Synthetic Biology: A New Tool for the Trade

Zakeri, Bijan

doi:10.1002/cbic.201500372

Cited by 24 publications

(21 citation statements)

References 59 publications

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“…Two proteins fused to SpyTag and SpyCatcher, respectively, can then be covalently linked by intermolecular isopeptide bond formation simply upon mixing the reactants. With the advent of this covalent split-protein system many applications have been demonstrated [20], which benefit from the system´s robustness and independence from most experimental conditions [21], e.g. cell surface labeling in fluorescence microscopy [18], enzyme resilience to boiling [22–25], bioactive hydrogels [26], and protein assembly [21, 27–32].…”

Section: Introductionmentioning

confidence: 99%

Probing the potential of CnaB-type domains for the design of tag/catcher systems

et al. 2017

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Building proteins into larger, post-translational assemblies in a defined and stable way is still a challenging task. A promising approach relies on so-called tag/catcher systems that are fused to the proteins of interest and allow a durable linkage via covalent intermolecular bonds. Tags and catchers are generated by splitting protein domains that contain intramolecular isopeptide or ester bonds that form autocatalytically under physiological conditions. There are already numerous biotechnological and medical applications that demonstrate the usefulness of covalent linkages mediated by these systems. Additional covalent tag/catcher systems would allow creating more complex and ultra-stable protein architectures and networks. Two of the presently available tag/catcher systems were derived from closely related CnaB-domains of Streptococcus pyogenes and Streptococcus dysgalactiae proteins. However, it is unclear whether domain splitting is generally tolerated within the CnaB-family or only by a small subset of these domains. To address this point, we have selected a set of four CnaB domains of low sequence similarity and characterized the resulting tag/catcher systems by computational and experimental methods. Experimental testing for intermolecular isopeptide bond formation demonstrated two of the four systems to be functional. For these two systems length and sequence variations of the peptide tags were investigated revealing only a relatively small effect on the efficiency of the reaction. Our study suggests that splitting into tag and catcher moieties is tolerated by a significant portion of the naturally occurring CnaB-domains, thus providing a large reservoir for the design of novel tag/catcher systems.

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Section: Introductionmentioning

confidence: 99%

Probing the potential of CnaB-type domains for the design of tag/catcher systems

et al. 2017

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“…However, DNA maintained in vivo can also be used for the communication of digital information, for example for genome watermarking applications [ 1 ]. In future experiments, we aim to utilize programmable post-translational protein assembly [ 21 – 24 ] to develop an addiction module that will allow for the intracellular dual maintenance of two plasmids with a common origin and selection marker, which would enable in vivo MuSE. We intend for these early explorations to stimulate the development of future DNA communication tools that in turn may further broaden adoption of DNA for communication, an increasing possibility with the development of portable sequencing devices [ 25 , 26 ].…”

Section: Discussionmentioning

confidence: 99%

Multiplexed Sequence Encoding: A Framework for DNA Communication

Zakeri

Carr

2016

PLoS ONE

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Synthetic DNA has great propensity for efficiently and stably storing non-biological information. With DNA writing and reading technologies rapidly advancing, new applications for synthetic DNA are emerging in data storage and communication. Traditionally, DNA communication has focused on the encoding and transfer of complete sets of information. Here, we explore the use of DNA for the communication of short messages that are fragmented across multiple distinct DNA molecules. We identified three pivotal points in a communication—data encoding, data transfer & data extraction—and developed novel tools to enable communication via molecules of DNA. To address data encoding, we designed DNA-based individualized keyboards (iKeys) to convert plaintext into DNA, while reducing the occurrence of DNA homopolymers to improve synthesis and sequencing processes. To address data transfer, we implemented a secret-sharing system—Multiplexed Sequence Encoding (MuSE)—that conceals messages between multiple distinct DNA molecules, requiring a combination key to reveal messages. To address data extraction, we achieved the first instance of chromatogram patterning through multiplexed sequencing, thereby enabling a new method for data extraction. We envision these approaches will enable more widespread communication of information via DNA.

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“…The SpyTag/SpyCatcher system originates from covalently-stabilized pilin polymers found in the pilus of Gram-positive bacteria which usually undergo intramolecular amide bond formation to impart mechanical and proteolytic stability to pili [60], precisely Streptococcus pyogenes, inspiring the name Spy Technology. When split and engineered, Zakeri and co-workers generated first generation isopeptags binding to pilin-N or -C [61,62], mediation of irreversible peptide-peptide interactions via SpyLigase [63], and finally, the SpyCatcher and SpyTag [64]. This approach makes use of the collagen adhesin domain (CnaB2) of fibronectin binding protein (FbaB) that possesses an internal isopeptide bond between N-and C-domains' amino acids Lys31 and Asp117.…”

Section: The Spytag/spycatcher Systemmentioning

confidence: 99%

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

Hofmann

Krah

Sellmann

et al. 2020

IJMS

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Recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. The plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. Although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a ‘plug and play’ manner. In this review, we provide an overview of the most relevant methodologies for bispecific screening purposes—the DuoBody concept, paired light chain single cell production approaches, Sortase A and Transglutaminase, the SpyTag/SpyCatcher system, and inteins—and elaborate on the benefits as well as drawbacks of the different technologies.

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Synthetic Biology: A New Tool for the Trade

Cited by 24 publications

References 59 publications

Probing the potential of CnaB-type domains for the design of tag/catcher systems

Probing the potential of CnaB-type domains for the design of tag/catcher systems

Multiplexed Sequence Encoding: A Framework for DNA Communication

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

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