Aggregation and folding of recombinant human creatine kinase.
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.
Hahn
Hwa-Sun
HS
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.
Park
Yong-Doo
YD
Lee
Jae-Rin
JR
Park
Kyung-Hee
KH
Kim
Tae Jin
TJ
Yang
Jun-Mo
JM
Hahn
Myong-Joon
MJ
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
D010710:Phosphates; D014508:Urea; D003402:Creatine Kinase; C018279:sodium phosphate