Tris-(Nitrilotriacetic Acid)-Decorated Polymer Conjugates as Tools for Immobilization and Visualization of His-Tagged Proteins

Abstract

Recombinant proteins are commonly expressed with artificial affinity tags for purification, immobilization and characterization. The most frequently used tag, His-tag, is a sequence of consecutive histidine residues fused to the protein of interest. Specialized small molecules that bind His-tag are primarily used for purification, while antibodies are used for protein analysis. However, various issues may be encountered with the use of antibodies. Low inherent stability, the difficulty of introducing chemical modifications, and often-unreliable batch-to-batch consistency are among the limiting factors that call for better alternatives. Recently described polymer conjugates of N-(2-hydroxypropyl) methacrylamide and low-molecular-weight functional ligands, so-called iBodies, are antibody mimetics capable of replacing antibodies in biochemical applications. We tailored this system for methods utilizing His-tag by accessorizing the polymer carrier with tris-nitrilotriacetic acid targeting ligands. These anti-polyHis iBodies are additionally accessorized with fluorophores, enabling detection, and biotin ligands, enabling immobilization. Here, we characterized anti-polyHis iBodies and explored their use as antibody mimetics. We tested their stability, as well as the influence of different metal mediators and His-tag lengths on binding. With high affinity and stability, iBodies represent a new alternative for immobilization and visualization of His-tagged proteins.