Sortase A-mediated multi-functionalization of protein nanoparticles

Abstract

We report here a new strategy to enable fast, covalent, and site-directed functionalization of protein nanoparticles using Sortase A-mediated ligation using functional proteins ranging from monomeric to large tetrameric structures. Easy purification of the modified E2 nanoparticles is achieved by functionalization with a thermo-responsive elastin-like-peptide. The resulting protein nanoparticles remained intact and active even after repeated phase transitions, suggesting their use in biocatalysis, biosensing, and imaging applications.

Protein nanoparticles as multifunctional biocatalysts and health assessment sensors

“…While many PNP systems cannot support direct fusions of large proteins as they can interfere with self-assembly [6,42], many platforms can afford the fusion of small functional peptides [6,8,36,44]. For instance, the tobacco mosaic virus (TMV) nanorod VLP monomers can be modified at the C-terminus with a FLAG tag [44]; however, an “IQ” peptide for Genhance 680 fluorophore binding was found to interfere with the folding and assembly.…”

“…Sortase A from Staphylococcus aureus is often used for its tail-to-head ligations between a small C-terminal LPXTG motif and small N-terminal poly-glycine motif [47]. Sortase A has been utilized for the functionalization of a triglycine modified E2 protein nanocage [6,22]. Our group demonstrated the ligation of LPETG modified elastin-like polypeptide (ELP) and a tetrameric β-galactosidase onto E2 [6].…”

“…It is well known that immobilization of functional biomolecules either onto the exterior or the inside of PNPs improves their stability and activity [2]. This feature when combined with the ability of PNPs to tolerate extreme conditions make PNPs an ideal platform for a wide range of bionanotechnology applications in biomedicine [2–4] and biocatalysis [5,6]. …”

“…Although most PNPs used today are based on biologically evolved protein assemblies such as viral capsids [5,7] or multimeric enzymes [6,8], there is a move toward engineering these protein nano-assemblies based on in silico design [9,10]. The ability to provide multi-functionalization is often highly challenging as the modifications may result in a loss of nanoparticle self-assembly or protein functionality.…”

Recent Advances in the Generation of Antibody–Nanomaterial Conjugates

“…Enzyme-based conjugation also offers superior control over the antibody orientation following attachment to the nanoparticle surface. [114] Despite these obvious advantages, there are reports showing limitations for the enzyme-based coupling approaches, which arise mainly by fusion proteins being too large and preparation procedure to undertake the coupling reaction being inefficient due to multiple steps. [115] Leung et al developed an alternative strategy to overcome some of these limitations using sortase A enzyme (Srt A) (Figure 13).…”