Abstract
HP0270, HP0555, HP0746, HP1493, HP1074. HP1209 or HP1448 gene of H. pylori 26695 was cloned into the 5’ position of histidine-tag site in pQE30 vector, respectively. After transforming individual recombinant vector into E. coli SG13009, antibiotic resistant clones were screened with corresponding primers of various targeted genes by PCR and 1 mM IPTG-induction in individual culture at 37℃ for 3 hours. Since clone #10 contains the right sequence of the HP1448 gene in vector and can produce more soluble protein than the others, it was used in the rest of this study. More recombinant (rec)- HP1448 protein was induced from bacteria culture after 0.5 mM IPTG addition (a) at 25℃ for 9 h, or (b) at 20℃ for 20 h, in comparison with those at 37℃ for 3 h. Approximately 1.5 mg HP1448 protein could be purified through Ni-NTA affinity column from 1 liter of bacteria culture containing IPTG. Newly prepared protein aggregated easily in Tris imidazole NaCl elution buffer at pH 8.0. If this elution buffer was replaced to pH of 6.5 for protein storage at 4℃, HP1448 protein would not aggregate for at least 1 month. Therefore, the HP1448 protein was stored in this buffer for further functional and structural studies in this research. The purified HP1448 protein was used as an antigen to produce antibodies in rabbits. Anti-HP1448 antibody from rabbit sera were prepared and tittered. About 16 ng of HP1448 protein could be detected in 1:4000 of anti-sera from rabbit after the 5th boosted injection. HP1448 protein expression was recognized in the H. pylori culture at different conditions by using antibodies and western analysis,. In comparison with those at pH 7.2, HP1448 protein expression, was found in the H. pylori, was slightly down-regulated after growth on Brucella agar plates at pH 5.5 for 48 hours. In contrast, more than 1.7-fold HP1448 protein appeared from samples cultured on plates containing 5mM urea at pH 5.5 than those at pH 7.2. Without urea in the medium at pH 5.5, the HP1448 protein was expressed only one half of those from control in the H. pylori. However, cultured in a medium containing urea for 24 h, bacteria at pH 5.5 showed 4-fold HP1448 expression to those samples at pH of 7.2. Since the HP1448 gene was annotated as a protein component of RNase P, its role was determined by means of in-vitro RNase P activity measurement. In this study, the substrate pre-tRNAPhe and the RNA component for RNase P assay were prepared by means of in-vitro transcription from the constructed pGEM vector inserted with a gene for pre-tRNA and a gene for RNA component of RNase P, respectively. This study indicated that the RNase P RNA component cleaved pre-tRNAPhe in the high salt buffer for RNase P assay. More studies should be conducted to determine the role of the recombinant HP1448 protein (as a cofactor for the assay of RNase P activity) by its ability to induce cleavage activity on pre-tRNAPhe in low salt condition.