AGGREGATION OF RECOMBINANT HEPATITIS-B SURFACE-ANTIGEN IN PICHIA-PASTORIS

The combination of immunoaffinity and size-exclusion chromatography (S EC) is a powerful tool to analyze multiprotein particle assembly. This approach was used to investigate the source of aggregation of recombi nant hepatitis B surface antigen (HBsAg) detected in purified material . As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the pr ocess. To test whether aggregation tabes place in vivo, crude yeast ex tract containing the expressed HBsAg was fractioned on a Sephacryl S-4 00 column just after cell disruption, and each fraction immunopurified individually. As a result, the HBsAg aggregate was isolated from a fr action corresponding to the elution of large particle aggregates only, not native HBsAg particles. It was biologically active, which demonst rates aggregate formation by specific assembly of partially or wholly folded HBsAg intermediates. (C) 1998 Published by Elsevier Science B.V ; All rights reserved.