Isolation and refolding of a mutant methionine-free interleukin-2-receptor alpha chain synthesized as a fusion protein in Escherichia coli.
A soluble domain of the interleukin(IL)-2 receptor, the alpha 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 alpha chain (synIL-2R alpha) after BrCN cleavage and renaturation of the crude cleavage material, although the alpha 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-2R alpha did not affect the renaturation yield of the receptor protein. Additionally a cysteine-free and methionine-free beta-galactosidase derivative was fused to the soluble synIL-2R alpha 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 alpha-subunit.
Institute of Organic Chemistry, J. W. Goethe University of Frankfurt/Main, Germany.
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Seipelt
J W
JW
Engels
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli; D005813:Genes, Synthetic; D006801:Humans; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D015375:Receptors, Interleukin-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004274:DNA, Recombinant; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D008715:Methionine; D001616:beta-Galactosidase