Name: Protein Engineering by Yeast Surface Display (Video) | JoVE Journal
Uploaded: 2024-11-29T13:40:00.000+00:00
Duration: 5 min 49 s
Description: Learn yeast surface display for protein engineering using flow cytometric sorting and controlled selection conditions. This method enables enrichment of antigen-binding variants for therapeutic development.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by the Austrian Science Fund (FWF Project W1224 - Doctoral Program on Biomolecular Technology of Proteins - BioToP and FWF Project ESP 465-B), the Federal Ministry for Digital and Economic Affairs of Austria, the National Foundation for Research, Technology and Development of Austria to the Christian Doppler Research Association (Christian Doppler Laboratory for Next Generation CAR T Cells), and by private donations to the St. Anna Children&#39;s Cancer Research Institute (Vienna, Austria). E.S. is a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the St. Anna Children&#39;s Cancer Research Institute. Figures were created with BioRender.com.

Optimizing Protein Functions Through Yeast Surface Display Techniques

M.W.T. receives funding from Miltenyi Biotec. All authors are inventors on patent applications for technologies and engineered proteins that were developed by using yeast surface display.

Yeast surface display has evolved as one of the key methods used in protein engineering. Although it is commonly employed for the engineering of affinity1,18,40,41, expression/stability24,27,42,43 and activity28,44, further uses like epitope mapping45,46 or characterization of the individual mutants on the surface of yeast cells9 are possible as well. In this protocol, we provide the basic steps for starting a yeast surface display selection campaign, including the selection with magnetic beads and by flow cytometric sorting as well as diversification of the yeast library by epPCR for affinity maturation. 
One essential requirement for conventional yeast surface display selections is the availability of soluble protein of sufficient quality. Starting with a well-folded target protein with high purity and a defined oligomerization state (i.e., monomeric protein should only be present as monomer) provides the highest success rate to select for a protein variant binding to the target antigen with high affinity. An alternative for difficult-to-express target proteins is cell-based selections, which present a reasonable strategy to circumvent this limitation47. However, yeast surface display offers many advantages, such as the possibility of characterizing resulting protein variants directly on the surface of yeast without the necessity to perform laborious and time-intensive cloning, expression in a soluble format, and protein purification. Both the affinity and the stability of the variants can be analyzed directly on the yeast surface9. 
In this protocol, we show how the G4 library of protein variants, more specifically of the 10th type III domain of human fibronectin, was selected for binding to the antigen hRBP4 in the presence of the small molecule A1120. The combination of bead selections and flow cytometric sorting yielded an enrichment of variants, which showed an increased binding to the target antigen throughout selection rounds (Figure 2B). We showed that using lower concentrations of antigen enables the selection of high-affinity protein variants (Figure 2C). Typically, affinities that can be achieved with yeast display selections are in the nanomolar or even picomolar range18. The final affinities depend on the target antigen, the number of selection rounds and affinity maturation, the binding scaffold used, and the applied gating strategy. The characterization of individual protein variants is not covered in this protocol but is explained in detail in our previous work9. Although yeast display was originally employed for the engineering of antibody fragments such as scFvs1,40, the method has been widely used for non-antibody-based proteins as well10. 
To sum up, yeast surface display is a powerful protein engineering tool that enables the generation of protein variants with novel or improved properties, such as binding to almost any target protein and/or increased stability.

