Inducible production of recombinant human Flt3 ectodomain variants in mammalian cells and preliminary crystallographic analysis of Flt3 ligand-receptor complexes.
Acta Cryst Sect F. 2011-02-23; 67(3): 325-331
DOI: 10.1107/S1744309111003319
Read on PubMed
1. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Mar 1;67(Pt 3):325-31.
doi: 10.1107/S1744309111003319. Epub 2011 Feb 23.
Inducible production of recombinant human Flt3 ectodomain variants in mammalian
cells and preliminary crystallographic analysis of Flt3 ligand-receptor
complexes.
Verstraete K(1), Remmerie B, Elegheert J, Lintermans B, Haegeman G, Vanhoenacker
P, Van Craenenbroeck K, Savvides SN.
Author information:
(1)Unit for Structural Biology, Laboratory for Protein Biochemistry and
Biomolecular Engineering (L-ProBE), Ghent University, 9000 Ghent, Belgium.
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, Flt3D1-D5 and
Flt3D1-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 Flt3D1-D4-FL and Flt3D1-D5-FL
complexes diffracted to 4.3 and 7.8 Å resolution, respectively, and exhibited
variable diffraction quality even within the same crystal. The resulting data led
to the successful structure determination of Flt3D1-D4-FL via a combination of
molecular-replacement and density-modification protocols exploiting the
noncrystallographic symmetry and high solvent content of the crystals.
DOI: 10.1107/S1744309111003319
PMCID: PMC3053156
PMID: 21393836 [Indexed for MEDLINE]