SpyTag/SpyCatcher Cyclization Confers Resilience to Boiling on a Mesophilic Enzyme

Schoene, Christopher; Fierer, Jacob O.; Bennett, Samantha; Howarth, Mark

doi:10.1002/anie.201402519

Cited by 152 publications

(115 citation statements)

References 31 publications

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“…Altogether, the SpyCatcher-SpyTag is a modular system can be used for a variety of applications to produce novel protein architectures, including joining functionalized polypeptide strands to proteins (Fig. 4C) at specific times during cell culture [230], provide an environment to improve viability of cells [232], enzyme stability [233], and vaccine optimization [234]. …”

Section: Targeting the Extracellular Space – Modules In Biomaterialsmentioning

confidence: 99%

Bottom-up approaches in synthetic biology and biomaterials for tissue engineering applications

Weisenberger

Deans

2018

Journal of Industrial Microbiology and Biotechnology

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Synthetic biologists use engineering principles to design and construct genetic circuits for programming cells with novel functions. A bottom-up approach is commonly used to design and construct genetic circuits by piecing together functional modules that are capable of reprogramming cells with novel behavior. While genetic circuits control cell operations through the tight regulation of gene expression, a diverse array of environmental factors within the extracellular space also has a significant impact on cell behavior. This extracellular space offers an addition route for synthetic biologists to apply their engineering principles to program cell-responsive modules within the extracellular space using biomaterials. In this review, we discuss how taking a bottom-up approach to build genetic circuits using DNA modules can be applied to biomaterials for controlling cell behavior from the extracellular milieu. We suggest that, by collectively controlling intrinsic and extrinsic signals in synthetic biology and biomaterials, tissue engineering outcomes can be improved.

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Section: Targeting the Extracellular Space – Modules In Biomaterialsmentioning

confidence: 99%

Bottom-up approaches in synthetic biology and biomaterials for tissue engineering applications

Weisenberger

Deans

2018

Journal of Industrial Microbiology and Biotechnology

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“…12 Among them, cyclic topology is the most commonly encountered nonlinear protein topology in nature and has also been extensively studied to date. 18−20 Various technologies have been developed to prepare cyclic proteins, both in vivo (21) and in vitro , 22 including intein technology, 23 SpyTag-SpyCatcher chemistry, 24,25 sortase/butlase-mediated ligation, 26−30 etc. Typical benefits from backbone cyclization include the dramatically improved stability against both thermal denaturation and proteolytic digestion.…”

Section: Introductionmentioning

confidence: 99%

Topology Engineering of Proteins in Vivo Using Genetically Encoded, Mechanically Interlocking SpyX Modules for Enhanced Stability

Liu

et al. 2017

ACS Cent. Sci.

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Recombinant proteins are traditionally limited to linear configuration. Herein, we report in vivo protein topology engineering using highly efficient, mechanically interlocking SpyX modules named AXB and BXA. SpyX modules are protein domains composed of p53dim (X), SpyTag (A), and SpyCatcher (B). The p53dim guides the intertwining of the two nascent protein chains followed by autocatalytic isopeptide bond formation between SpyTag and SpyCatcher to fulfill the interlocking, leading to a variety of backbone topologies. Direct expression of AXB or BXA produces protein catenanes with distinct ring sizes. Recombinant proteins containing SpyX modules are obtained either as mechanically interlocked obligate dimers if the protein of interest is fused to the N- or C-terminus of SpyX modules, or as star proteins if the protein is fused to both N- and C-termini. As examples, cellular syntheses of dimers of (GB1)2 (where GB1 stands for immunoglobulin-binding domain B1 of streptococcal protein G) and of four-arm elastin-like star proteins were demonstrated. Comparison of the catenation efficiencies in different constructs reveals that BXA is generally much more effective than AXB, which is rationalized by the arrangement of three domains in space. Mechanical interlocking induces considerable stability enhancement. Both AXB and BXA have a melting point ∼20 °C higher than the linear controls and the BXA catenane has a melting point ~2 °C higher than the cyclic control BX’A. Notably, four-arm elastin-like star proteins demonstrate remarkable tolerance against trypsin digestion. The SpyX modules provide a convenient and versatile approach to construct unconventional protein topologies via the “assembly-reaction” synergy, which opens a new horizon in protein science for stability enhancement and function reinforcement via topology engineering.

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“…32,[38][39][40][41][42] It has also recently been demonstrated that cyclic protein or tadpole protein can be synthesized in cells in situ by highly reactive genetically encoded chemistry with improved stability. 41,43 While the integration of cyclic polymer with other topological macromolecules has been explored in depth, only a few examples of macrocycle-nanoparticle conjugates are known. 44 Considering that introducing macrocyclic segment into giant molecules could greatly increase the structural complexity of whole macromolecules, it is thus highly desirable to construct a series of cyclic polymer-based giant surfactants for further study on their structure-property relationships.…”

Section: Introductionmentioning

confidence: 99%

Precision synthesis of macrocyclic giant surfactants tethered with two different polyhedral oligomeric silsesquioxanes at distinct ring locations via four consecutive “click” reactions

Feng

et al. 2015

Polym. Chem.

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The combined utilization of chemoselective "click" chemistry allows for the preparation of well-defined macromolecules with complex compositions and architectures. In this article, we employed the sequential "click" strategy to further expand the scope of synthetically available giant molecules by precisely constructing new giant surfactants based on polyhedral oligomeric silsesquioxane (POSS) tethered cyclic polymers. The general synthetic approach involves sequentially performed strain-promoted azide-alkyne cycloaddition (SPAAC) as a method for bimolecular homobifunctional ring closure, copper-catalyzed azide-alkyne cycloaddition (CuAAC) for POSS-polymer conjugation, and thiol-Michael/thiol-ene reactions for POSS surface functionalization. Specifically, a cyclic polymer tethered with two POSS cages of distinct surface chemistry at different locations of the chain has been prepared. This work promises to afford numerous cyclic polymers-based giant surfactants with diverse structural variations for further investigation on unexpected physical properties.

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SpyTag/SpyCatcher Cyclization Confers Resilience to Boiling on a Mesophilic Enzyme

Cited by 152 publications

References 31 publications

Bottom-up approaches in synthetic biology and biomaterials for tissue engineering applications

Bottom-up approaches in synthetic biology and biomaterials for tissue engineering applications

Topology Engineering of Proteins in Vivo Using Genetically Encoded, Mechanically Interlocking SpyX Modules for Enhanced Stability

Precision synthesis of macrocyclic giant surfactants tethered with two different polyhedral oligomeric silsesquioxanes at distinct ring locations via four consecutive “click” reactions

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