Yeast surface display is one of the key technologies in the field of protein engineering. It enables the selection of protein variants with desired properties such as improved affinity or stability. First introduced in 19971, it is one of the most commonly used display technologies besides phage display2,3, ribosome display4, and mammalian cell display5,6,7. The protein of interest (POI) is displayed on the surface of yeast cells by fusing it to anchor proteins. A range of different anchor proteins is available, and most commonly, the POI is fused to the C-terminus of the yeast agglutinin mating protein Aga2p1,8. Additionally, the POI is typically flanked by two tags, such as a hemagglutinin tag (HA-tag) and c-myc tag, which enables detection of the display level by using fluorescently labeled antibodies and flow cytometry (Figure 1A). Typical yeast selection campaigns involve a combination of magnetic bead selections and flow cytometric sorting. The bead selections enable the handling of large cell numbers and enrichment of protein variants binding to the target antigen also with low affinities since multivalent interactions with the antigen-loaded beads lead to avidity effects and, therefore, prevent the loss of low-affinity variants (Figure 1B). Flow cytometric analysis and selection offer the advantage of visualizing the binding of the displayed POI variants to the labeled antigen. Consequently, the binding populations can be sorted and cultivated, leading to the enrichment of protein variants with desired characteristics throughout several sorting rounds. Moreover, additional rounds of random mutagenesis can be performed to further increase the diversity and, hence, the likelihood of finding additional mutations that contribute to the affinity and/or stability of the protein.
Yeast surface display presents certain advantages, such as (a) eukaryotic expression machinery, enabling oxidative protein folding as well as eukaryotic post-translational modifications (such as N-glycosylation), (b) expression normalization due to the detection of the two peptide tags flanking the protein, (c) visual inspection of the selection progress by flow cytometry (e.g., percentage of binding cells and binding intensity) and (d) the possibility of analyzing individual protein mutants on yeast (e.g., analyzing thermostability as well as affinity), presenting a time-saving alternative to laborious protein expression and purification9. In fact, both affinities (KD values) as well as stabilities (T50 values) of yeast surface displayed proteins have shown good correlations with data obtained using biophysical methods and soluble proteins9,10,11,12. Yeast surface display has been employed for the engineering of a variety of proteins, e.g., antibody fragments13,14,15,16, the 10th type III fibronectin domain17,18, rcSso7d19,20, or knottins21. Similarly, extensive research has been undertaken to optimize yeast library designs by altering the randomized positions as well as amino acid codon usage17,22,23. Yeast surface display has been proven successful for the engineering of stability14,15,24,25, affinity18,26,27, enzymatic activity28,29,30,31, and protein expression32. Additionally, more sophisticated applications like conditional binding in the presence or absence of a small molecule were accomplished using yeast surface display20.
In this protocol, we describe all essential steps for a selection campaign with yeast surface display with the example of the G4 library (based on the 10th type III fibronectin domain, Fn3) selected against the antigen human retinol-binding protein 4 (hRBP4) in the presence of the small molecule A112020. This selection was conducted to yield a protein-protein interaction which is dependent on a small molecule that can be used as a molecular switch. Of note, while alternative approaches are possible with yeast surface display, typical yeast selections usually aim for binding to a target antigen without any previous binding affinity. We cover all steps of a yeast selection campaign, involving the cultivation of a yeast library, bead selections, flow cytometric sorting, and affinity maturation by error-prone PCR (epPCR). Therefore, this protocol complements previous yeast surface display protocols33,34 and can be used as a basis for yeast surface display selections (Figure 1) with any given yeast library and target antigen of choice.
