Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2020258 | Protein Expression and Purification | 2016 | 7 Pages |
Abstract
Successfully recovering proinsulin's native conformation from inclusion body is the crucial step to guarantee high efficiency for insulin's manufacture. Here, two by-products of disulfide-linked oligomers and disulfide-isomerized monomers were clearly identified during proinsulin aspart's refolding through multiple analytic methods. Arginine and urea are both used to assist in proinsulin refolding, however the efficacy and possible mechanism was found to be different. The oligomers formed with urea were of larger size than with arginine. With the urea concentrations increasing from 2Â M to 4Â M, the content of oligomers decreased greatly, but simultaneously the refolding yield at the protein concentration of 0.5Â mg/mL decreased from 40% to 30% due to the increase of disulfide-isomerized monomers. In contrast, with arginine concentrations increasing up to 1Â M, the refolding yield gradually increased to 50% although the content for oligomers also decreased. Moreover, it was demonstrated that not redox pairs but only oxidant was necessary to facilitate the native disulfide bonds formation for the reduced denatured proinsulin. An oxidative agent of selenocystamine could increase the yield up to 80% in the presence of 0.5Â M arginine. Further study demonstrated that refolding with 2Â M urea instead of 0.5Â M arginine could achieve similar yield as protein concentration is slightly reduced to 0.3Â mg/mL. In this case, refolded proinsulin was directly purified through one-step of anionic exchange chromatography, with a recovery of 32% and purity up to 95%. All the results could be easily adopted in insulin's industrial manufacture for improving the production efficiency.
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Authors
Ying Chen, Qi Wang, Chun Zhang, Xiunan Li, Qiang Gao, Changqing Dong, Yongdong Liu, Zhiguo Su,