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Expression and Purification of native TL1A (TNFSF15) to generate TL1A Aptamers.
Author(s) -
Majid Ayaz Mahmood,
Deyev Vadim,
Podack Eckhard R
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.676.9
Subject(s) - maltose binding protein , fusion protein , aptamer , secretion , microbiology and biotechnology , recombinant dna , chemistry , affinity chromatography , biology , biochemistry , gene , enzyme
The TNF‐like cytokine TL1A (TNFSF15) has gained significant immunological interest since its characterization as a ligand for TNFRSF25. We have shown that TL1A is intimately involved in the pathogenesis of inflammatory lung disease. Moreover, TL1A up‐regulation on macrophages and lymphocytes has been implicated in human inflammatory bowel disease (IBD) and rheumatoid arthritis. The pMAL‐2 protein fusion and purification system (New England Biolabs) was initially invented for obtaining recombinant protein targets in E. coli. We have devised a strategy to utilize that system in eukaryotic cells to express native TL1A. Since active TL1A is proposed to be in a soluble form, an expression cassette containing the IgK secretion signal, maltose binding protein (MBP) and human TL1A (hTL1A) was created in a pBMG‐Neo plasmid. Transfection and selection of NIH 3T3 cells or 293SF suspension cells resulted in active secretion of MBP‐hTL1A as shown by an ‘in‐house’ TL1A ELISA, and MBP‐based Western blots. Secreted MBP‐TL1A proteins were purified on Amylose columns under serum free conditions and analyzed by SDS‐PAGE or Agilent protein electrophoresis. Furthermore, an enterokinase cleavage site allowed cleavage of hTL1A from MBP. Chromatography techniques were applied to further investigate native TL1A monomers and trimeric complexes. These targets will be utilized to generate TL1A aptamers using SELEX based or capillary electrophoresis applications. Selected aptamers may allow further understanding of TL1A immunology and provide useful immunotherapeutic interventions. This work was supported by NIH grants; AI061807 and AI068515.