Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine----serine analogs.
The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.
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Amgen Incorporated, Thousand Oaks, California 91320.
Clogston
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Narhi
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Merewether
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Pearl
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Boone
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D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry; D019327:Copper Sulfate; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012694:Serine; D013056:Spectrophotometry, Ultraviolet
D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D012694:Serine; D003300:Copper; D003545:Cysteine; D019327:Copper Sulfate