Secretion of recombinant proteins via the chaperone/usher pathway in Escherichia coli.
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.
Zavialov
A V
AV
Finnish-Russian Joint Biotechnology Laboratory, University of Turku, BioCity 6A, FIN-20520 Turku, Finland. azaviabo@abo.fi
Batchikova
N V
NV
Korpela
T
T
Petrovskaya
L E
LE
Korobko
V G
VG
Kersley
J
J
MacIntyre
S
S
Zav'yalov
V P
VP
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D001426:Bacterial Proteins; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012795:Sialoglycoproteins; C086725:caf1 protein, Yersinia pestis; C070195:CAf1M protein, Yersinia pestis; D016178:Granulocyte-Macrophage Colony-Stimulating Factor