Construction of recombinant Prevotella intermedia DnaK protein

Abstract

We previously demonstrated that the expression of DnaK, one of the major heat shock proteins (HSPs), was up-regulated in biofilm-forming Prevotella intermedia strain 17 (strain 17). The entire encoding sequence for the strain 17 DnaK protein was cloned into pET and expressed in Escherichia coli (E. coli) BL21 (DE3) pLysS. The DnaK was expressed in a soluble form at a high level in the E. coli cytoplasm. This recombinant DnaK protein was used as an immunogen to obtain rabbit antibodies against the wild-type DnaK of this organism. The alignment of 10 amino-acid residues from an N-terminus of this recombinant protein was identical to that of the wild-type DnaK protein. The rabbit polyclonal antibodies recognized 70 kD protein whose molecular weight corresponds to that of the wild-type DnaK in the sonicate of strain 17 cells as well as in the culture supernatant. These results suggest that the recombinant P. intermedia DnaK and the antibodies obtained in this study can be used to study the function of this HSP in the biofilm formation of this organism.