High-level expression of a truncated wall-associated protein A from the dental cariogenic Streptococcus mutans.
Streptococcus mutans plays a primary role in the formation of dental caries. Previously, in our laboratory, an S. mutans genomic library was prepared, and the wapA gene was cloned into the shuttle vector, pSA4/4B2. To generate overexpression of wapA and to facilitate efficient purification of the WapA protein for use as an immunogen, an expression vector with the strong tac promoter was used. In order to answer questions regarding the optimization of solubility and expression based on gene size or the hydrophobicity of the protein product, 12 truncated constructs of the wapA gene were prepared using PCR. The truncated products were subcloned into the pGEX-6P-1 glutathione S-transferase (GST) fusion vector and expressed in E. coli BL21. The fusion proteins were analyzed by SDS-PAGE and confirmed by analysis with anti-GST and anti-WapA antibodies. Our study suggests that abrogation of the wapA promoter is necessary for expression of this gene in this expression system. Deletion of the signal peptide and the hydrophobic C terminus of WapA increased expression compared with the full-length construct, and truncation at the protease cleavage site of the C-terminal region greatly increased the stability of the protein without a loss in reactivity with the anti-WapA antibody. Western immunoblot analysis with anti-WapA antiserum clearly showed that the majority of the epitopes of the GST-WapA fusions are located in the N-terminal region of WapA. The immunogenicity of the various WapA fusion products is being examined in mice and rats to further map the immunologically dominant regions of the protein. This method effectively increased the expression of WapA and should contribute to the further understanding of gene expression of E. coli, as well as aid in the characterization of this protein for future immunologic evaluation.
Yoder
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Department of Biology, University of South Florida, Tampa, USA.
Cao
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Ugen
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Dao
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D000942:Antigens, Bacterial; D001426:Bacterial Proteins / Q000235:genetics*; D015153:Blotting, Western; D003731:Dental Caries / Q000382:microbiology*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013295:Streptococcus mutans / Q000235:genetics*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C081813:wapA protein, Bacillus subtilis; D005982:Glutathione Transferase