Synthetic protein switches: Combinatorial linker engineering with iFLinkC

Gräwe, Alexander; Ranglack, Jan; Weyrich, Anastasia; Stein, Viktor

doi:10.1016/bs.mie.2020.09.009

Cited by 4 publications

(8 citation statements)

References 45 publications

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“…In the context of the iFLinkC-X assembly framework, α- and ω-plasmids strictly feature Gly at their N- and C-terminal ends (Figure D). In this way, the resultant linker, once assembled, is readily compatible with the parent iFLinkC assembly process , (Figure A). Apart from that, the α- and ω-plasmids generally code for the two lowest indices of a linker motif, while the bridging amino acid X on the inside can be flexibly chosen to connect arbitrary linker fragments depending on the predominant amino acid in a polymeric linker repeat (Figure D).…”

Section: Resultsmentioning

confidence: 99%

“…We recently developed a DNA assembly process termed iterative functional linker cloning (iFLinkC) which enables the fusion of any two protein coding sequences, for example, a functional domain with a linker element, via a single Gly residue , (Figure A). Assembly is achieved through the combined action of several type IIS restriction enzymes and T4 DNA ligase.…”

Section: Resultsmentioning

confidence: 99%

“…(D) Detailed depiction of α-, μ-, and ω-assembly junctions. single Gly residue 6,18 (Figure 1A). Assembly is achieved through the combined action of several type IIS restriction enzymes and T4 DNA ligase.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Furthermore, it highlights a need for empiric optimization and readily motivates further studies in the context of alternative protein sensors, switches, and other protein assemblies including biocatalytic reaction cascades 3 and polyvalent protein therapeutics. 10,11 ■ CONCLUSIONS Building on the iFLinkC logic, 6,18 a scalable framework was devised to engineer co-polymeric linkers of variable lengths, resolution, and�except for the identity of the bridging amino acid�composition too. Inspired by the logic of numeral systems that are capable of representing a large set of numbers based on a limited number of digits, iFLinkC-X now provides a scalable route to assemble a large set of linker elements based on a limited number of linker fragments that are stored in sequence-verified, and therefore reusable, entry plasmids.…”

Section: ■ Introductionmentioning

confidence: 99%

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iFLinkC-X: A Scalable Framework to Assemble Bespoke Genetically Encoded Co-polymeric Linkers of Variable Lengths and Amino Acid Composition

2022

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Linker engineering is rapidly gaining prominence as protein engineers and synthetic biologists construct increasingly sophisticated protein assemblies capable of executing complex molecular functions in the context of biosensing, biocatalysis, or biotherapeutics. Depending on the application, the structural and functional requirements imposed on the underlying linkers can differ vastly. At the same time, there is a distinct lack of methods to effectively code linkers at the level of DNA and tailor them to the functional requirements of different fusion proteins. Addressing these limitations, a scalable framework is presented to compose copolymeric linkers of variable lengths and amino acid composition based on a limited number of linker fragments stored in sequence-verified entry plasmids. The assembly process is exemplified for Pro-rich linkers in the context of a Zn 2+ -responsive dual-readout BRET/FRET sensor while examining how linker composition impacts key functional properties such as ligand affinity, dynamic range, and their ability to separate structurally distinct domains.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

See 2 more Smart Citations

iFLinkC-X: A Scalable Framework to Assemble Bespoke Genetically Encoded Co-polymeric Linkers of Variable Lengths and Amino Acid Composition

2022

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“…Engineering Recombinant Nanopore Assemblies. Finally, a set of recombinant nanopores was engineered by recombining the truncated S 21 56 variant with ring-shaped oligomeric protein structures and linkers of varying length and flexibility 46,47 (Figure 6A). Ring-shaped oligomeric protein structures were drawn from members of the Lsm protein family AfSm2, 48 AfSm1, 49 and ScLsm3 50 featuring defined hexa-, hepta-, and octameric geometries.…”

Section: ■ Resultsmentioning

confidence: 99%

Functional Nanopore Screen: A Versatile High-Throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli

et al. 2022

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Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the functional nanopore (FuN) screen now provides a quantitative and dynamic readout of nanopore assembly and function in the context of the inner membrane of Escherichia coli. The assay is based on genetically encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca 2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S 21 68 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S 21 68 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta-, and octameric geometries. Library screening highlights substantial plasticity in the ability of the S 21 68 transmembrane module to oligomerize in alternative geometries, while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology, and synthetic biology.

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Dissecting the Permeability of the Escherichia coli Cell Envelope to a Small Molecule Using Tailored Intensiometric Fluorescent Protein Sensors

Kemp,

Weber,

Desczyk

et al. 2023

ACS Omega

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Membranes provide a highly selective barrier that defines the boundaries of any cell while providing an interface for communication and nutrient uptake. However, despite their central physiological role, our capacity to study or even engineer the permeation of distinct solutes across biological membranes remains rudimentary. This especially applies to Gram-negative bacteria, where the outer and inner membrane impose two permeation barriers. Addressing this analytical challenge, we exemplify how the permeability of the Escherichia coli cell envelope can be dissected using a small-molecule-responsive fluorescent protein sensor. The approach is exemplified for the biotechnologically relevant macrolide rapamycin, for which we first construct an intensiometric rapamycin detector (iRapTor) while comprehensively probing key design principles in the iRapTor scaffold. Specifically, this includes the scope of minimal copolymeric linkers as a function of topology and the concomitant need for gate post residues. In a subsequent step, we apply iRapTors to assess the permeability of the E. coli cell envelope to rapamycin. Despite its lipophilic character, rapamycin does not readily diffuse across the E. coli envelope but can be enhanced by recombinantly expressing a nanopore in the outer membrane. Our study thus provides a blueprint for studying and actuating the permeation of small molecules across the prokaryotic cell envelope.

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Synthetic protein switches: Combinatorial linker engineering with iFLinkC

Cited by 4 publications

References 45 publications

iFLinkC-X: A Scalable Framework to Assemble Bespoke Genetically Encoded Co-polymeric Linkers of Variable Lengths and Amino Acid Composition

iFLinkC-X: A Scalable Framework to Assemble Bespoke Genetically Encoded Co-polymeric Linkers of Variable Lengths and Amino Acid Composition

Functional Nanopore Screen: A Versatile High-Throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli

Dissecting the Permeability of the Escherichia coli Cell Envelope to a Small Molecule Using Tailored Intensiometric Fluorescent Protein Sensors

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