title: Aggregation and folding of recombinant human creatine kinase.
abstract: The processes of aggregation and refolding of recombinant human creatine kinase (rHCK) were studied. Most of the rHCK expressed in E. coli was present in the insoluble traction and it could be solubilized in 6 M urea solution. Unfolding of rHCK in 6 M urea showed biphasic kinetic courses (kappa1 = 6.5 x 10(-3) s(-1); kappa2 = 0.54 x 10(-3) s(-1)) as observed by maximum fluorescence wavelength change. During refolding of the rHCK dissolved in urea, significant aggregation was noticed following first-order kinetics. Aggregation rate constants were influenced by the concentration of NaCl, which increased the difference in transition-free energy (deltadeltaG), showing that stabilization of folding intermediates by NaCl could efficiently reduce the formation of insoluble aggregates. Formations of aggregate were also reduced by adjusting temperature, pH, and concentration of rHCK. Refolding of rHCK under the optimized condition which prevented the aggregation also showed multi-kinetic phases (kappa1 = 3.0 x 10(-3) s(-1); kappa2 = 0.64 x 10(-3) s(-1)). Under optimized conditions applied in this study, rHCK could correctly refold retrieving the high specific enzymatic activity.
authors.affiliation: Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.
authors.forename: Hwa-Sun
authors.initials: HS
authors.lastname: Hahn
authors.forename: Yong-Doo
authors.initials: YD
authors.lastname: Park
authors.forename: Jae-Rin
authors.initials: JR
authors.lastname: Lee
authors.forename: Kyung-Hee
authors.initials: KH
authors.lastname: Park
authors.forename: Tae Jin
authors.initials: TJ
authors.lastname: Kim
authors.forename: Jun-Mo
authors.initials: JM
authors.lastname: Yang
authors.forename: Myong-Joon
authors.initials: MJ
authors.lastname: Hahn
mesh_terms: D003402:Creatine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry / Q000187:drug effects; D007700:Kinetics; D010710:Phosphates / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation; D012995:Solubility; D013696:Temperature; D014508:Urea / Q000494:pharmacology
chemical_list: D010710:Phosphates; D014508:Urea; D003402:Creatine Kinase; C018279:sodium phosphate