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  • A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment.

A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment.

Biomolecules (2021-10-24)
Gang Chen, Yu Kong, You Li, Ailing Huang, Chunyu Wang, Shanshan Zhou, Zhenlin Yang, Yanling Wu, Jianke Ren, Tianlei Ying
要旨

Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin-proteasome and autophagy-lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.

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Sigma-Aldrich
モノクローナル抗FLAG® M2抗体-ペルオキシダーゼ(HRP)標識 マウス宿主抗体, clone M2, purified immunoglobulin, buffered aqueous glycerol solution
Roche
ABTS溶液, solution (ready-to-use), suitable for ELISA