Self-assembling nitrilotriacetic acid nanofibers for tracking and enriching His-tagged proteins in living cells

Abstract

Specific and expeditious identification and enrichment of target proteins in living cells is often a challenging task. The hexahistidine (6His) tag is frequently used to label artificially engineered proteins produced in prokaryotic or eukaryotic cells. Utilizing the interaction between 6His-tag and nitrilotriacetic acid (NTA) mediated by divalent metal ions (Ni²⁺, Cu²⁺, Zn²⁺ or Co²⁺), we designed and synthesized a series of Nap-G/Biotin/ANA-FFpYGK-NTA probes that, assisted by alkaline phosphatase (ALP), self-assemble into nanofibers. The probe consists of an NTA group that specifically binds to 6His-tag, an FFpY group that promotes self-assembly facilitated by ALP, and a hydrophobic (Nap-G/ANA/Biotin) capping group for various applications. We demonstrate that the ANA-FFpYGK-NTA(Ni²⁺) nanofibers are fit for real-time tracking of His-tagged protein in living cells, and the Biotin-FFpYGK-NTA(Ni²⁺) nanofibers are for isolating His-tagged proteins and other proteins that they interact with.