Improved folding yields of a model protein using protein disulfide isomerase

• Published in: Pharmaceutical research Vol. 15; no. 12; pp. 1808 - 1815
• Main Authors: CHENGAN DU, YE, J. M, WOLFE, J. L
• Format: Journal Article
• Language: English
• Published: New York, NY Springer 01.12.1998; Springer Nature B.V
• Subjects: Biological and medical sciences; Biotechnology; Chromatography, High Pressure Liquid - methods; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Glutathione - analysis; Glutathione - metabolism; Humans; Inclusion Bodies - chemistry; Interleukin-2 - chemistry; Interleukin-2 - genetics; Methods. Procedures. Technologies; Mutagenesis - physiology; Oxidation-Reduction; Protein Disulfide-Isomerases - analysis; Protein Disulfide-Isomerases - chemistry; Protein Disulfide-Isomerases - genetics; Protein engineering; Protein Folding; Recombinant Proteins - analysis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Temperature; Human; Temperature; Intramolecular oxidoreductases; Enzyme; Cytokine; Environmental factor; Gene expression; Isomerases; Interleukin 2; Disulfide bond; Protein disulfide-isomerase; Inclusion body; Redox potential; Recombinant protein; Folding; Glutathione
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1023/A:1011941603339

To study the effects of recombinant human protein disulfide isomerase (rhPDI) concentration, reduced glutathione:oxidized glutathione ratio (GSH:GSSG) and temperature on the efficiency of oxidative folding of a model protein, recombinant human interleukin 2 (C125A mutation) (C125A rhIL-2). C 125A rhIL-2 inclusion bodies were reduced and denatured by guanidium hydrochloride (Gdm.Cl) and 100 mM GSH. The solution was diluted 10 times into folding buffer, allowing C125A rhIL-2 to fold either in the absence or presence of rhPDI. The renatured and unfolded C125A rhIL-2 species were quantitated by reversed phase-HPLC. The initial folding rate of C125A rhIL-2 linearly increased with rhPDI:C125A rhIL-2 molar ratio in the first 2.5 minutes, and reached the highest rate when the rhPDI:C125A rhIL-2 ratio was 1:1. The oxidative folding of C125A rhIL-2 linearly increased as the GSH:GSSG molar ratio decreased from 10:0 to 10:3. The folding of C125A rhIL-2 was also dependent on temperature, and optimum folding was realized at 23 degrees C. These results demonstrate that under optimal redox potential and temperature, rhPDI enhances the oxidative folding of C125A rhIL-2. In the oxidative folding of C125A rhIL-2, rhPDI exerts its effect on folding by the acceleration of thiol/disulfide interchange.