Cloning of the fimA gene of Porphyromonas gingivalis and its expression and purification in Escherichia coli / 华西口腔医学杂志

ABSTRACT

<p><b>UNLABELLED</b>OBJECTIVE; To clone the fimA gene of Porphyromonas gingivalis (P. gingivalis) and detect its expression in Escherichia coli (E. coli).</p><p><b>METHODS</b>The fimA gene was obtained by PCR from the genome of P. gingivalis to construct a prokaryotic expression plasmid pT-BAD/fimA. pT-BAD/fimA was transformed into E. coli BL21 (DE3) competent cells and the recombination protein was characterized by means of matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. The bound protein was eluted with different concentrations of imidazole (250, 200, 150, 100, 50 micromol x L(-1)) respectively.</p><p><b>RESULTS</b>DNA sequencing showed that the fragment was 99.9% consistent with that of the published. After induction with L-arabinose, a new 3.8 x 10(4) protein appeared on SDS-PAGE gel. The protein was further identified by MALDI-TOF-MS. Purity of 95% of the target protein was purified by Ni-NTA Purification System after eluted with 100 micromol x L(-1) imidazole.</p><p><b>CONCLUSION</b>The fimA gene of P. gingivalis was cloned successfully and its protein was expressed correctly in E. coli. A high purity of protein FimA was obtained and it could be applied for follow-up researches.</p>