Abstract
Biofilm-associated protein (Bap) is a large surface protein (~238 kDa) that plays a significant role in the development of Staphylococcus biofilms. Surface proteins in S. aureus are functionally redundant, which implies that a null mutant that affects one surface protein might only be partially defective in the studied function. Therefore, the objective of this study was to clone, overexpress and purify the full Bap protein in E. coli to enable us to characterize the protein in detail for future experiments. The challenging part of this study was to resolve the problem of plasmid instability of recombinant construct, which is speculated to be due the large size of the gene and the presence of 13 direct tandem repeats, when conventional E. coli strains such as DH5-α and XL1-Blue were used as a cloning host and optimizing the parameters for overexpression of the gene. The full bap gene (~6.8 kb) was amplified by long-range Taq polymerase and cloned in an expression vector pET21b in E. coli stbl2 and in BL21(DE3)-pLysS for over expression. DNA sequencing of the cloned gene confirms 100% identity with bap gene in-situ (S. aureus V329). Successful expression of the full length of Bap protein in E. coli BL21(DE3)-pLysS was confirmed by the SDS-PAGE and Western blotting using Anti-His tag antibody. To the best of our knowledge, it is first attempt to clone and overexpress full-length Bap protein in E. coli. The use of recombinant Bap gene will allow us to study and aid in its biophysical characterization.
Highlights
Staphylococcus aureus is one of the most common root causes of nosocomial infections because of its dominant biofilm forming property
It plays a crucial role in bacterial infection process even in the absence of ica operon, which is responsible for polysaccharide intercellular adhesion (PIA)/polyβ-1,6-N-acetylglucosamine (PNAG) synthesis [3]
In one of our previous studies, we showed that how Biofilm Associated Surface Protein (Bap) is important in S. aureus biofilm stability and how non-antibiotic factors such as Calcium ion concentration and proteinase K can be used to modulate the S. aureus biofilms [9,10,11]
Summary
Staphylococcus aureus is one of the most common root causes of nosocomial infections because of its dominant biofilm forming property. One of the factors is the expression of Biofilm-associated protein which confers the capacity to form biofilm It plays a crucial role in bacterial infection process even in the absence of ica operon, which is responsible for polysaccharide intercellular adhesion (PIA)/polyβ-1,6-N-acetylglucosamine (PNAG) synthesis [3]. A number of surface proteins are reported to have structural homology with bap and constitute Biofilm-associated proteins family (Bap family). Such proteins have been identified in different organisms’ viz., Bap in Staphylococcus aureus, Esp in Enterococcus faecalis [6], LapA in Pseudomonas putida [7] and BapA in Salmonella [8]. It is necessary to decode how these proteins perform in their adhesive functions
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