Cloning, expression, and purification of c-di-AMP metabolism-related genes from Porphyromonas gingivalis / 华西口腔医学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To clone, express, and purify cyclic diadenosine monophosphate (c-di-AMP) metabolism-related genes from Porphyromonas gingivalis (P. gingivalis) ATCC33277.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) from the genome of P. gingivalis ATCC33277 amplified, the coding regions of pgn0523, pgn1187, and pgn2003 genes. The amplified DNA fragments were ligated with a prokaryotic expression vector pET28a to construct the recombinant expression plasmids pET-pgn0523, pET-pgn1187, and pET-pgn2003. These recombinant plasmids were transformed into Escherichia coli (E. coli) BL21 (DE3) competent cells. The expression of recombinant proteins was induced by isopropyl-β-D-thiogalactoside and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were purified using a Ni²⁺ matrix column, and their concentrations were determined by a BCA Protein Quantitative Kit.</p><p><b>RESULTS</b>The c-di-AMP metabolism-related genes from P. gingivalis ATCC33277 were amplified successfully with the correct molecular size. The recombinant expression vectors were constructed by ligating enzyme-digested PCR products and pET28a vector, and verified by PCR and sequencing. After induction and purification, recombinant proteins were expressed successfully and obtained with the correct molecular size (19.5 x 10³, 39.9 x 10³, 66.0 x 10³). The final protein concentrations were 0.708, 0.523, and 0.861 mg · mL⁻¹ after dialysis.</p><p><b>CONCLUSION</b>The c-di-AMP metabolism-related genes from P. gingivalis ATCC33277 are cloned successfully, and their coding products are expressed correctly in E. coli. High-purity proteins are finally obtained. The cloning and purification of these important proteins will help us to further investigate the physiological function and regulatory mechanism of c-di-AMP signaling system in P. gingivalis.</p>