Applications of Yeast Surface Display for Protein Engineering

Cherf, Gerald Maxwell; Cochran, Jennifer R.

doi:10.1007/978-1-4939-2748-7_8

Cited by 193 publications

(160 citation statements)

References 132 publications

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“…4) (Boder & Wittrup, 1997). YSD as a technology to engineer proteins has been reviewed extensively (Chen et al, 2013; Cherf & Cochran, 2015; Colby et al, 2004; Moore & Cochran, 2012). YSD is also a useful platform for performing characterization of binding interactions with a soluble target, obviating the need for large-scale expression and purification of a protein of interest (Gai & Wittrup, 2007).…”

Section: Measuring Binding On the Surface Of Yeastmentioning

confidence: 99%

Cell-Binding Assays for Determining the Affinity of Protein–Protein Interactions

Hunter

Cochran

2016

Methods in Enzymology

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Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein–protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities—time to equilibrium and ligand depletion—will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented.

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Section: Measuring Binding On the Surface Of Yeastmentioning

confidence: 99%

Cell-Binding Assays for Determining the Affinity of Protein–Protein Interactions

Hunter

Cochran

2016

Methods in Enzymology

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“…Compared to existing technologies, our approach has several advantages. Although yeast and phage display can powerfully discriminate between 'bound' and 'unbound' peptide populations 38,39 for > 10 8 protein-peptide interactions, these high-throughput screening methods cannot quantitatively measure affinities and are unable to probe the effects of PTMs or unnatural amino acids with therapeutic potential. The MRBLE-pep assay is also faster and requires less in material than alternative methods: measuring interaction affinities for 384 peptides using MRBLE-pep requires ~400x, ~700x, and ~9600x less purified protein than surface plasmon resonance, fluorescence polarization, and isothermal calorimetry, respectively, with savings increasing with library size (Table S6).…”

Section: Discussionmentioning

confidence: 99%

Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

Nguyen

Roy

Harink

et al. 2018

Preprint

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Weak, transient binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drive cellular signaling. Mapping the energy landscape of such interactions is essential for deciphering signaling networks and developing therapeutic inhibitors, but is complicated by technical challenges associated with quantitatively measuring weak interactions in high-throughput. Here, we synthesize peptide libraries on spectrally encoded beads with each peptide sequence uniquely linked to a spectral code (MRBLE-pep), allowing high-throughput quantification of protein-peptide binding via imaging. Using computational modeling and MRBLEpep assays, we map the affinity landscape for human calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, interacting with a known SLiM (PxIxIT). We find that PxIxIT recognition depends critically on flanking residues and is regulated by post-translational modifications. Using this information, we designed PxIxIT peptides with unprecedented affinity and therapeutic potential that strongly inhibit CN function in vivo.

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“…Typically, scFv retain most of the positive characteristics of their parental immunoglobulin while eliminating Fc-mediated immune responses and clearance mechanisms. Moreover, the recombinant construction of scFv and their expression in microbial systems allows efficient scale up of production, straightforward introduction of sequence modifications, direct fusion to therapeutic cargo, and the potential for affinity maturation via display techniques[8,21–24]. …”

Section: Introductionmentioning

confidence: 99%

Molecular engineering of high affinity single-chain antibody fragment for endothelial targeting of proteins and nanocarriers in rodents and humans

Greineder

Hood

Yao

et al. 2016

Journal of Controlled Release

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Endothelial cells (EC) represent an important target for pharmacologic intervention, given their central role in a wide variety of human pathophysiologic processes. Studies in lab animal species have established that conjugation of drugs and carriers with antibodies directed to surface targets like the Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, a highly expressed endothelial transmembrane protein) help to achieve specific therapeutic interventions in ECs. To translate such “vascular immunotargeting” to clinical practice, it is necessary to replace antibodies by advanced ligands that are more amenable to use in humans. We report the molecular design of a single chain variable antibody fragment (scFv) that binds with high affinity to human PECAM-1 and cross-reacts with its counterpart in rats and other animal species, allowing parallel testing in vivo and in human endothelial cells in microfluidic model. Site-specific modification of the scFv allows conjugation of protein cargo and liposomes, enabling their endothelial targeting in these models. This study provides a template for molecular engineering of ligands, enabling studies of drug targeting in animal species and subsequent use in humans.

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Applications of Yeast Surface Display for Protein Engineering

Cited by 193 publications

References 132 publications

Cell-Binding Assays for Determining the Affinity of Protein–Protein Interactions

Cell-Binding Assays for Determining the Affinity of Protein–Protein Interactions

Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

Molecular engineering of high affinity single-chain antibody fragment for endothelial targeting of proteins and nanocarriers in rodents and humans

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