Secretion of recombinant proteins via the chaperone/usher pathway in Escherichia coli

Fl antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/C af1A 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 coil. Despite correct processing of a chimeric prot ein composed of a modified Caf1 signal peptide, mature human interleukin-1 beta (hIL-1 beta), and mature Can, the processed product (hIL-1 beta :Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M c haperone led to the accumulation of soluble hIL-1 beta :Caf1 in the peripla sm. Soluble hIL-1 beta :Caf1 reacted,vith monoclonal antibodies directed ag ainst structural epitopes of hIL-1 beta. The results indicate that Caf1M-in duced release of hIL-1 beta :Caf1 from the inner membrane promotes folding of the hIL-1 beta domain. Similar results were obtained with the fusion of Caf1 to hIL-1 beta receptor antagonist or to human granulocyte-macrophage c olony-stimulating factor. Following coexpression of the hIL-1 beta :Caf1 pr ecursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL -1 beta :Caf1 could he detected on the cell surface of E. coli. These resul ts demonstrate for the first time the potential application of the chaperon e/usher secretion pathway in the transport of subunits with large heterogen eous 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.