Cloning, Expression, Purification, and Crystallization of P. aeruginosa ICMP ()
Affiliation(s)
1West China Hospital Emergency Department (WCHED), State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China.
2Chengdu Institute of biological Products Co., Ltd., Chengdu, China.
3National Engineering Research Center of Immunological Products, Department of Microbiology and Biochemical Pharmacy, College of Pharmacy, Third Military Medical University, Chongqing, China.
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 Å resolution 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.
Share and Cite:
Pi, R. , Gu, J. and Lu, G. (2018) Cloning, Expression, Purification, and Crystallization of
P. aeruginosa ICMP.
American Journal of Molecular Biology,
8, 195-204. doi:
10.4236/ajmb.2018.84017.