Purification and crystallization of recombinant human TNF-like ligand TL1A
Introduction
The tumor necrosis factor (TNF) superfamily of cytokines and their receptors regulate diverse biological processes, including cell proliferation, immune regulation, inflammation, cell death and cell differentiation (for review, see [1], [2]). The TNF ligand superfamily has at least 19 members and the TNF receptor superfamily has at least 29 members. The structures of several members have been determined [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]. However, some TNF superfamily ligands are known to specifically associate with only one receptor while other TNF superfamily ligands can bind up to five receptors. In addition, most TNF family receptors bound to one to three ligands [13]. Thus, structural information is required for understanding the specificity of the interactions among the TNF superfamily ligands and receptors.
TNFSF15 is a recently identified member of the tumor necrosis factor (ligand) superfamily. Several variants of this cytokine exist. Those are the products of different transcripts generated with the use of cryptic splice sites and alternate exons. The first TNFSF15 variant isolated contains 174 amino acids and is also known as TL1 (for TNF-like ligand 1) [14] and VEGI (for vascular endothelial growth inhibitor) that was reported to inhibit endothelial cell proliferation [15]. VEGI/TL1, as an angiogenesis inhibitor, was also known to suppress the growth of colon carcinomas in vivo. Hence, structural and functional information on TNFSF15 may be highly valuable for angiogenesis-based cancer therapy [15].
The longest and most abundant form of the TNFSF15 protein contains 251 amino acids and is also known as TL1A [16]. TL1A binds to death receptor 3 (DR3, or TNFRSF25), activates NF-κB, induces c-IAP2 production and regulates DR3 mediated apoptosis in TF-1 cells [17]. TL1A also interacts with decoy receptor 3 (DcR3, also known as TR6, or TNFRSF21), which has an opposing effect on the signal transduction and immune response triggered by TL1A–DR3 association.
TL1A can be post-translationally processed to a soluble protein that consists of its C-terminus 180 amino acids [16]. Soluble recombinant TL1A was shown to act as a costimulator in T cells that increases IL-2 responsiveness and secretion of IFN-γ and GM-CSF, both in vitro and in vivo [16], [18]. Another TNFSF15 variant that consists of 192 amino acids was also reported [19]. The C-terminus 151 amino acids from all known forms of TL1A are the same and this part of the protein, when expressed alone, induced apoptosis of endothelial cells within 36 h [20]. However, in contrast with the short forms of TNFSF15, TL1A had no anti-angiogenic activity.
We sought to obtain information on the structural basis for the differences in the protein–protein interaction and in the function of the variants of TL1A and among different members of the TNF superfamily. Here, we report our results of the expression, purification and characterization of TL1A.
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Plasmid construction
The coding sequence for mature TL1A (starting from Leu72 to the end) was PCR-amplified from pcDNA3-TL1A [21] with primers ggaaaacctgtacttccagggtgccatgggtctaaaaggacaggagtttgcac (forward) and tgggtctcgagggcccgcctatagtaagaaggctccaaaga (reverse). A secondary PCR reaction was performed to introduce attB attachment sites to use the Gateway™ (Invitrogen, Carlsbad, CA) system. The primers for the second PCR are ggaatggggacaagtttgtacaaaaaagcaggctcggaaaacctgtacttccagggtatg (forward) and
TL1A purification
To purify enough protein with very high purity, un-tagged TL1A was tested for its ability to bind immobilized divalent ions in a column mode. As shown in Fig. 1, TL1A has an affinity to the Ni2+-column. After washing with the binding buffer that contained 5 mM imidazole, TL1A can be eluted with a much lower imidazole concentration compared to that used for eluting His-tag proteins. This IMAC step is very effective in TL1A purification. As shown in Fig. 2, after IMAC purification, over 78% of the
Discussion
Purification of E. coli expressed recombinant TL1A was reported previously [16]. The reported procedure used anion exchange, hydrophobic interaction and size exclusion chromatography. However, the yield and the purity of the protein were not reported. We sought to determine the structure of TL1A. Using a procedure similar to that reported for TL1A purification, we failed to get highly pure protein required for crystallization (data not shown). Purification of another member of the TNF ligand
Acknowledgments
This work was supported by a fund from Illinois Institute of Technology. X-ray diffraction data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, United State Argonne National Laboratory. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract W-31-109-Eng-38.
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