scholarly journals Characterizing Protein-Protein Interactions Using Deep Sequencing Coupled to Yeast Surface Display

2018 ◽  
pp. 101-121 ◽  
Author(s):  
Angelica V. Medina-Cucurella ◽  
Timothy A. Whitehead
Keyword(s):  
Deep Sequencing ◽  
Protein Interactions ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Protein Protein Interactions ◽  
Yeast Surface

scholarly journals Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications

2022 ◽  
Vol 9 ◽  
Author(s):  
Karla V. Teymennet-Ramírez ◽  
Fernando Martínez-Morales ◽  
María R. Trejo-Hernández
Keyword(s):  
Protein Interactions ◽  
Genetic Manipulation ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Yeast Cells ◽  
Biotechnological Applications ◽  
Protein Protein Interactions ◽  
Post Translational Modifications ◽  
Yeast Surface ◽  
Antibody Design

Yeast surface display (YSD) is a “whole-cell” platform used for the heterologous expression of proteins immobilized on the yeast’s cell surface. YSD combines the advantages eukaryotic systems offer such as post-translational modifications, correct folding and glycosylation of proteins, with ease of cell culturing and genetic manipulation, and allows of protein immobilization and recovery. Additionally, proteins displayed on the surface of yeast cells may show enhanced stability against changes in temperature, pH, organic solvents, and proteases. This platform has been used to study protein-protein interactions, antibody design and protein engineering. Other applications for YSD include library screening, whole-proteome studies, bioremediation, vaccine and antibiotics development, production of biosensors, ethanol production and biocatalysis. YSD is a promising technology that is not yet optimized for biotechnological applications. This mini review is focused on recent strategies to improve the efficiency and selection of displayed proteins. YSD is presented as a cutting-edge technology for the vectorial expression of proteins and peptides. Finally, recent biotechnological applications are summarized. The different approaches described herein could allow for a better strategy cascade for increasing protein/peptide interaction and production.

scholarly journals Generating quantitative binding landscapes through fractional binding selections, deep sequencing and data normalization

2019 ◽  
Author(s):  
Michael Heyne ◽  
Niv Papo ◽  
Julia Shifman
Keyword(s):  
Free Energy ◽  
Protein Interactions ◽  
Binding Free Energy ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Protein Protein Interactions ◽  
Protein Mutants ◽  
Display Technology ◽  
Data Points ◽  
Affinity Interaction

AbstractQuantifying the effects of various mutations on binding free energy is crucial for understanding the evolution of protein-protein interactions and would greatly facilitate protein engineering studies. Yet, measuring changes in binding free energy (ΔΔGbind) remains a tedious task that requires expression of each mutant, its purification, and affinity measurements. We developed a new approach that allows us to quantify ΔΔGbindfor thousands of protein mutants in one experiment. Our protocol combines protein randomization, Yeast Surface Display technology, Next Generation Sequencing, and a few experimental ΔΔGbinddata points on purified proteins to generate ΔΔGbindvalues for the remaining numerous mutants of the same protein complex. Using this methodology, we comprehensively map the single-mutant binding landscape of one of the highest-affinity interaction between BPTI and Bovine Trypsin. We show that ΔΔGbindfor this interaction could be quantified with high accuracy over the range of 12 kcal/mol displayed by various BPTI single mutants.

scholarly journals Quantitative yeast-yeast two hybrid for discovery and binding affinity estimation of protein-protein interactions

2020 ◽  
Author(s):  
Kaitlyn Bacon ◽  
Abigail Blain ◽  
John Bowen ◽  
Matthew Burroughs ◽  
Nikki McArthur ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Protein Interactions ◽  
Quantitative Estimation ◽  
Surface Display ◽  
Combinatorial Libraries ◽  
Yeast Surface Display ◽  
Binding Affinities ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Two Hybrid

AbstractQuantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two hybrid (qYY2H) system that not only enables discovery of specific protein-protein interactions, but also efficient, quantitative estimation of their binding affinities (KD). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semi-empirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of the apparent KD of yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.

Parallelized identification of on- and off-target protein interactions

2020 ◽  
Vol 5 (1) ◽  
pp. 349-357
Author(s):  
Jiayi Dou ◽  
Inna Goreshnik ◽  
Cassie Bryan ◽  
David Baker ◽  
Eva-Maria Strauch
Keyword(s):  
Protein Interactions ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Target Protein ◽  
Yeast Surface

Yeast surface display using multi target selections enables monitoring of specificity profiles for thousands of proteins in parallel.

scholarly journals Evaluation of the yeast surface display system for screening of functional nanobodies

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kaho Kajiwara ◽  
Wataru Aoki ◽  
Mitsuyoshi Ueda
Keyword(s):  
Surface Display ◽  
Yeast Surface Display ◽  
Display System ◽  
Yeast Surface

scholarly journals Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 757
Author(s):  
Huiyi Shang ◽  
Danni Yang ◽  
Dairong Qiao ◽  
Hui Xu ◽  
Yi Cao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Large Scale ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Fusion Partner ◽  
Whole Cell ◽  
Food Industries ◽  
Yeast Surface ◽  
The Stability

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

scholarly journals Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

2009 ◽  
Vol 284 (24) ◽  
pp. 16369-16376 ◽  
Author(s):  
Xuebo Hu ◽  
Sungkwon Kang ◽  
Xiaoyue Chen ◽  
Charles B. Shoemaker ◽  
Moonsoo M. Jin
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Coiled Coil ◽  
Affinity Maturation ◽  
Antibody Binding ◽  
Soluble Form ◽  
Protein Protein Interactions ◽  
Yeast Surface ◽  
Two Hybrid

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

Yeast Surface Display in Combination with Fluorescence‐activated Cell Sorting Enables the Rapid Isolation of Antibody Fragments Derived from Immunized Chickens

2018 ◽  
Vol 14 (4) ◽  
pp. 1800466 ◽  
Author(s):  
Julius Grzeschik ◽  
Desislava Yanakieva ◽  
Lukas Roth ◽  
Simon Krah ◽  
Steffen C. Hinz ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Surface Display ◽  
Antibody Fragments ◽  
Yeast Surface Display ◽  
Fluorescence Activated Cell Sorting ◽  
Rapid Isolation ◽  
Yeast Surface
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