期刊
JOURNAL OF BIOTECHNOLOGY
卷 142, 期 2, 页码 170-178出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2009.04.002
关键词
hTNF; Single domain antibody; VHH; Transglutaminase; Enbrel; Nanobody
资金
- Deutsche Forschungsgemeinschaft [SFB415]
Tumor necrosis factor (TNF) plays an important role in chronic inflammatory disorders, such as Rheumatoid Arthritis and Crohn's disease. Recently, monoclonal Camelidae variable heavy-chain domain-only antibodies (VHH) were developed to antagonize the action of human TNF (hTNIF). Here. we show that hTNF-VHH does not interfere with hTNF trimerization, but competes with hTNF for hTNF-receptor binding. Moreover, we describe posttranslational dimerization and multimerization of hTNF-VHH molecules in vitro catalyzed by microbial transglutaminases (MTG). The ribonuclease S-tag-peptide was shown to act as a peptidyl substrate in covalent protein cross-linking reactions catalyzed by MTG from Streptomyces mobaraensis. The S-tag sequence was C-terminally fused to the hTNF-VHH and the fusion protein was expressed and purified from Escherichia coli culture supernatants. hTNF-VHH-S-tag fusion proteins were efficiently dimerized and multimerized by MTG whereas hTNF-VHH was not susceptible to protein cross-linking. Cell cytotoxicity assays, using hTNF as apoptosis inducing cytokine, revealed that dimerized and multimerized hTNF-VHH proteins were much more active than the monomeric hTNF-VHH. We hypothesize that improved inhibition by dimeric and multimeric single chain hTNF-VHH proteins is caused by avidity effects. (C) 2009 Elsevier B.V. All rights reserved.
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