Biology subjects

Bacon, K.

Publications and source records attributed to Bacon, K..

3 recordsLinked to original sources

bioRxiv2020

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

Quantifying 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.

biochemistry↗

bioRxiv2020

Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display

Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated crosslinking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides by magnetic selection and fluorescence activated cell sorting (FACS), to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ~ 1.2 - 3.7 {micro}M). The isolated peptides bound lysozyme selectively, and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides could be used to efficiently obtain quantitative estimates of binding affinity, without chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that binds IL-17 in its receptor binding region with moderate affinity (KD ~ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.

synthetic biology↗

bioRxiv2019

Screening yeast display libraries against magnetized yeast cell targets enables efficient isolation of membrane protein binders.

When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is co-expressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization. Subsequently, binder cells that interact with the magnetized target cells can be isolated using a magnet. Using a known binder-target pair with modest binding affinity (KD ~ 400 nM), we showed that a binder present at low frequency (1 in 105) could be enriched >100-fold, in a single round of screening, suggesting feasibility of screening combinatorial libraries. Subsequently, we screened yeast display libraries of Sso7d and nanobody variants against yeast displayed targets to isolate binders specific to the cytosolic domain of the mitochondrial membrane protein TOM22 (KD ~ 271-2009 nM) and the extracellular domain of the c-Kit receptor (KD ~ 131 to KD >2000 nM). Additional studies showed that the TOM22 binders identified using this approach could be used for the enrichment of mitochondria from cell lysates, thereby confirming binding to the native mitochondrial protein. The ease of expressing a membrane protein or a domain thereof as a yeast cell surface fusion - in contrast to recombinant soluble expression - makes the use of yeast-displayed targets particularly attractive. Therefore, we expect the use of magnetized yeast cell targets will enable efficient isolation of binders to membrane proteins.\n\nO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioengineering↗