Refolding of α₁-antitrypsin expressed as inclusion bodies in Escherichia coli: characterization of aggregation

Abstract

Recombinant α1-antitrypsin (α1AT) produced as inclusion bodies in Escherichia coli was purified via several steps including solubilization of the inclusion bodies in 8 M urea and refolding by direct dilution of denaturant, followed by ion-exchange chromatography. The purified recombinant α1AT has an activity comparable to human plasma α1AT. During refolding, prolonged incubation of the α1AT polypeptides at intermediate urea concentration favored production of inactive but soluble aggregates, which could regain activity after denaturation and renaturation. Nondenaturing polyacrylamide gel electrophoresis of the aggregates revealed the existence of dimers and higher oligomers. Immunological approach to characterize conformation showed that the oligomers were distinct from the native, the cleaved, or the denatured form, but was similar to the polymers induced from the native structure in mild denaturing condition. These results suggest that the oligomers are formed through specific interactions between aggregation-competent species which are stabilized at intermediate denaturant concentration.