title: High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa.
abstract: The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17 kDa chaperone (PlcR2) and secreted as a fully folded heterodimer (PlcHR2) of approximately 95 kDa, by the twin arginine translocase (TAT) via the cytoplasmic membrane and through the outer membrane, by the Xcp (TypeII) secretory system. PlcHR2 has been shown to be an important virulence factor in model P. aeruginosa infections and is selectively cytotoxic, at picomolar concentrations to mammalian endothelial cells. Here we report how the various challenges starting from protein overexpression in the native organism P. aeruginosa, the use of detergents in the crystallization and data collection using the most advanced μ-focus synchrotron beam lines were overcome. Native diffraction data of this heterodimeric protein complex were collected up to a resolution of 4Å, whereas needle-shaped crystals of l-selenomethionine substituted PlcHR2 with a maximum diameter of 10 micron were used to collect data sets with a maximum resolution of 2.75Å.
authors.affiliation: Swiss Light Source, Paul Scherrer Institute, 5232 Villigen, Switzerland.
authors.forename: Daphné
authors.initials: D
authors.lastname: Truan
authors.forename: Adriana
authors.initials: A
authors.lastname: Vasil
authors.forename: Martin
authors.initials: M
authors.lastname: Stonehouse
authors.forename: Michael L
authors.initials: ML
authors.lastname: Vasil
authors.forename: Ehmke
authors.initials: E
authors.lastname: Pohl
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000378:metabolism; D012645:Selenomethionine / Q000737:chemistry; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D037521:Virulence Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D037521:Virulence Factors; D012645:Selenomethionine; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase