A two-plasmid Escherichia coli system for expression of Dr adhesins.
This paper presents a very efficient expression system for production of Dr adhesins. The system consists of two plasmids. One is the pACYCpBAD-DraC-C-His, which contains the draC gene under the control of the arabinose promoter (pBAD), encoding the DraC usher. The second is the pET30b-syg-DraBE, which contains the draB and draE genes under the control of the T7lac promoter, encoding the DraB chaperone and the DraE adhesin, respectively. Those plasmids have different origin of replication and can therefore coexist in one cell. Since different promoters are present, the protein expression can be controlled. The Dr adhesion expression system constructed opens up a lot of possibilities, and could be very useful in experiments focusing on understanding the biogenesis of Gram-negative bacteria adhesins. For this purpose we showed that the AfaE-III adhesin (98.1% identity between the DraE and the AfaE-III adhesins, with three divergent amino acids within the sequences) was able to pass through the DraC channel in the Escherichia coli BL21(DE3) strain. Immunoblotting analysis and immunofluorescence microscopy showed the presence of AfaE-III on the bacterial cell surface. In addition, the system described can be useful for displaying the immune-relevant sectors of foreign proteins on the bacterial cell. The heterologous epitope sequence of the HSV1 glycoprotein D was inserted into the draE gene in place of the N-terminal region of surface exposed domain 2. Chimeric proteins were exposed on the bacterial surface as evidenced by immunoblotting and immunofluorescence microscopy. The effective display of peptide segments on Dr fimbriae expressed at the bacterial cell surface, can be used for the development of a fimbrial vaccine.
Kur
Marta
M
Gdańsk University of Technology, Chemical Faculty, Department of Microbiology, Narutowicza 11/12, 80-952 Gdańsk, Poland.
Piatek
Rafał
R
Kur
Józef
J
D018830:Adhesins, Escherichia coli / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D010861:Fimbriae, Bacterial; D008856:Microscopy, Fluorescence; D010957:Plasmids
D018830:Adhesins, Escherichia coli; D017931:DNA Primers