Cloning, Expression, Purification, and Crystallization of P. aeruginosa ICMP - American Journal of Molecular Biology

AJMB > Vol.8 No.4, October 2018

Cloning, Expression, Purification, and Crystallization of P. aeruginosa ICMP ()

HTML XML

Download as PDF (Size: 568KB) PP. 195-204

DOI: 10.4236/ajmb.2018.84017 833 Downloads 1,628 Views Citations

Author(s)

Ruliang Pi^1,2*, Jiang Gu³, Guangwen Lu^1*

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 Å^3.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.

KEYWORDS

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

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.

Journals Menu

Follow SCIRP

	+1 323-425-8868
	customer@scirp.org
	+86 18163351462(WhatsApp)
	1655362766

	Paper Publishing WeChat

Journals Menu

Home

About SCIRP

Service

Policies