Improved folding yields of a model protein using protein disulfide isomerase.
OBJECTIVE
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).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Du
C
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Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis 38163, USA.
Ye
J M
JM
Wolfe
J L
JL
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005978:Glutathione / Q000032:analysis / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics; D016296:Mutagenesis / Q000502:physiology; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D013696:Temperature
D007376:Interleukin-2; D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases; D005978:Glutathione