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Heterologous Expression and Purification of a 238 kDa Large Biofilm Associated Surface Protein (Bap) in Escherichia coli

Sudhir K. Shukla and T. Subba Rao

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.