Crystallization and preliminary X-ray crystallographic studies of dipeptidyl peptidase 11 fromPorphyromonas gingivalis

Abstract

Dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) preferentially cleaves substrate peptides with Asp and Glu at the P1 position [NH2-P2-P1(Asp/Glu)-P1'-P2'...]. For crystallographic studies, PgDPP11 was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 1.82 Å resolution were collected from an orthorhombic crystal form belonging to space group C2221, with unit-cell parameters a = 99.33, b = 103.60, c = 177.33 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress.

Structural and mutational analyses of dipeptidyl peptidase 11 from Porphyromonas gingivalis reveal the molecular basis for strict substrate specificity

“…PgDPP11 was expressed and purified as described elsewhere 48 . A synthetic gene coding for full-length PgDPP11 (residues 1-720), codon-optimised for expression in E. coli , was purchased from Genscript (Piscataway, USA) and cloned into the pET22b expression plasmid (Merck, Darmstadt, Germany).…”

“…To obtain peptide-free PgDPP11 crystals 48 , the samples were crystallised using the hanging-drop method, in which 1 μl of protein solution (10 mg/ml PgDPP11 in 80 mM Tris-HCl, pH 8.5) was mixed with the same volume of reservoir solution (20% (v/v) glycerol, 16% (m/v) PEG8000 and 0.16 M tri-potassium citrate) and incubated at 293 K. The drops were suspended over 200 μl of reservoir solution in 48-well plates. Peptide-free crystals were also obtained using a counter-diffusion crystallisation method 49 under a microgravity environment in the Japanese Experimental Module “Kibo” at the International Space Station (ISS) 47 .…”