Purification and crystallization of recombinant human TNF-like ligand TL1A.
TL1A is a recently discovered TNF-like ligand. Because of the interests in the structural basis of the specificity of the bindings of the TNF ligands to the TNF receptors, we sought to crystallize the mature soluble form of human TL1A. To prepare recombinant human TL1A, the coding sequence for mature human soluble TL1A (aa72-aa251) was cloned into Escherichia coli expression vector pDEST14 and the protein was purified in a succession of immobilized metal affinity, hydrophobic interaction, ion exchange and size exclusion chromatography, indicating that mature TL1A may have a metal ligand. The functional activity of recombinant TL1A was confirmed by its ability to bind to DcR3, a soluble decoy receptor of the TNF receptor family that has been previously reported to bind to TL1A. Single crystals of TL1A were obtained in a screen with a crystal screen kit using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. TL1A crystals belong to the tetragonal space group P4(1)2(1)2, with unit cell parameter of a=b=116.734, c=118.927A. The TL1A crystals diffracted to at least 3.2A. Self-rotation functions showed that there are three molecules in the asymmetry unit. Assuming an average partial specific volume of 0.74cm(3)g(-1) for proteins, the water content of the crystal is 62.8%. A preliminary molecular replacement solution was obtained with three TL1A molecules in the asymmetric unit. The three protomers are related by a non-crystallographic 3-fold axis, like those of other TNF ligand family members.
Jin
Tengchuan
T
Department of Biology, Illinois Institute of Technology, 3101 South Dearborn Street, LS 182, Chicago, IL 60616, USA.
Kim
Sunghee
S
Guo
Feng
F
Howard
Andrew
A
Zhang
Yu-Zhu
YZ
D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D053380:Tumor Necrosis Factor Ligand Superfamily Member 15 / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; C117026:TNFSF15 protein, human; D053380:Tumor Necrosis Factor Ligand Superfamily Member 15