FUSIONS TO THE CHOLERA-TOXIN-B SUBUNIT - INFLUENCE ON PENTAMERIZATIONAND GM1 BINDING

The cholera toxin B (CTB) subunit has been used extensively in vaccine research as a carrier for peptide immunogens due to its immunopotenti ating properties, where coupling has been obtained either by genetic f usion or chemical conjugation. For genetically fused immunogens both N - and C-terminal fusions have been used. Only shorter extensions have previously been evaluated and in some reports these fusions have impai red the biological functions of CTB, such as the ability to form penta mers and to adhere to its cell receptor, the GM1 ganglioside. Here we report the first systematic study where the same fusion partner has be en used for either C-terminal, N-terminal or dual fusions to CTB. The serum albumin binding region (BB, approximately 25 kDa) from streptoco ccal protein G, which is known to fold independently of N- or C-termin al fusions, was selected as fusion partner. The three fusion proteins CTB-BB, BB-CTB and BB-CTB-BB were expressed in Escherichia coli, where they were efficiently secreted to the periplasmic space, and could be purified by affinity chromatography on human serum albumin (HSA) colu mns. The CTB fusion proteins were compared for their ability to form p entamers, by gel electrophoresis and size-exclusion chromatography, an d it was concluded that all three fusion proteins were able to pentame rize. Interestingly, the C-terminal fusion to CTB showed most efficien t pentamerization, while the dual fusion was much less efficient. Puri fied pentamer fractions from all three fusions where found to react to a monoclonal antibody described to react only to pentameric forms of CTB, Ln addition, the purified pentamer fractions were analyzed in an enzyme-linked immunosorbent assay (ELISA) for their ability to bind GM 1, and it was found that the C-terminal fusion (CTB-BB) showed signifi cant GM1-binding, but that also the N-terminal and dual CTB fusion pro teins bound GM1, although less efficiently, The implications of the re sults for the design and use of CTB fusion proteins as subunit vaccine s are discussed. (C) 1997 Elsevier Science B.V.