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Title: Expression, purification, crystallization and preliminary X-ray diffraction analysis of an essential lipoprotein implicated in cell-wall biosynthesis in Mycobacteria

Abstract

A lipoprotein implicated in mycobacterial cell-wall biosynthesis, LpqW, was expressed in E. coli. Crystals were obtained that diffracted to 2.4 Å resolution. Mycobacterium tuberculosis is a renewed cause of devastation in the developing world. Critical to the success of this re-emerging pathogen is its unusual waxy cell wall, which is rich in rare components including lipoarabinomannan (LAM) and its precursors, the phosphatidylinositol mannosides (PIMs). Balanced synthesis of these related glycolipids is intrinsic to both cell-wall integrity and virulence in M. tuberculosis and presents a promising, albeit poorly defined, therapeutic target. Here, the expression, purification and crystallization of an essential 600-amino-acid lipoprotein, LpqW, implicated in this process are reported. Crystals of LpqW were grown using 20–24%(w/v) PEG 4000, 8–16%(v/v) 2-propanol, 100 mM sodium citrate pH 5.5 and 10 mM DTT. A complete data set was collected at 2.4 Å using synchrotron radiation on a crystal belonging to space group C222, with unit-cell parameters a = 188.57, b = 312.04, c = 104.15 Å. Structure determination is under way.

Authors:
; ;  [1];  [2]; ;  [3];  [2];  [1];  [2]
  1. The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia)
  2. (Australia)
  3. ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 (Australia)
Publication Date:
OSTI Identifier:
22356203
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 61; Journal Issue: Pt 12; Other Information: PMCID: PMC1978156; PMID: 16511240; PUBLISHER-ID: bw5120; OAI: oai:pubmedcentral.nih.gov:1978156; Copyright (c) International Union of Crystallography 2005; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CITRATES; CRYSTALLIZATION; CRYSTALS; PRECURSOR; PROPANOLS; RESOLUTION; SODIUM COMPOUNDS; SPACE GROUPS; SYNCHROTRON RADIATION; X-RAY DIFFRACTION

Citation Formats

Marland, Zara, Beddoe, Travis, Zaker-Tabrizi, Leyla, ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, Coppel, Ross L., Crellin, Paul K., E-mail: paul.crellin@med.monash.edu.au, Department of Microbiology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Rossjohn, Jamie, E-mail: paul.crellin@med.monash.edu.au, and ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800. Expression, purification, crystallization and preliminary X-ray diffraction analysis of an essential lipoprotein implicated in cell-wall biosynthesis in Mycobacteria. United Kingdom: N. p., 2005. Web. doi:10.1107/S1744309105037541.
Marland, Zara, Beddoe, Travis, Zaker-Tabrizi, Leyla, ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, Coppel, Ross L., Crellin, Paul K., E-mail: paul.crellin@med.monash.edu.au, Department of Microbiology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Rossjohn, Jamie, E-mail: paul.crellin@med.monash.edu.au, & ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800. Expression, purification, crystallization and preliminary X-ray diffraction analysis of an essential lipoprotein implicated in cell-wall biosynthesis in Mycobacteria. United Kingdom. doi:10.1107/S1744309105037541.
Marland, Zara, Beddoe, Travis, Zaker-Tabrizi, Leyla, ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, Coppel, Ross L., Crellin, Paul K., E-mail: paul.crellin@med.monash.edu.au, Department of Microbiology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Rossjohn, Jamie, E-mail: paul.crellin@med.monash.edu.au, and ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800. Thu . "Expression, purification, crystallization and preliminary X-ray diffraction analysis of an essential lipoprotein implicated in cell-wall biosynthesis in Mycobacteria". United Kingdom. doi:10.1107/S1744309105037541.
@article{osti_22356203,
title = {Expression, purification, crystallization and preliminary X-ray diffraction analysis of an essential lipoprotein implicated in cell-wall biosynthesis in Mycobacteria},
author = {Marland, Zara and Beddoe, Travis and Zaker-Tabrizi, Leyla and ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 and Coppel, Ross L. and Crellin, Paul K., E-mail: paul.crellin@med.monash.edu.au and Department of Microbiology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 and Rossjohn, Jamie, E-mail: paul.crellin@med.monash.edu.au and ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800},
abstractNote = {A lipoprotein implicated in mycobacterial cell-wall biosynthesis, LpqW, was expressed in E. coli. Crystals were obtained that diffracted to 2.4 Å resolution. Mycobacterium tuberculosis is a renewed cause of devastation in the developing world. Critical to the success of this re-emerging pathogen is its unusual waxy cell wall, which is rich in rare components including lipoarabinomannan (LAM) and its precursors, the phosphatidylinositol mannosides (PIMs). Balanced synthesis of these related glycolipids is intrinsic to both cell-wall integrity and virulence in M. tuberculosis and presents a promising, albeit poorly defined, therapeutic target. Here, the expression, purification and crystallization of an essential 600-amino-acid lipoprotein, LpqW, implicated in this process are reported. Crystals of LpqW were grown using 20–24%(w/v) PEG 4000, 8–16%(v/v) 2-propanol, 100 mM sodium citrate pH 5.5 and 10 mM DTT. A complete data set was collected at 2.4 Å using synchrotron radiation on a crystal belonging to space group C222, with unit-cell parameters a = 188.57, b = 312.04, c = 104.15 Å. Structure determination is under way.},
doi = {10.1107/S1744309105037541},
journal = {Acta Crystallographica. Section F},
number = Pt 12,
volume = 61,
place = {United Kingdom},
year = {Thu Dec 01 00:00:00 EST 2005},
month = {Thu Dec 01 00:00:00 EST 2005}
}