Mechanism of pH-sensitive polymer-assisted protein refolding and its application in TGF-beta1 and KGF-2.
Refolding of proteins at high concentrations often results in non-productive aggregation. This study, through a unique combination of spectroscopic and chromatographic analyzes, provides biomolecular evidence to demonstrate the ability of Eudragit S-100, a pH-responsive polymer, to enhance refolding of denatured-reduced lysozyme at high concentrations. The addition of Eudragit in the refolding buffer significantly increases lysozyme refolding yield to 75%, when dilution refolding was conducted at 1 mg/mL lysozyme. This study shows evidence of an electrostatic interaction between oppositely charged lysozyme and the Eudragit polymer during refolding. This ionic complexing of Eudragit and lysozyme appears to shield exposed hydrophobic residues of the lysozyme refolding intermediates, thus minimizing hydrophobic-driven aggregation of the molecules. Importantly, results from this study show that the Eudragit-lysozyme bioconjugation does not compromise refolded protein structure, and that the polymer can be readily dissociated from the protein by ion exchange chromatography. The strategy was also applied to refolding of TGF-beta1 and KGF-2.
Huang
Zhifeng
Z
Nanjing University of Science and Technology, China. cpu_son@163.com
Ni
Chunyan
C
Zhou
Xiangtian
X
Liu
Yanlong
Y
Tan
Yi
Y
Xiao
Jian
J
Feng
Wenke
W
Li
Xiaokun
X
Yang
Shulin
S
D000818:Animals; D002942:Circular Dichroism; D051526:Fibroblast Growth Factor 10 / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D011109:Polymethacrylic Acids / Q000737:chemistry*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D053773:Transforming Growth Factor beta1 / Q000737:chemistry* / Q000378:metabolism
D051526:Fibroblast Growth Factor 10; D011109:Polymethacrylic Acids; D053773:Transforming Growth Factor beta1; C038300:methylmethacrylate-methacrylic acid copolymer; C421864:hen egg lysozyme; D009113:Muramidase