Expression of peptidylarginine deiminase from Porphyromonas gingivalis in Escherichia coli: enzyme purification and characterization.
Porphyromonas gingivalis peptidylarginine deiminase (PAD) catalyzes the deimination of peptidylarginine residues of various peptides to produce peptidylcitrulline and ammonia. P. gingivalis is associated with adult-onset periodontitis and cardiovascular disease, and its proliferation depends on secretion of PAD. We have expressed two recombinant forms of the P. gingivalis PAD in Escherichia coli, a truncated form with a 43-amino acid N-terminal deletion and the full-length form of PAD as predicted from the DNA sequence. Both forms contain a poly-His tag and Xpress epitope at the N-terminus to aid in detection and purification. The activities and stabilities of these two forms have been evaluated. PAD is cold sensitive; it aggregates within 30 min at 4 degrees C, and optimal storage conditions are at 25 degrees C in the presence of a reducing agent. PAD is not a metalloenzyme and does not need a cofactor for catalysis or stability. Multiple l-arginine analogs, various arginine-containing peptides, and free l-arginine were used to evaluate substrate specificity and determine kinetic parameters.
Rodríguez
Sofía B
SB
Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Stitt
Barbara L
BL
Ash
David E
DE
D000595:Amino Acid Sequence; D055162:Biocatalysis; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007097:Imines / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics; D000076342:Protein-Arginine Deiminases; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D003067:Coenzymes; D007097:Imines; D008670:Metals; D006867:Hydrolases; D000076342:Protein-Arginine Deiminases