Monomeric Covalent‐Avidin for Rapid and Covalent Labeling of Quantum Dots to Cell Surface Proteins

Abstract

With high brightness and photostability, quantum dots (QDs) are potent probes for long‐term imaging of dynamic cell surface proteins, but practical methods to covalently label QDs to target proteins for stable imaging are largely lacking. Here, a small covalent‐bond forming protein (Covalent‐avidin)/peptide pair is introduced, which provides a recombinant protein‐based rapid and covalent QD labeling strategy. Covalent‐avidin is constructed by optimized fusion of circular permuted monomeric avidin to SpyCatcher, which forms an isopeptide bond with the SpyTag peptide. Covalent‐avidin‐conjugated QDs allow for strong and irreversible QD labeling to the biotinylated SpyTag‐fused adrenergic receptor on live cells in 2 min. In addition, QDs with only a minimum number of conjugated Covalent‐avidin show more stable receptor labeling than commercially available streptavidin‐conjugated QDs, also with minimal unwanted clustering of labeled receptors. Monomeric Covalent‐avidin will be a valuable protein linker for diverse other nanolabeling structures with beneficial properties such as covalent linkages and facile valency control.

Sequential, all-bioorthogonal reaction cascade catalyzed by a dual functional artificial metalloenzyme inside encapsulin

“…With a TON above 400 its catalytic activity is comparable to the previously described gold-NHC complexes. 14 The monomeric rhizavidin analogue cpMA 21 shows noncompetitive binding to biotinylated TM-complexes and is a suitable alternative to previously described avidin derivatives. 51 As a fusion protein with HaloTag, cpMA is suitable for designing dual functional ArMs, even though it does not express in soluble form.…”

“…17 Several monomeric analogues of avidin-type proteins have been described, but only one report of a monomeric avidin based ArM exists. [18][19][20][21] In the present work, two different guest proteins with either monomeric streptavidin (mSA) 22 or circular permuted monomeric avidin (cpMA) 21 fused to HaloTag were investigated. The first step of the presented reaction cascade involves the alloc-deprotection of a secondary amine catalyzed by a ruthenium catalyst (RuH, see Figure 2) covalently bound to HaloTag.…”

A dual-metal catalyzed sequential cascade reaction in an engineered protein cage

“…Its catalytic activity is comparable to those of previously described gold-NHC complexes. [20] The monomeric rhizavidin analogue cpMA [30] shows noncompetitive binding to biotinylated TM-complexes and is a suitable alternative to previously described avidin derivatives. [49] Recent investigations in our group focus on studying the tertiary sphere effects provided by the encapsulin shell by introducing several pore mutated variants.…”

“…With two compatible TM-complexes in hand, a protein scaffold for immobilizing the catalysts inside encapsulin was developed. For our design, we chose to fuse cpMA with our established HaloTag construct [17] for several reasons: a) cpMA allows for the immobilization of biotin-containing small molecules with high affinity, [30] b) no potentially interfering moieties are released during the immobilization process (e.g., as with SNAPtag, the release of benzylguanine), [39] c) cpMA is a monomeric 16 kDa protein. With a total of 53 kDa and a calculated approximate volume of 120 nm³ the fusion protein Halo-cpMA is well suited to fit into the inside of encapsulin (14 nm inner diameter, 1400 nm³ calculated volume).…”

“…[26][20,27] The resulting reaction cascade combines both major mechanisms of (bio)-precursor prodrug activation: an initial bond cleavage step is followed by a subsequent bond forming step. [28] For stable immobilization of the two orthogonal transition metal (TM) complexes inside Enc, we designed a dual functional protein chimera based on HaloTag [29] (Halo) fused to monomeric rhizavidin [30] (circular permuted monomeric avidin, cpMA).…”