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Title: Expression, Purification and Crystallization of Human 5-Lipoxygeanse-Activating Protein with Leukotriene-Biosynthesis Inhibitors

Abstract

The nuclear membrane protein 5-lipoxygenase-activating protein (FLAP) plays an essential role in leukotriene synthesis. Recombinant full-length human FLAP with a C-terminal hexahistidine tag has been expressed and purified from the cytoplasmic membrane of Escherichia coli. Diffraction-quality crystals of FLAP in complex with leukotriene-synthesis inhibitor MK-591 and with an iodinated analogue of MK-591 have been grown using the sitting-drop vapor-diffusion method. The crystals exhibit tetragonal symmetry (P4212) and diffracted to a resolution limit of 4 Angstroms .

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
960092
Report Number(s):
BNL-83078-2009-JA
TRN: US201016%%1236
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica Section F: Structural Biology and Crystallization Communications; Journal Volume: 63
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; CRYSTALLIZATION; ESCHERICHIA COLI; MEMBRANE PROTEINS; MEMBRANES; PROTEINS; PURIFICATION; RESOLUTION; SYMMETRY; SYNTHESIS; CRYSTALS; national synchrotron light source

Citation Formats

Xu,S., McKeever, B., Wisniewski, D., Miller, D., Spencer, R., Chu, L., Ujjainwalla, F., Yamin, T., Evans, J., and et al. Expression, Purification and Crystallization of Human 5-Lipoxygeanse-Activating Protein with Leukotriene-Biosynthesis Inhibitors. United States: N. p., 2007. Web. doi:10.1107/S1744309107055571.
Xu,S., McKeever, B., Wisniewski, D., Miller, D., Spencer, R., Chu, L., Ujjainwalla, F., Yamin, T., Evans, J., & et al. Expression, Purification and Crystallization of Human 5-Lipoxygeanse-Activating Protein with Leukotriene-Biosynthesis Inhibitors. United States. doi:10.1107/S1744309107055571.
Xu,S., McKeever, B., Wisniewski, D., Miller, D., Spencer, R., Chu, L., Ujjainwalla, F., Yamin, T., Evans, J., and et al. Mon . "Expression, Purification and Crystallization of Human 5-Lipoxygeanse-Activating Protein with Leukotriene-Biosynthesis Inhibitors". United States. doi:10.1107/S1744309107055571.
@article{osti_960092,
title = {Expression, Purification and Crystallization of Human 5-Lipoxygeanse-Activating Protein with Leukotriene-Biosynthesis Inhibitors},
author = {Xu,S. and McKeever, B. and Wisniewski, D. and Miller, D. and Spencer, R. and Chu, L. and Ujjainwalla, F. and Yamin, T. and Evans, J. and et al},
abstractNote = {The nuclear membrane protein 5-lipoxygenase-activating protein (FLAP) plays an essential role in leukotriene synthesis. Recombinant full-length human FLAP with a C-terminal hexahistidine tag has been expressed and purified from the cytoplasmic membrane of Escherichia coli. Diffraction-quality crystals of FLAP in complex with leukotriene-synthesis inhibitor MK-591 and with an iodinated analogue of MK-591 have been grown using the sitting-drop vapor-diffusion method. The crystals exhibit tetragonal symmetry (P4212) and diffracted to a resolution limit of 4 Angstroms .},
doi = {10.1107/S1744309107055571},
journal = {Acta Crystallographica Section F: Structural Biology and Crystallization Communications},
number = ,
volume = 63,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • The expression, purification and crystallization of human 5-lipoxygenase-activating protein in complex with two leukotriene-biosynthesis inhibitors is decribed. The processes that were used to generate diffraction quality crystals are presented in detail. The nuclear membrane protein 5-lipoxygenase-activating protein (FLAP) plays an essential role in leukotriene synthesis. Recombinant full-length human FLAP with a C-terminal hexahistidine tag has been expressed and purified from the cytoplasmic membrane of Escherichia coli. Diffraction-quality crystals of FLAP in complex with leukotriene-synthesis inhibitor MK-591 and with an iodinated analogue of MK-591 have been grown using the sitting-drop vapor-diffusion method. The crystals exhibit tetragonal symmetry (P42{sub 1}2) and diffractedmore » to a resolution limit of 4 Å.« less
  • The purification, crystallization and preliminary structural characterization of human MAWD-binding protein (MAWDBP) are described. MAWDBP is the only representative of the phenazine biosynthesis-like protein family in the human genome. Its expression is elevated in several disease processes, including insulin resistance, folate deficiency and hypotension, and it may also be involved in carcinogenesis. The exact molecular function of MAWDBP is unknown. Native and seleno-l-methionine-labelled MAWDBP were expressed in Escherichia coli and crystallized at room temperature from precipitants containing 10 mM KF, 14%(w/v) PEG 3350 and 0.1 M sodium citrate pH 5.4. Crystals belong to space group H32, with unit-cell parameters amore » = b = 187, c = 241 Å, indicative of three to five monomers per asymmetric unit. Crystals were cryoprotected with 15%(v/v) glycerol and data have been collected to 2.7 Å resolution.« less
  • Leukotriene C{sub 4} is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C{sub 4} synthase (LTC{sub 4}S) participate in its biosynthesis. We report evidence that LTC{sub 4}S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC{sub 4}S. FLAP bound to the N-terminal part/first hydrophobic region of LTC{sub 4}S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC{sub 4}S. Fluorescent FLAP- and LTC{sub 4}S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC{sub 4}S co-localized withmore » a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC{sub 4}S. Direct interaction of 5-LO and LTC{sub 4}S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC{sub 4}S.« less
  • The protein SH3BGRL, containing both SH3-binding and Homer EVH1-binding motifs, has been crystallized using the hanging-drop vapour-diffusion method. The protein SH3BGRL, containing both SH3-binding and Homer EVH1-binding motifs, has been crystallized using the hanging-drop vapour-diffusion method. The crystals diffract to 0.88 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 28.8886, b = 34.9676, c = 98.0016 Å. Preliminary analysis indicates that the asymmetric unit contains one molecule and has a solvent content of about 34%.