Mechanism of pH-sensitive polymer-assisted protein refolding and its application in TGF- Delta b1 and KGF-2

• Published in: Biotechnology progress Vol. 25; no. 5; pp. 1387 - 1395
• Main Authors: Huang, Zhifeng, Ni, Chunyan, Zhou, Xiangtian, Liu, Yanlong, Tan, Yi, Xiao, Jian, Feng, Wenke, Li, Xiaokun, Yang, Shulin
• Format: Journal Article
• Language: English
• Published: 01.09.2009
• ISSN: 1520-6033
• DOI: 10.1002/btpr.218

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- Delta *b1 and KGF-2. [copy 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009