Programmable polyproteams built using twin peptide superglues

Veggiani, Gianluca; Nakamura, Tomohiko; Brenner, Michael D.; Gayet, Raphaël V.; Yan, Jun; Robinson, Carol V.; Howarth, Mark

doi:10.1073/pnas.1519214113

Cited by 264 publications

(344 citation statements)

References 68 publications

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“…The SpyTag/SpyCatcher interaction has been used to prepare cross-linked hydrogels, 27 to control protein topology, 28 to analyze expression and localization of proteins, 29,30 and to create long, extended proteins by linking together multiple polypeptides. 31 Each of the associative protein domains was genetically fused at its C -terminus to an elastin-like polypeptide bearing a C -terminal cysteine residue for site-specific conjugation to particle surfaces. Hexahistidine tags were added to N - and C -termini of each protein to facilitate purification.…”

Section: Resultsmentioning

confidence: 99%

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Protein-Mediated Colloidal Assembly

2017

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Programmable colloidal assembly enables the creation of mesoscale materials in a bottom-up manner. Although DNA oligonucleotides have been used extensively as the programmable units in this paradigm, proteins, which exhibit more diverse modes of association and function, have not been widely used to direct colloidal assembly. Here we use protein–protein interactions to drive controlled aggregation of polystyrene microparticles, either through reversible coiled-coil interactions or through intermolecular isopeptide linkages. The sizes of the resulting aggregates are tunable and can be controlled by the concentration of immobilized surface proteins. Moreover, particles coated with different protein pairs undergo orthogonal assembly. We demonstrate that aggregates formed by association of coiled-coil proteins, in contrast to those linked by isopeptide bonds, are dispersed by treatment with chemical denaturants or soluble competing proteins. Finally, we show that protein–protein interactions can be used to assemble complex core–shell aggregates. This work illustrates a versatile strategy for engineering colloidal systems for use in materials science and biotechnology.

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Section: Resultsmentioning

confidence: 99%

“…The strategy is general and easily expanded, owing to the diversity of associative protein domains. 31,35,36 The preparation of complex colloidal aggregates in a scalable, programmable manner should find application in catalysis, health technologies and environmental remediation. 37–39 …”

Section: Resultsmentioning

confidence: 99%

Protein-Mediated Colloidal Assembly

2017

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“…Although, our current assembly was accomplished with EGFP fusions, we do not foresee any difficulty fusing other proteins onto the Tag-Catchers as shown from previous studies [11, 12, 15]. To further enhance yields, solid phase addition, as demonstrated by SnoopTag [16], or step-wise purifications could also be utilized.…”

Section: Resultsmentioning

confidence: 99%

“…The new Tag-Catcher pair will be used alongside SpyTag-SpyCatcher partners, to demonstrate kinetically controlled directed protein ligation, domain specific protein circularization and macromolecular assembly. During our preparation of this manuscript, Dr. M. Howarth at University of Oxford have also published an alternative Tag-Catcher system, SnoopTag-Catcher, that can also be utilized with SpyTag-Catcher [16]. …”

Section: Introductionmentioning

confidence: 99%

Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly

Tan¹,

Hoon²,

Wong³

2016

PLoS ONE

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Over the last few years, a number of different protein assembly strategies have been developed, greatly expanding the toolbox for controlling macromolecular assembly. One of the most promising developments is a rapid protein ligation approach using a short polypeptide SpyTag and its partner, SpyCatcher derived from Streptococcus pyogenes fibronectin-binding protein, FbaB. To extend this technology, we have engineered and characterized a new Tag-Catcher pair from a related fibronectin-binding protein in Streptococcus dysgalactiae. The polypeptide Tag, named SdyTag, was constructed based on the native Cna protein B-type (CnaB) domain and was found to be highly unreactive to SpyCatcher. SpyCatcher has 320-fold specificity for its native SpyTag compared to SdyTag. Similarly, SdyTag has a 75-fold specificity for its optimized Catcher, named SdyCatcherDANG short, compared to SpyCatcher. These Tag-Catcher pairs were used in combination to demonstrate specific sequential assembly of tagged proteins in vitro. We also demonstrated that the in vivo generation of circularized proteins in a Tag-Catcher specific manner where specific Tags can be left unreacted for use in subsequent ligation reactions. From the success of these experiments, we foresee the application of SdyTags and SpyTags, not only, for multiplexed control of protein assembly but also for the construction of novel protein architectures.

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“…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.…”

mentioning

confidence: 99%

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

Wang

Yang

Luo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

137

120

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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

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Programmable polyproteams built using twin peptide superglues

Cited by 264 publications

References 68 publications

Protein-Mediated Colloidal Assembly

Protein-Mediated Colloidal Assembly

Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly

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

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Programmable polyproteams built using twin peptide superglues

Cited by 264 publications

References 68 publications

Protein-Mediated Colloidal Assembly

Protein-Mediated Colloidal Assembly

Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly

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

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Product

Resources

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