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Inducible production of recombinant human Flt3 ectodomain variants in mammalian cells and preliminary crystallographic analysis of Flt3 ligand-receptor complexes

Kenneth Verstraete UGent, Bert Remmerie UGent, Jonathan Elegheert UGent, Béatrice Lintermans UGent, Guy Haegeman UGent, Peter Vanhoenacker, Kathleen Van Craenenbroeck UGent and Savvas Savvides UGent (2011) ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS. 67(3). p.325-331
abstract
The extracellular complex between the haematopoietic receptor Flt3 and its cytokine ligand (FL) is the cornerstone of signalling cascades that are central to early haematopoiesis and the immune system. Here, efficient protocols for the production of two ectodomain variants of human Flt3 receptor, Flt3(D1-D5) and Flt3(D1-D4), for structural studies are reported based on tetracycline-inducible stable cell lines in HEK293S cells deficient in N-acetylglycosaminyltransferase I (GnTI-/-) that can secrete the target proteins with limited and homogeneous N-linked glycosylation to milligram amounts. The ensuing preparative purification of Flt3 receptor-ligand complexes yielded monodisperse complex preparations that were amenable to crystallization. Crystals of the Flt3(D1-D4)-FL and Flt3(D1-D5)-FL complexes diffracted to 4.3 and 7.8 A resolution, respectively, and exhibited variable diffraction quality even within the same crystal. The resulting data led to the successful structure determination of Flt3(D1-D4)-FL via a combination of molecular-replacement and density-modification protocols exploiting the noncrystallographic symmetry and high solvent content of the crystals.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
Fms-like tyrosine kinase 3 receptor, Flt3, DENDRITIC CELLS, STRUCTURAL BASIS, PROTEIN CRYSTALS, DENSITY MODIFICATION, JUXTAMEMBRANE DOMAIN, MOLECULAR-REPLACEMENT, CONSTITUTIVE ACTIVATION, TYROSINE KINASE, INTERNAL TANDEM DUPLICATION, ACUTE MYELOID-LEUKEMIA, Flt3 ligand, ectodomains
journal title
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS
Acta Crystallogr. F-Struct. Biol. Cryst. Commun.
volume
67
issue
3
pages
325 - 331
Web of Science type
Article
Web of Science id
000288225100007
JCR category
CRYSTALLOGRAPHY
JCR impact factor
0.506 (2011)
JCR rank
21/25 (2011)
JCR quartile
4 (2011)
ISSN
1744-3091
DOI
10.1107/S1744309111003319
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
1234451
handle
http://hdl.handle.net/1854/LU-1234451
date created
2011-05-24 16:06:44
date last changed
2011-08-02 13:38:29
@article{1234451,
  abstract     = {The extracellular complex between the haematopoietic receptor Flt3 and its cytokine ligand (FL) is the cornerstone of signalling cascades that are central to early haematopoiesis and the immune system. Here, efficient protocols for the production of two ectodomain variants of human Flt3 receptor, Flt3(D1-D5) and Flt3(D1-D4), for structural studies are reported based on tetracycline-inducible stable cell lines in HEK293S cells deficient in N-acetylglycosaminyltransferase I (GnTI-/-) that can secrete the target proteins with limited and homogeneous N-linked glycosylation to milligram amounts. The ensuing preparative purification of Flt3 receptor-ligand complexes yielded monodisperse complex preparations that were amenable to crystallization. Crystals of the Flt3(D1-D4)-FL and Flt3(D1-D5)-FL complexes diffracted to 4.3 and 7.8 A resolution, respectively, and exhibited variable diffraction quality even within the same crystal. The resulting data led to the successful structure determination of Flt3(D1-D4)-FL via a combination of molecular-replacement and density-modification protocols exploiting the noncrystallographic symmetry and high solvent content of the crystals.},
  author       = {Verstraete, Kenneth and Remmerie, Bert and Elegheert, Jonathan and Lintermans, B{\'e}atrice and Haegeman, Guy and Vanhoenacker, Peter and Van Craenenbroeck, Kathleen and Savvides, Savvas},
  issn         = {1744-3091},
  journal      = {ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS},
  keyword      = {Fms-like tyrosine kinase 3 receptor,Flt3,DENDRITIC CELLS,STRUCTURAL BASIS,PROTEIN CRYSTALS,DENSITY MODIFICATION,JUXTAMEMBRANE DOMAIN,MOLECULAR-REPLACEMENT,CONSTITUTIVE ACTIVATION,TYROSINE KINASE,INTERNAL TANDEM DUPLICATION,ACUTE MYELOID-LEUKEMIA,Flt3 ligand,ectodomains},
  language     = {eng},
  number       = {3},
  pages        = {325--331},
  title        = {Inducible production of recombinant human Flt3 ectodomain variants in mammalian cells and preliminary crystallographic analysis of Flt3 ligand-receptor complexes},
  url          = {http://dx.doi.org/10.1107/S1744309111003319},
  volume       = {67},
  year         = {2011},
}

Chicago
Verstraete, Kenneth, Bert Remmerie, Jonathan Elegheert, Béatrice Lintermans, Guy Haegeman, Peter Vanhoenacker, Kathleen Van Craenenbroeck, and Savvas Savvides. 2011. “Inducible Production of Recombinant Human Flt3 Ectodomain Variants in Mammalian Cells and Preliminary Crystallographic Analysis of Flt3 Ligand-receptor Complexes.” Acta Crystallographica Section F-structural Biology and Crystallization Communications 67 (3): 325–331.
APA
Verstraete, Kenneth, Remmerie, B., Elegheert, J., Lintermans, B., Haegeman, G., Vanhoenacker, P., Van Craenenbroeck, K., et al. (2011). Inducible production of recombinant human Flt3 ectodomain variants in mammalian cells and preliminary crystallographic analysis of Flt3 ligand-receptor complexes. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 67(3), 325–331.
Vancouver
1.
Verstraete K, Remmerie B, Elegheert J, Lintermans B, Haegeman G, Vanhoenacker P, et al. Inducible production of recombinant human Flt3 ectodomain variants in mammalian cells and preliminary crystallographic analysis of Flt3 ligand-receptor complexes. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS. 2011;67(3):325–31.
MLA
Verstraete, Kenneth, Bert Remmerie, Jonathan Elegheert, et al. “Inducible Production of Recombinant Human Flt3 Ectodomain Variants in Mammalian Cells and Preliminary Crystallographic Analysis of Flt3 Ligand-receptor Complexes.” ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 67.3 (2011): 325–331. Print.