title: [Cloning, expression, and purification of c-di-AMP metabolism-related genes from Porphyromonas gingivalis].
abstract: OBJECTIVE
  To clone, express, and purify cyclic diadenosine monophosphate (c-di-AMP) metabolism-related genes from Porphyromonas gingivalis (P. gingivalis) ATCC33277.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Qiu
authors.initials: Q
authors.lastname: Wei
authors.forename: Cheng
authors.initials: C
authors.lastname: Xingqun
authors.forename: Zhou
authors.initials: Z
authors.lastname: Xuedong
authors.forename: Li
authors.initials: L
authors.lastname: Yuqing
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D015226:Dinucleoside Phosphates; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D010957:Plasmids; D016133:Polymerase Chain Reaction; D016966:Porphyromonas gingivalis / Q000235:genetics*; D011994:Recombinant Proteins
chemical_list: D001426:Bacterial Proteins; D015226:Dinucleoside Phosphates; D011994:Recombinant Proteins; C528998:cyclic diadenosine phosphate