Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine----serine analogs.

摘要: Abstract The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the process, two intermediates, I1 I2, were detected by kinetic studies using high performance liquid chromatography. exists transiently disappears quickly with concomitant formation I2. In contrast, I2 requires a longer time to fold into final oxidized form, N. CuSO4 catalysis increases rate I1, while elevated temperature (37 degrees C) have dramatic effect on N These observations suggest following sequential oxidative pathway. [sequence: see text] Peptide map analysis iodoacetate-labeled intermediates revealed that represents fully reduced containing 5 free cysteines; is partially species single Cys36-Cys42 disulfide bond; N, folded has bonds. physicochemical properties biological activities several Cys----Ser analogs made site-directed mutagenesis further guanidine hydrochloride-induced denaturation studies, disulfide-reduced missing either bonds are conformationally less stable than those wild type molecule or analog Cys at position 17 changed Ser. Recombinant colony stimulating lacking bond both overall secondary tertiary structures different exhibits lower activity. show crucial for maintaining in properly biologically active form.