Recombinant PPAD and Enolase Production

The full-length PPAD coding sequence was amplified from P gingivalis W83 DNA (ATCC) and cloned into pET-28a(+) (Novagen), generating a fusion protein with N-terminal His-tag. Enzymatically inactive PPAD was generated by site-directed mutagenesis to replace cysteine 351 at the active site of the protein with serine (PPAD^C351S).^{36 (link)} N-terminal truncated PPAD (rPPAD^Ntx) was generated by amplifying the coding sequence for amino acids 44 to 556 of full-length PPAD with primers containing a 5’ Kozak sequence. The PCR product was cloned into pET-28a(+) to encode for a C-terminal His-tagged fusion protein. Alpha-enolase encoding cDNA was cloned into pET-28a(+). Recombinant PPAD, PPAD^C351S, PPAD^Ntx, and enolase were expressed in E coli BL21 (DE3) (Agilent), and purified from the soluble fraction of cell lysates prepared in 20 mM Tris pH 7.6, 400 mM NaCl, 5 mM imidazole, 20 mM β-mercaptoethanol, 1% Triton X by Ni-NTA affinity chromatography (Qiagen). Purity of rPPAD and rPPAD^C351S used in ELISA assays exceeded 95% (see online supplementary figure S1A). Enolase was citrullinated in vitro using human rPAD4 as previously described.^{37 (link)}