Synthesis of bioactive protein hydrogels by genetically encoded SpyTag-SpyCatcher chemistry

Abstract

Protein-based hydrogels have emerged as promising alternatives to synthetic hydrogels for biomedical applications, owing to the precise control of structure and function enabled by protein engineering. Nevertheless, strategies for assembling 3D molecular networks that carry the biological information encoded in fulllength proteins remain underdeveloped. Here we present a robust protein gelation strategy based on a pair of genetically encoded reactive partners, SpyTag and SpyCatcher, that spontaneously form covalent isopeptide linkages under physiological conditions. The resulting "network of Spies" may be designed to include celladhesion ligands, matrix metalloproteinase-1 cleavage sites, and full-length globular proteins [mCherry and leukemia inhibitory factor (LIF)]. The LIF network was used to encapsulate mouse embryonic stem cells; the encapsulated cells remained pluripotent in the absence of added LIF. These results illustrate a versatile strategy for the creation of information-rich biomaterials.protein biomaterials | stem cell encapsulation | cell fate control H ydrogels made from natural or synthetic polymers have been investigated for many years as scaffolds for encapsulated cells (1). The past decade has witnessed a growing trend in hydrogel design that calls for systems capable of controlling the behavior of encapsulated cells by providing, sensing, and responding to biological signals (2). This design criterion demands a new level of craftsmanship from scientists and engineers who wish to incorporate biomolecular species, which may range in size and complexity from small molecules to multidomain proteins, into biomaterials (3). A promising approach to this challenge uses bio-orthogonal chemistry to introduce the species of interest with spatial and temporal control (4-7).Here we discuss an alternative approach, based on the design and expression of artificial proteins that are programmed to form covalent molecular networks, which offers important advantages in the engineering of dynamic biomaterials systems (8, 9). The method requires no chemical reagents, proceeds efficiently under mild conditions (e.g., in aqueous solution at physiological pH, in the presence of ambient levels of oxygen, and at temperatures ranging from 4 to 37°C), allows introduction of biological information in modular fashion, and enables encapsulation of cells without loss of viability.The recent discovery of naturally occurring isopeptide bonds in Gram-positive bacterial adhesins (10) inspired Howarth and coworkers (11, 12) to design a pair of reactive protein partners (SpyTag and SpyCatcher) by splitting the second immunoglobulinlike collagen adhesin domain (CnaB2) of the fibronectin-binding protein (FbaB) of Streptococcus pyogenes. SpyTag-SpyCatcher chemistry forms a specific isopeptide bond between Asp-117 of SpyTag and Lys-31 of SpyCatcher. In a previous study, we demonstrated that placing SpyTag and SpyCatcher at carefully chosen locations within elastin-like proteins (ELPs) enabled efficient synthesis of unusual nonlinea...

Programmable polyproteams built using twin peptide superglues

“…Polyproteams should be a powerful platform to dissect the spatial requirements for cellular signaling, such as in immunity and differentiation (51)(52)(53). Other applications of this simple route to new biological architectures may include vaccination (2), biomaterials (34,(54)(55)(56), multienzyme organization (9), and enhancing capture of circulating tumor cells (57).…”

Building Spatial Synthetic Biology with Compartments, Scaffolds, and Communities

“…For example, a SpyCatcher/SpyTag cross-linked elastin network that incorporates leukemiainhibitory factor has been used as a growth medium for mouse embryonic stem cells; this medium maintains stem-cell pleuripotency (Sun et al 2014). In theory, this matrix could be genetically encoded.…”

B 12 -dependent photoresponsive protein hydrogels for controlled stem cell/protein release

“…Assembling genetically engineered proteins into molecular networks represents an alternative strategy to make hydrogels with well-controlled properties (16)(17)(18)(19). Although natural evolution has led to numerous functional protein domains that can sense and respond to a variety of environmental stimuli, such as light, oxidative stress, pH, small molecules, metal ions, etc., such ecological diversity has yet to be fully tapped to develop responsive biomaterials with dynamically tunable properties.…”

“…Because of its high efficiency and modularity, this chemistry has led to a number of applications, including control of biomacromolecular topology, synthesis of bioactive and "living" materials, and biomolecular imaging (16,18,(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). It has proven to be a powerful method for constructing complex biomolecular architectures both in vitro and in vivo.…”