Open AccessIsolation and refolding of a mutant methionine‐free interleukin‐2‐receptor α chain synthesized as a fusion protein in Escherichia coli
Author(s) -
SEIPELT Irene,
ENGELS Joachim W.
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb18993.x
Subject(s) - cyanogen bromide , chemistry , cysteine , methionine , fusion protein , biochemistry , alpha chain , receptor , residue (chemistry) , escherichia coli , amino acid , stereochemistry , peptide sequence , recombinant dna , enzyme , gene
A soluble domain of the interleukin(IL)‐2 receptor, the α chain synthesized in Escherichia coli , was employed to study expression and refolding of the protein. The results showed that it is possible to obtain biologically active synthetic methionine‐free IL‐2 receptor α chain (synIL‐2Rα) after BrCN cleavage and renaturation of the crude cleavage material, although the α chain is expressed as a deglycosylated, methionine‐free protein. The soluble receptor comprises amino acids 1–219 and forms 5 disulfide bonds in its biologically active state. Biological activity has been analysed by affinity chromatography and ELISA with mutant [Ala125]IL‐2 and monoclonal antibodies as ligands. Renaturation yield is limited mainly by the high aggregation rate of incorrectly folded protein. Aggregation could be limited by varying the oxidation conditions. The deletion of a non‐bridging cysteine at position 192 in the synIL‐2Rα did not affect the renaturation yield of the receptor protein. Additionally a cysteine‐free and methionine‐free β‐galactosidase derivative was fused to the soluble synIL‐2Rα derivatives to prevent reoxidation of incorrect disulfide bonds in the crude BrCN‐cleavage material. It is suggested that cysteine impurities from cyanogen‐bromide‐cleaved peptides might interfere seriously with the refolding process of the synthetic IL‐2 receptor α‐subunit.