Recombinant P.g. Peptidyl-Arginine-Deiminase, a tool for evaluating rheumatic patient antibodies

Objectives: Porphyromonas gingivalis derived peptidyl-arginine-deiminase (PPAD) catalyze the arginine-to-citrulline conversion in proteins, leading to refolding and apparition of neoepitopes. A positive relationship between anti-PPAD antibodies presence and rheumatoid arthritis (RA) activity has been reported, therefore this work was aimed to obtain a recombinant PPAD for titrating the serum antibodies in RA patients by an ELISA test.
Methods: Both 3 and 5 regions of PPAD gene were amplified and cloned in pGEM-T-plasmid, then excised and directionally subcloned in the expression vector pET-CT and transformed again. Positive colonies were confirmed by PCR and sequencing and the protein expression induced with IPTG. Using the bacterial inclusion bodies, the N- and C-terminal fragments were purified using a Ni-coupled resin and confirmed by anti-His Western blot. PPAD fragments were used to standardize and titrate anti-PPAD antibodies from RA patients previously evaluated by rheumatology, periodontology and microbiology services. The dilution above the defined cutoff were registered and data analyzed (Exact Chi square test; Spearman correlation test).
Results: Recombinant N- and C-terminal fragments with an approximate 25 kDa molecular mass were obtained early after IPTG induction and easily purified. Seventy per cent of patients were female (mean age 58.1±4.5) and most of them were positive in the ELISA test. There was not a significant relationship between anti-PPAD antibodies and periodontal disease status nor with pocket P. gingivalis positivity nor with anti-P. gingivalis antibodies titer. Despite the number of painful joints and simple disease activity index (SDAI) were larger in those patients with higher anti-PPAD titers, these differences were non-significant (p=0.053 and p=0.082 respectively), indicating a putative positive relationship.
Conclusions: Recombinant fragments of PPAD were efficiently produced and purified, which were a suitable tool for evaluating antibodies anti-PPAD in RA patients with periodontal disease.