Engineered Calcium-Precipitable Restriction Enzyme

Hendrix, Josephina R.; Read, Timothy D.; Lalonde, Jean‐François; Jensen, Phillip K.; Heymann, William; Lovelace, Elijah; Zimmermann, Sarah; Brasino, Michael; Rokicki, Joseph; Dowell, Robin D.

doi:10.1021/sb500042m

Cited by 5 publications

(5 citation statements)

References 6 publications

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“…Pure and active recombinant proteins were obtained via calcium-induced precipitation of the proteins from the E. coli cell lysate, followed by the protease cleavage of the BRT17 tag [ 65 ]. This platform was used by an undergraduate iGEM team for the purification of a tagged Eco RI restriction enzyme, where RTX-tagged enzyme retained its endonuclease activity after purification via calcium-mediated precipitation [ 81 ].…”

Section: Exploring the Order Of Rtx Domain Repeat Sequence Lead Tomentioning

confidence: 99%

Block V RTX Domain of Adenylate Cyclase from Bordetella pertussis: A Conformationally Dynamic Scaffold for Protein Engineering Applications

Bulutoglu

Banta

2017

Toxins

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The isolated Block V repeats-in-toxin (RTX) peptide domain of adenylate cyclase (CyaA) from Bordetella pertussis reversibly folds into a β-roll secondary structure upon calcium binding. In this review, we discuss how the conformationally dynamic nature of the peptide is being engineered and employed as a switching mechanism to mediate different protein functions and protein-protein interactions. The peptide has been used as a scaffold for diverse applications including: a precipitation tag for bioseparations, a cross-linking domain for protein hydrogel formation and as an alternative scaffold for biomolecular recognition applications. Proteins and peptides such as the RTX domains that exhibit natural stimulus-responsive behavior are valuable building blocks for emerging synthetic biology applications.

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Section: Exploring the Order Of Rtx Domain Repeat Sequence Lead Tomentioning

confidence: 99%

Block V RTX Domain of Adenylate Cyclase from Bordetella pertussis: A Conformationally Dynamic Scaffold for Protein Engineering Applications

Bulutoglu

Banta

2017

Toxins

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“…A series of purification tags comprised 5, 9, 13, and 17 repeats of the nonapeptide, and 13-and 17-repeat domains precipitated when calcium concentrations exceeded 25 mM. 27 Genetic fusion of RTX tags to globular proteins of interest enables purification by calcium-driven precipitation, 27,[195][196][197] and consensus RTX tags included an enzymatic cleavage site for later removal from target proteins. This purification approach allows for quick and simple precipitation of proteins at lower temperature and ionic strength compared to other phase-changing polypeptides.…”

Section: Repeats-in-toxin Proteins: Calcium-responsive Motifs With Reversible Foldingmentioning

confidence: 99%

“…The reversible structural change of RTX domains has enabled controlled changes in separation between proteins, 192,193 tunable gelation behavior, 178,188,194 and precipitation based on Ca 2+ signals. 27,[195][196][197] This stimuli-responsive behavior makes the RTX motif an emerging domain of interest for biotechnological applications from bioseparations to drug delivery to tissue engineering.…”

Section: Repeats-in-toxin Proteins: Calcium-responsive Motifs With Reversible Foldingmentioning

confidence: 99%

Monomer‐scale design of functional protein polymers using consensus repeat sequences

Chang

Huang

Danielle

2021

Journal of Polymer Science

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Protein-based polymers possess chemically defined sequences that can encode diverse properties and functions into a new class of biopolymeric materials.However, sequence variation that emerges from evolution can obscure the sequence-function relationships of naturally derived polymers. One strategy to clarify these relationships is to identify common sequences between proteins with similar functions. These conserved sequences often emerge from repeat proteins, and "consensus repeat sequences" provide a convenient platform for systematic investigations of biopolymer sequence-property relationships. In this review, we highlight recent approaches to engineer tunable polymeric materials using monomer-scale design of consensus repeat proteins. We explore established and emerging protein-based materials with mechanical resilience, thermodynamic phase behavior, chemical responsiveness, biomolecular transport, and hierarchical structure. Overall, recent advances in the monomer-scale design of repetitive protein polymers present exciting fundamental and translational opportunities for polymer scientists and engineers.

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“…Indeed, there was a noticeable boost in iGEM team publications in when the journal published several papers as part of an iGEM special issue (http://pubs.acs.org/toc/asbcd6/3/12). Several of these publications were particularly relevant to research involving bacteria (Atanaskovic, Bencherif, Deyell et al 2014;Buren, Karrenbelt, Lingemann et al 2014;Daszczuk, Dessalegne, Drenth et al 2014;Hendrix, Read, Lalonde et al 2014;Libis, Bernheim, Basier et al 2014;Nielsen, Madsen, Seppala et al 2014;Wang, Ding, Chen et al 2014;Wu, Wang, Cao et al 2014).…”

Section: Responsible Research and Innovation In Synthetic Biologymentioning

confidence: 99%

Promoting microbiology education through the iGEM synthetic biology competition

Kelwick

Bowater

Yeoman

et al. 2015

FEMS Microbiology Letters

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Synthetic biology has developed rapidly in the 21st century. It covers a range of scientific disciplines that incorporate principles from engineering to take advantage of and improve biological systems, often applied to specific problems. Methods important in this subject area include the systematic design and testing of biological systems and, here, we describe how synthetic biology projects frequently develop microbiology skills and education. Synthetic biology research has huge potential in biotechnology and medicine, which brings important ethical and moral issues to address, offering learning opportunities about the wider impact of microbiological research. Synthetic biology projects have developed into wide-ranging training and educational experiences through iGEM, the International Genetically Engineered Machines competition. Elements of the competition are judged against specific criteria and teams can win medals and prizes across several categories. Collaboration is an important element of iGEM, and all DNA constructs synthesized by iGEM teams are made available to all researchers through the Registry for Standard Biological Parts. An overview of microbiological developments in the iGEM competition is provided. This review is targeted at educators that focus on microbiology and synthetic biology, but will also be of value to undergraduate and postgraduate students with an interest in this exciting subject area.

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Engineered Calcium-Precipitable Restriction Enzyme

Cited by 5 publications

References 6 publications

Block V RTX Domain of Adenylate Cyclase from Bordetella pertussis: A Conformationally Dynamic Scaffold for Protein Engineering Applications

Block V RTX Domain of Adenylate Cyclase from Bordetella pertussis: A Conformationally Dynamic Scaffold for Protein Engineering Applications

Monomer‐scale design of functional protein polymers using consensus repeat sequences

Promoting microbiology education through the iGEM synthetic biology competition

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