OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT HtrA PROTEIN FROM STREPTOCOCCUS PNEUMONIAE
Yasser Musa Ibrahim1,2 - Ahmed Megahed Abouwarda1
1General Department of Basic Medical Sciences, Microbiology Department, National Organization for Drug Control and Research (NODCAR), Giza, Egypt. 2Department of Pharmaceutics, College of Pharmacy, King Khalid University, Abha, Kingdom of Saudi Arabia.
Analysis of the complete genome sequence of Streptococcus pneumoniae revealed a single copy of the gene for High Temperature Requirement A (HtrA) protein. HtrA is indispensable for survival of the pneumococcus at conditions of stress. It was also reported as a virulence factor of this bacterium. Pneumococcal proteins involved in virulence are potential candidates for new vaccine development to protect against this life-threatening organism. We cloned the gene for HtrA in the expression vector pET33b with N-terminal His tag. The construction of the expression vector was confirmed by restriction digestion and DNA sequencing. Escherichia coli BL21 (DE3) was used for the expression of the protein. HiTrapTM Chelating HP columns were used in the purification of the histidine-tagged HtrA protein. The purified protein was confirmed by SDS-PAGE and western immunoblotting analyses. We also report here that the proteolytic activity of HtrA is temperature-dependent.