Construction of recombinant Prevotella intermedia GroEL protein

Abstract

Previously we used microarray analysis and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to demonstrate that expression of groEL, which is a highly-conserved heat shock protein gene, was up-regulated in biofilm-forming Prevotella intermedia (P. intermedia) strain 17 as compared to the biofilm non-forming variant strain 17-2. The entire encoding sequence for the P. intermedia GroEL protein was cloned into pET and expressed in Escherichia coli (E. coli) BL 21 (DE 3) pLysS or BL 21 (DE 3) pLysE. GroEL was expressed in a soluble form at a high level in the E. coli cytoplasm. Recombinant GroEL was transferred from the SDS electrophoresis gel to PVDF membrane, and applied to N-terminus amino-acid sequencing. N-terminus amino-acid sequencing revealed that the alignment of 10 amino-acid residues from an N-terminus of this recombinant protein was identical to that of wild-type GroEL protein. These results show a way to generate recombinant P. intermedia GroEL that can be used for further investigation of the function of this heat shock protein in biofilm formation and for study of the development of inflammatory diseases caused by this organism.