title: Improving the refolding efficiency for proinsulin aspart inclusion body with optimized buffer compositions.
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 2 M to 4 M, the content of oligomers decreased greatly, but simultaneously the refolding yield at the protein concentration of 0.5 mg/mL decreased from 40% to 30% due to the increase of disulfide-isomerized monomers. In contrast, with arginine concentrations increasing up to 1 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 0.5 M arginine. Further study demonstrated that refolding with 2 M urea instead of 0.5 M arginine could achieve similar yield as protein concentration is slightly reduced to 0.3 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.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
authors.forename: Chun
authors.initials: C
authors.lastname: Zhang
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
authors.forename: Xiunan
authors.initials: X
authors.lastname: Li
authors.affiliation: Novo Nordisk Research Center China, Beijing 102206, PR China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Gao
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China.
authors.forename: Changqing
authors.initials: C
authors.lastname: Dong
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China. Electronic address: ydliu@ipe.ac.cn.
authors.forename: Yongdong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China. Electronic address: Zgsu@ipe.ac.cn.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry*; D002021:Buffers; D003538:Cystamine / Q000031:analogs & derivatives* / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D016566:Organoselenium Compounds / Q000737:chemistry*; D010084:Oxidation-Reduction; D011384:Proinsulin / Q000737:chemistry* / Q000235:genetics; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014508:Urea / Q000737:chemistry*
chemical_list: D002021:Buffers; D004220:Disulfides; D016566:Organoselenium Compounds; D011994:Recombinant Proteins; C014807:selenocystamine; D014508:Urea; D011384:Proinsulin; D001120:Arginine; D003538:Cystamine
keywords: Additive; Aggregate; Diselenide; Inclusion bodies; Oxidative refolding; Proinsulin