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has been cited by the following article:

• TITLE: Cloning, Expression, Purification, and Crystallization of P. aeruginosa ICMP

  AUTHORS: Ruliang Pi, Jiang Gu, Guangwen Lu

  KEYWORDS: Pseudomonas aeruginosa, ICMP, Metallopeptidase, Imelysin/Imelysin-Like Protein, Crystallographic Analysis

  JOURNAL NAME: American Journal of Molecular Biology, Vol.8 No.4, September 29, 2018

  ABSTRACT: Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic human pathogen that can lead to severe diseases in immunocompromised patients. The insulin-cleaving membrane protease (ICMP) of P. aeruginosa plays a vital role in the pathogenesis of the bacterium and is therefore characterized as an important bacterial virulence factor. In addition, ICMP also serves as a founding member of the M75 peptidase family and represents a prototype of the imelysin/imelysin-like proteins. Despite of its functional importance in the pathogenesis of P. aeruginosa and of a root position as the prototypic imelysin/imelysin-like member, the structural features of the protein remain uninvestigated. Since preparation of homogeneous and crystallizable protein species is the prerequisite for structural studies by crystallography, we reported the successful expression, purification, and crystallization of P. aeruginosa ICMP in this study. The protein was over-expressed in Escherichia coli as a GST-fusion protein, cleaved to remove the fusion tag, and then purified to homogeneity. Diffractable crystals were obtained using the sitting-drop vapour-diffusion method. The crystals diffracted to 2.5 Åresolution and belonged to space group P212121, with unit-cell parameters a = 54.47, b = 158.98, c = 162.84 Å, α = β = γ = 90°. Preliminary analysis of the diffraction data revealed high-quality crystallographic statistics with a Matthews coefficient of about 2.61 Å3.Da-1 and a solvent content of about 52.58%, indicating the presence of three ICMP molecules in the asymmetric unit. The current work therefore paved the way for future studies aiming to delineate the characteristics of ICMP at the atomic level.