Yeast Surface Display in Protein Engineering and Analysis

Hackel, Benjamin J.; Wittrup, K. Dane

doi:10.1002/9783527634026.ch26

Cited by 4 publications

(2 citation statements)

References 71 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…When using a centromeric plasmid (CEN6/ARS) for cell surface display, about 10-30% cells do not show any display by flow cytometry (Boder and Wittrup 1997, Hackel and Wittrup 2008, Lee, DeLoache et al 2015. Moreover, transcription noise is introduced due to plasmid copy number variation (4-8 copies per cell) (Jahn, Vorpahl et al 2016, Shao, Rammohan et al 2021, Fu, Patel et al 2022).…”

Section: Integrative Protease Activity Reporters Enhance Reliability ...mentioning

confidence: 99%

Modular and integrative activity reporters enhance biochemical studies in the yeast ER

Martinusen,

Slaton,

Nelson

et al. 2023

Preprint

View full text Add to dashboard Cite

The yeast endoplasmic reticulum sequestration and screening (YESS) system is a generalizable platform that has become highly useful to investigate post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.

show abstract

Section: Integrative Protease Activity Reporters Enhance Reliability ...mentioning

confidence: 99%

Modular and integrative activity reporters enhance biochemical studies in the yeast ER

Martinusen,

Slaton,

Nelson

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Common high-throughput methods, such as yeast − and phage − display technologies linking genotype to phenotype, coupled with selections via magnetic bead capture − and fluorescence activated cell sorting (FACS), , have been used to address this demand. Experimental screening strategies require the use of the biomarker to isolate binding ligands; however, many clinically relevant biomarkers possess hydrophobic transmembrane domains, making them difficult to work with in an aqueous environment.…”

Section: Introductionmentioning

confidence: 99%

Magnetic Bead-Immobilized Mammalian Cells Are Effective Targets to Enrich Ligand-Displaying Yeast

Lown

Hackel

2020

ACS Comb. Sci.

Self Cite

View full text Add to dashboard Cite

Yeast surface display empowers selection of protein binding ligands, typically using recombinant soluble antigens. However, ectodomain fragments of transmembrane targets may fail to recapitulate their true, membrane-bound form. Direct selections against adhered mammalian cells empower enrichment of genuine binders yet benefit from high target expression, robustly adherent mammalian cells, and nanomolar affinity ligands. This study evaluates a modified format with mammalian cells immobilized to magnetic beads; yeast-displayed fibronectin domain and affibody ligands of known affinities and cells with expression ranges of epidermal growth factor receptor (EGFR) and CD276 elucidate important parameters to ligand enrichment and yield in cell suspension panning with comparison to adherent panning. Cell suspension panning is hindered by significant background of nondisplaying yeast but exhibits yield advantages in model EGFR systems for a high affinity (K D = 2 nM) binder on cells with both high (10 6 per cell) target expression (9.6 ± 0.6% vs 3.2 ± 0.4%, p < 0.0001) and mid (10 5 ) target expression (2.3 ± 0.5% vs 0.41 ± 0.09%, p = 0.0008), as well as for a low affinity (K D > 600 nM) binder on high target expression cells (2.0 ± 0.5% vs 0.017 ± 0.005%; p = 0.001). Significant enrichment was observed for all EGFR systems except the low-affinity, high expression system. The CD276 system failed to provide significant enrichment, indicating that this technique may not be suitable for all targets. Collectively, this study highlights new approaches that yield successful enrichment of yeast-displayed ligands via panning on immobilized mammalian cells.

show abstract

Extended yeast surface display linkers enhance the enrichment of ligands in direct mammalian cell selections

Lown

Cai

Ritter

et al. 2021

Protein Engineering, Design and Selection

View full text Add to dashboard Cite

Selections of yeast-displayed ligands on mammalian cell monolayers benefit from high target expression and nanomolar affinity, which are not always available. Prior work extending the yeast–protein linker from 40 to 80 amino acids improved yield and enrichment but is hypothesized to be below the optimal length, prompting evaluation of an extended amino acid linker. A 641-residue linker provided enhanced enrichment with a 2-nM affinity fibronectin ligand and 105 epidermal growth factor receptors (EGFR) per cell (14 ± 2 vs. 8 ± 1, P = 0.008) and a >600-nM affinity ligand, 106 EGFR per cell system (23 ± 7 vs. 0.8 ± 0.2, P = 0.004). Enhanced enrichment was also observed with a 310-nM affinity affibody ligand and 104 CD276 per cell, suggesting a generalizable benefit to other scaffolds and targets. Spatial modeling of the linker suggests that improved extracellular accessibility of ligand enables the observed enrichment under conditions not previously possible.

show abstract

Yeast Surface Display in Protein Engineering and Analysis

Cited by 4 publications

References 71 publications

Modular and integrative activity reporters enhance biochemical studies in the yeast ER

Modular and integrative activity reporters enhance biochemical studies in the yeast ER

Magnetic Bead-Immobilized Mammalian Cells Are Effective Targets to Enrich Ligand-Displaying Yeast

Extended yeast surface display linkers enhance the enrichment of ligands in direct mammalian cell selections

Contact Info

Product

Resources

About