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High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa.

Truan, D. and Vasil, A. and Stonehouse, M. and Vasil, M.L. and Pohl, E. (2013) 'High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa.', Protein expression and purification., 90 (1). pp. 40-46.

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 4 Å, 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 2.75 Å.

Item Type:Article
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Available under License - Creative Commons Attribution.
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Status:Peer-reviewed
Publisher Web site:http://dx.doi.org/10.1016/j.pep.2012.11.005
Publisher statement:This is an Open Access article distributed under the terms of the Creative Commons CC BY license.
Date accepted:No date available
Date deposited:09 October 2015
Date of first online publication:July 2013
Date first made open access:No date available

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