A Shorter Route to Antibody Binders via Quantitative in vitro Bead-Display Screening and Consensus Analysis

Mankowska, Sylwia A; Gatti‐Lafranconi, Pietro; Chodorge, Matthieu; Sridharan, Sridha; Minter, Ralph; Hollfelder, Florian

doi:10.1038/srep36391

Cited by 15 publications

(9 citation statements)

References 60 publications

(85 reference statements)

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“…Here, we found the throughput to be limited by bacterial transformation efficiency using cloned DNA. By coupling the PEN CSR technology and an in vitro enzyme expression platform 51 , a wide range of toxic proteins could be targeted, while also avoiding the transformation bottleneck.…”

Section: Discussionmentioning

confidence: 99%

Directed Evolution of Enzymes based on in vitro Programmable Self-Replication

Dramé-Maigné

Espada

McCallum

et al. 2021

Preprint

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High-throughput directed evolution, implemented in well-controlled in vitro conditions, provides a powerful route for enzyme engineering. Most existing technologies are based on activity screening and require the sequential observation and sorting of each individual variant. By contrast, approaches based on autonomous feedback loops, linking phenotype to genotype replication, enable autonomous selection without screening. However, these approaches are only possible in vivo, or applicable to very specific activities, such as polymerases or ligases. Here, we leverage synthetic molecular networks to create a programmable in vitro feedback loop linking a target enzymatic activity to gene amplification. After encapsulation and lysis of up to 10^7 transformed variants, the genes present in each droplet are amplified according to the activity of the encoded enzyme, resulting in the autonomous enrichment of interesting sequences. Applied to a nicking enzyme with thermal or kinetic selection pressures, this method reveals detailed mutational landscapes and provides improved variants.

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Section: Discussionmentioning

confidence: 99%

Directed Evolution of Enzymes based on in vitro Programmable Self-Replication

Dramé-Maigné

Espada

McCallum

et al. 2021

Preprint

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“…In addition, given the flexibility of labelling and of screening, bacterial cell display offers a unique opportunity to systematically optimise all steps in method development – a challenge regularly highlighted in the field and not easily accessible to all selection platforms. Typically, the success of selections can be assessed by PCR enrichment in a model system (Figures B and 5D), and while simple, this approach masks the complexity of parameters known to be involved in selection.…”

Section: Discussionmentioning

confidence: 99%

Bacterial Cell Display as a Robust and Versatile Platform for Engineering Low‐Affinity Ligands and Enzymes

2020

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Directed evolution has been remarkably successful at expanding the chemical and functional boundaries of biology. That progress is heavily dependent on the robustness and flexibility of the available selection platforms, given the significant cost to (re)develop a given platform to target a new desired function. Bacterial cell display has a significant track record as a viable strategy for the engineering of mesophilic enzymes, as enzyme activity can be probed directly and free from interference from the cellular milieu, but its adoption has lagged behind other display-based methods. Herein, we report the development of SNAP as a quantitative reporter for bacterial cell display, which enables fast troubleshooting and the systematic development of the display-based selection platform, thus improving its robustness. In addition, we demonstrate that even weak interactions between displayed proteins and nucleic acids can be harnessed for the specific labelling of bacterial cells, allowing functional characterisation of DNA binding proteins and enzymes, thus making it a highly flexible platform for these biochemical functions. Together, this establishes bacterial display as a robust and flexible platform, ideally suited for the systematic engineering of ligands and enzymes needed for XNA molecular biology.

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“…When present at sufficiently low concentration, assay components can be encapsulated on a single-molecule or singleparticle basis (Collins et al 2015;Mankowska et al 2016). This enables the high-throughput study of phenomena relevant to PPIs on the single-molecule level, for example in the directed evolution of peptide binders against MDM2, a negative regulator of the tumour suppressor protein p53 (Iwakuma and Lozano 2003;Cui et al 2016) (Fig.…”

Section: Analysis Of Ppis Following Microdroplet Compartmentalisationmentioning

confidence: 99%

Microfluidic approaches for the analysis of protein–protein interactions in solution

et al. 2020

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Exploration and characterisation of the human proteome is a key objective enabling a heightened understanding of biological function, malfunction and pharmaceutical design. Since proteins typically exhibit their behaviour by binding to other proteins, the challenge of probing protein-protein interactions has been the focus of new and improved experimental approaches. Here, we review recently developed microfluidic techniques for the study and quantification of protein–protein interactions. We focus on methodologies that utilise the inherent strength of microfluidics for the control of mass transport on the micron scale, to facilitate surface and membrane-free interrogation and quantification of interacting proteins. Thus, the microfluidic tools described here provide the capability to yield insights on protein–protein interactions under physiological conditions. We first discuss the defining principles of microfluidics, and methods for the analysis of protein–protein interactions that utilise the diffusion-controlled mixing characteristic of fluids at the microscale. We then describe techniques that employ electrophoretic forces to manipulate and fractionate interacting protein systems for their biophysical characterisation, before discussing strategies that use microdroplet compartmentalisation for the analysis of protein interactions. We conclude by highlighting future directions for the field, such as the integration of microfluidic experiments into high-throughput workflows for the investigation of protein interaction networks.

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A Shorter Route to Antibody Binders via Quantitative in vitro Bead-Display Screening and Consensus Analysis

Cited by 15 publications

References 60 publications

Directed Evolution of Enzymes based on in vitro Programmable Self-Replication

Directed Evolution of Enzymes based on in vitro Programmable Self-Replication

Bacterial Cell Display as a Robust and Versatile Platform for Engineering Low‐Affinity Ligands and Enzymes

Microfluidic approaches for the analysis of protein–protein interactions in solution

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