Overexpression in Escherichia coli of a methionine-free designed interleukin-2 receptor (Tac protein) based on a chemically cleavable fusion protein.
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.
Hüsken
D
D
Institute of Organic Chemistry, J.W. Goethe University of Frankfurt am Main, Federal Republic of Germany.
Beckers
T
T
Engels
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005696:Galactosidases / Q000096:biosynthesis / Q000235:genetics*; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011817:Rabbits; D015375:Receptors, Interleukin-2 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D008715:Methionine; D005696:Galactosidases; D001616:beta-Galactosidase; D003488:Cyanogen Bromide