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...

Control of molecular topology constitutes a fundamental challenge in macromolecular chemistry. Here we describe the synthesis and characterization of artificial elastinlike proteins (ELPs) with unconventional nonlinear topologies including circular, tadpole, star, and H-shaped proteins using genetically encoded SpyTag−SpyCatcher chemistry. SpyTag is a short polypeptide that binds its protein partner SpyCatcher and forms isopeptide bonds under physiological conditions. Sequences encoding SpyTag and SpyCatcher can be strategically placed into ELP genes to direct post-translational topological modification in situ. Placement of SpyTag at the N-terminus and SpyCatcher at the C-terminus directs formation of circular ELPs. Induction of expression at 16 °C with 10 μM IPTG yields 80% monomeric cyclic protein. When SpyTag is placed in the middle of the chain, it exhibits an even stronger tendency toward cyclization, yielding up to 94% monomeric tadpole proteins. Telechelic ELPs containing either SpyTag or SpyCatcher can be expressed, purified, and then coupled spontaneously upon mixing in vitro. Block proteins, 3-arm or 4-arm star proteins, and H-shaped proteins have been prepared, with the folded CnaB2 domain that results from the SpyTag−SpyCatcher reaction as the molecular core or branch junction. The modular character of the SpyTag−SpyCatcher strategy should make it useful for preparing nonlinear macromolecules of diverse sequence and structure.

Protein posttranslational modifications (PTMs), particularly phosphorylation, dramatically expand the complexity of cellular regulatory networks. Although cysteine (Cys) in various proteins can be subject to multiple PTMs, its phosphorylation was previously considered a rare PTM with almost no regulatory role assigned. We report here that phosphorylation occurs to a reactive cysteine residue conserved in the staphylococcal accessary regulator A (SarA)/MarR family global transcriptional regulator A (MgrA) family of proteins, and is mediated by the eukaryotic-like kinase-phosphatase pair Stk1-Stp1 in Staphylococcus aureus. Cys-phosphorylation is crucial in regulating virulence determinant production and bacterial resistance to vancomycin. Cell wall-targeting antibiotics, such as vancomycin and ceftriaxone, inhibit the kinase activity of Stk1 and lead to decreased Cys-phosphorylation of SarA and MgrA. An in vivo mouse model of infection established that the absence of stp1, which results in elevated protein Cys-phosphorylation, significantly reduces staphylococcal virulence. Our data indicate that Cys-phosphorylation is a unique PTM that can play crucial roles in bacterial signaling and regulation.

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Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive "smart" hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB 12 )-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarH C ) proteins using genetically encoded SpyTagSpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarH C polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarH C hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.hydrogels | cell encapsulation | drug delivery | photoresponsive materials | protein engineering