(634d) Enhanced Protein Refolding and Purification From Inclusion Bodies with Like-Charged Ion-Exchange Resin

Wang, G., Tianjin University

Protein refolding is a crucial step in the production of therapeutic proteins expressed in bacteria as inclusion bodies. In vitro protein refolding is severely impeded by the aggregation of folding intermediates during the folding process, so inhibition of the aggregation is the most effective approach to high-efficiency protein refolding. We have found that electrostatic repulsion between the protein to be refolded and like-charged ion exchange resins can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native protein recovery [1]. This finding has been extensively demonstrated with three aggregation-prone proteins, lysozyme, bovine serum albumin (BSA) and bovine carbonic anhydrase II, and four kinds of ion-exchange resins when the protein and ion-exchange gel were either positively or negatively charged at the refolding conditions. It is remarkable that the enhancing effect was significant at very high protein concentrations. The working mechanism of the like-charged resin is considered due to the charge repulsion that could induce oriented alignment of protein molecules near the charged surface, leading to the significant decrease of intermolecular protein attraction [1].
    The refolding method with like-charged ion exchangers is simple to perform, and the key material (ion-exchange resin) is easy to separate for recycling. Moreover, because ion exchangers can work as adsorbents of oppositely charged impurities, an operation of simultaneous protein refolding and purification is possible. This was demonstrated by the refolding of recombinant proteins, such as enhanced green fluorescent protein (EGFP), expressed as inclusion bodies in E. coli. Compared with refolding without an ion exchange resin, the refolding yield of negatively charge EGFP was about two-fold increased by refolding with like-charged SP Sepharose FF as an additive. Moreover, impurities in the solubilized and denatured protein solution were partially adsorbed onto the adsorbent particles, so purification of the target protein was simultaneously achieved during the refolding process. This proved that the method of protein refolding with like-charged ion-exchange resin is promising for preparative refolding and purification of valuable proteins expressed in bacteria as inclusion bodies.

[1] G.-Z. Wang, X.-Y. Dong, Y. Sun: Ion-exchange resins greatly facilitate refolding of like-charged proteins at high concentrations. Biotechnology and Bioengineering, 2011, 108 (5): 1068-1077.