iFLinkC: an iterative functional linker cloning strategy for the combinatorial assembly and recombination of linker peptides with functional domains

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

doi:10.1093/nar/gkz1210

Cited by 58 publications

(40 citation statements)

References 56 publications

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“…68 Alternatively, proline-rich linkers were beneficial in auto-inhibited biosensors as proline increases the rigidity and thus persistence length of the linkers. 69 Similarly, tandem repeats of small folded domains expand the reach of antibodies more efficiently than flexible linkers. 70…”

Section: Discussionmentioning

confidence: 99%

Estimation of effective concentrations enforced by complex linker architectures from conformational ensembles

Kjærgaard

2021

Preprint

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Proteins and protein assemblies often tether interaction partners to strengthen interactions, to regulate activity through auto-inhibition or -activation, or to boost enzyme catalysis. Tethered reactions are regulated by the architecture of tether, which define an effective concentration of the interactors. Effective concentrations can be estimated theoretically for simple linkers via polymer models, but there is currently no general method for estimating effective concentrations for complex linker architectures consisting of both flexible and folded domains. We describe how effective concentrations can be estimated computationally for any protein linker architecture by defining a realistic conformational ensemble. We benchmark against prediction from a worm-like chain and values measured by competition experiments, and find minor differences likely due to excluded volume effects. Systematic variation of the properties of flexible and folded segments show that the effective concentration is mainly determined by the combination of the total length of flexible segments and the distance between termini of the folded domains. We show that a folded domain in a disordered linker can increase the effective concentration beyond what can be achieved by a fully disordered linker by focusing the end-to-end distance at the appropriate spacing. This suggest that complex linker architecture may have advantages over simple flexible linker, and emphasize that annotation as a linker should depend on the molecular context.

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Section: Discussionmentioning

confidence: 99%

Estimation of effective concentrations enforced by complex linker architectures from conformational ensembles

Kjærgaard

2021

Preprint

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“…Constructs were cloned into suitable expression vectors for recombinant protein production in E. coli either on their own or as a fusion protein by means of the iFLinkC DNA assembly process. [ 35 ] The complete amino acid sequences of individual expression constructs are outlined in the Supporting Information. Porcine pancreatic α‐Amy was purchased commercially (Catalogue no.…”

Section: Methodsmentioning

confidence: 99%

Ultrasensitive and Selective Protein Recognition with Nanobody‐Functionalized Synthetic Nanopores

Duznovic

Gräwe

Weber

et al. 2021

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The development of flexible and reconfigurable sensors that can be readily tailored toward different molecular analytes constitutes a key goal and formidable challenge in biosensing. In this regard, synthetic nanopores have emerged as potent physical transducers to convert molecular interactions into electrical signals. Yet, systematic strategies to functionalize their surfaces with receptor proteins for the selective detection of molecular analytes remain scarce. Addressing these limitations, a general strategy is presented to immobilize nanobodies in a directional fashion onto the surface of track‐etched nanopores exploiting copper‐free click reactions and site‐specific protein conjugation systems. The functional immobilization of three different nanobodies is demonstrated in ligand binding experiments with green fluorescent protein, mCherry, and α‐amylase (α‐Amy) serving as molecular analytes. Ligand binding is resolved using a combination of optical and electrical recordings displaying quantitative dose–response curves. Furthermore, a change in surface charge density is identified as the predominant molecular factor that underlies quantitative dose–responses for the three different protein analytes in nanoconfined geometries. The devised strategy should pave the way for the systematic functionalization of nanopore surfaces with biological receptors and their ability to detect a variety of analytes for diagnostic purposes.

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“…Fusion proteins with multiple or new functions can be encoded by combining open reading frames. The length and amino acid sequence of the linker affects the activity and interaction of domains, so optimisation may be needed; linker libraries and combinatorial approaches are available (Gräwe et al, 2020;G. Li et al, 2016).…”

Section: Variants and Fusion Proteinsmentioning

confidence: 99%

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Young

Haines

Storch

et al. 2021

Metabolic Engineering

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Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.

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iFLinkC: an iterative functional linker cloning strategy for the combinatorial assembly and recombination of linker peptides with functional domains

Cited by 58 publications

References 56 publications

Estimation of effective concentrations enforced by complex linker architectures from conformational ensembles

Estimation of effective concentrations enforced by complex linker architectures from conformational ensembles

Ultrasensitive and Selective Protein Recognition with Nanobody‐Functionalized Synthetic Nanopores

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

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