A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Secretion of recombinant proteins via the chaperone/usher pathway in Escherichia coli
Tekijät: Zavialov AV, Batchikova NV, Korpela T, Petrovskaya LE, Korobko VG, Kersley J, MacIntyre S, Zav'yalov VP
Kustantaja: AMER SOC MICROBIOLOGY
Julkaisuvuosi: 2001
Tietokannassa oleva lehden nimi: APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Lehden akronyymi: APPL ENVIRON MICROB
Vuosikerta: 67
Numero: 4
Aloitussivu: 1805
Lopetussivu: 1814
Sivujen määrä: 10
ISSN: 0099-2240
DOI: https://doi.org/10.1128/AEM.67.4.1805-1814.2001
Tiivistelmä
Fl 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-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 chaperone led to the accumulation of soluble hIL-1 beta :Caf1 in the periplasm. Soluble hIL-1 beta :Caf1 reacted,vith monoclonal antibodies directed against structural epitopes of hIL-1 beta. The results indicate that Caf1M-induced 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 colony-stimulating factor. Following coexpression of the hIL-1 beta :Caf1 precursor 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 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.
Fl 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-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 chaperone led to the accumulation of soluble hIL-1 beta :Caf1 in the periplasm. Soluble hIL-1 beta :Caf1 reacted,vith monoclonal antibodies directed against structural epitopes of hIL-1 beta. The results indicate that Caf1M-induced 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 colony-stimulating factor. Following coexpression of the hIL-1 beta :Caf1 precursor 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 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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