Secretion of recombinant proteins via the chaperone/usher pathway in Escherichia coli |
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Authors: | Zavialov A V Batchikova N V Korpela T Petrovskaya L E Korobko V G Kersley J MacIntyre S Zav'yalov V P |
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Affiliation: | Finnish-Russian Joint Biotechnology Laboratory, University of Turku, BioCity 6A, FIN-20520 Turku, Finland. azaviabo@abo.fi |
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Abstract: | F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1beta (hIL-1beta), and mature Caf1, the processed product (hIL-1beta:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1beta:Caf1 in the periplasm. Soluble hIL-1beta:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1beta. The results indicate that Caf1M-induced release of hIL-1beta:Caf1 from the inner membrane promotes folding of the hIL-1beta domain. Similar results were obtained with the fusion of Caf1 to hIL-1beta receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1beta:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1beta:Caf1 could be detected on the cell surface of E. coli. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains. |
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