title: Structure of unfolded and refolded recombinant derived [Ala125]interleukin 2.
abstract: Naturally occurring interleukin 2 (IL-2) contains an odd number (three) of cysteinyl residues and thus is susceptible to the formation of a variety of intramolecular and intermolecular disulfide bonds. The cysteine at residue 125 has been replaced with an alanine residue by site-directed mutagenesis, and hence, this analogue can form only one intrachain disulfide bond. When expressed at high levels in Escherichia coli, this recombinant DNA derived IL-2 analogue is insoluble, reduced, and inactive. The protein was solubilized by denaturants and, after purification, was oxidized to form an intramolecular disulfide bond. Circular dichroism (CD) has been used to investigate the effects of various denaturants on the unfolding-refolding process of the purified, oxidized protein. A similar conformation is obtained when [Ala125]interleukin 2 [IL-2(Ala-125)] is refolded from 6 M guanidine hydrochloride, 8 M urea, or 5% acetic acid. The resultant protein, refolded from these denaturants, is monomeric and has activity comparable to or greater than that reported for naturally derived IL-2. In addition to this form, aggregates, as evidenced from gel filtration, are obtained. The specific activities of these are greatly reduced, and CD spectra indicated that they have much less helical content than the monomeric form of the protein. CD spectra also showed that the tertiary structure of IL-2(Ala-125) is entirely different in the presence of sodium dodecyl sulfate (SDS) from that of the monomeric form in the absence of SDS.
mesh_terms: D000085:Acetates; D002942:Circular Dichroism; D004220:Disulfides / Q000032:analysis; D019791:Guanidine; D006146:Guanidines; D007376:Interleukin-2 / Q000031:analogs & derivatives* / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins; D014508:Urea
chemical_list: D000085:Acetates; C051703:Ala(125)-interleukin 2; D004220:Disulfides; D006146:Guanidines; D007376:Interleukin-2; D011994:Recombinant Proteins; D014508:Urea; D019791:Guanidine