interleukin‐2‐receptor α subunit (IL‐2Rα or Tac protein) were constructed. They were designed in order to be cleavable by cyanogen bromide. Thus, the original internal methionines of the IL‐2Rα 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 β‐galactosidase and portions thereof did not result in substantial amounts of the expected construct. Ternary fusions consisting of β‐galactosidase domains N‐ and C‐terminally fused to the mutant synthetic methionine‐free interleukin‐2 receptor α subunit (synIL‐2Rα) 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 β‐galactosidase structure models. The fusion protein can be cleaved with cyanogen bromide, isolated and the resulting synIL‐2Rα detected by Western blot analysis.