<img alt="Yeast display diagram with affinity maturation flowchart for protein library screening and enrichment." class="xfigimg" src="/files/ftp_upload/66994/66994fig1v5.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Principle of yeast surface display and a typical workflow for yeast surface display selections. (A) The POI is cloned into a yeast surface display vector and typically flanked by an N-terminal HA- and a C-terminal c-myc-tag. The construct is fused to the yeast mating protein Aga2p for display on the surface. The protein depicted is the engineered binder "RS3" from PDB ID: 6QBA20. (B) Flowchart illustrating a typical workflow for yeast surface display selection campaigns, which combine enrichment of protein variants with desired properties by bead selections and flow cytometric sorting, as well as epPCR for affinity maturation. <a href="https://www.jove.com/files/ftp_upload/66994/66994fig1largev5.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>10-beta electrocompetent &lt;em&gt;E. coli&lt;/em&gt;&amp;nbsp;</td><td>NEB</td><td>C3020K</td><td></td></tr><tr><td>Agar-Agar, Kobe I</td><td>Carl Roth</td><td>5210.5</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Carl Roth</td><td>K029.2</td><td></td></tr><tr><td>Anti-c-myc antibody, clone 9E10, AF488</td><td>Invitrogen</td><td>MA1-980-A488 (Thermo Fisher)</td><td></td></tr><tr><td>Anti-c-myc antibody, clone 9E10, AF647</td><td>Invitrogen</td><td>MA1-980-A647 (Thermo Fisher)</td><td></td></tr><tr><td>Anti-HA antibody, clone 16B12, AF488</td><td>BioLegend</td><td>901509 (Biozym)</td><td></td></tr><tr><td>Anti-HA antibody, clone 16B12, AF647</td><td>BioLegend</td><td>682404 (Biozym)</td><td></td></tr><tr><td>BamHI-HF</td><td>NEB</td><td>R3136S</td><td></td></tr><tr><td>Bovine serum albumin, cold ethanol fraction</td><td>Sigma-Aldrich</td><td>A4503</td><td></td></tr><tr><td>Citric acid monohydrate</td><td>Sigma-Aldrich</td><td>C1909</td><td></td></tr><tr><td>D-Galactose</td><td>Carl Roth</td><td>4987.2</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Difco yeast nitrogen base</td><td>Becton Dickinson (BD)&amp;nbsp;</td><td>291940</td><td></td></tr><tr><td>Di-Sodium hydrogen phosphate heptahydrate</td><td>Carl Roth</td><td>X987.3</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>D-Sorbitol</td><td>Carl Roth</td><td>6213.1</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (10x)</td><td>Thermo Scientific</td><td>14190169</td><td></td></tr><tr><td>Dynabeads Biotin Binder</td><td>Invitrogen</td><td>11047 (Fisher Scientific)</td><td></td></tr><tr><td>DynaMag-2 Magnet</td><td>Thermo Fisher</td><td>12321D</td><td></td></tr><tr><td>EBY100</td><td>ATCC</td><td>MYA-4941</td><td></td></tr><tr><td>Electroporation cuvette 1 mm (for &lt;em&gt;E.coli&lt;/em&gt;)</td><td>VWR</td><td>732-1135</td><td></td></tr><tr><td>Electroporation cuvette 2 mm (for yeast)</td><td>VWR</td><td>732-1136</td><td></td></tr><tr><td>Ethanol absolute</td><td>MERCK</td><td>1070172511</td><td></td></tr><tr><td>GeneMorph II Random Mutagenesis Kit</td><td>Agilent Technologies</td><td>200550</td><td></td></tr><tr><td>Gibco Bacto Casamino Acids</td><td>Becton Dickinson (BD)</td><td>223120</td><td></td></tr><tr><td>Glycerol</td><td>AppliChem</td><td>131339.1211</td><td></td></tr><tr><td>LE agarose&amp;nbsp;</td><td>Biozym</td><td>840004</td><td></td></tr><tr><td>Lithium acetate dihydrate</td><td>Sigma-Aldrich</td><td>L4158</td><td></td></tr><tr><td>Monarch DNA Gel Extraction Kit</td><td>NEB</td><td>T1020S</td><td></td></tr><tr><td>Monarch PCR &amp;amp; DNA Cleanup Kit</td><td>NEB</td><td>T1030S</td><td></td></tr><tr><td>Multifuge 1S-R</td><td>Heraeus</td><td></td><td></td></tr><tr><td>NheI-HF</td><td>NEB</td><td>R3131S</td><td></td></tr><tr><td>Outgrowth medium</td><td>NEB</td><td>B9035S</td><td></td></tr><tr><td>pCTCON2</td><td>Addgene</td><td>#41843</td><td></td></tr><tr><td>Penicillin G sodium salt</td><td>Sigma-Aldrich</td><td>P3032</td><td></td></tr><tr><td>Penta-His antibody, AF488</td><td>Qiagen</td><td>35310</td><td></td></tr><tr><td>Penta-His antibody, AF647</td><td>Qiagen</td><td>35370</td><td></td></tr><tr><td>Peptone ex casein tryptically digested</td><td>Carl Roth</td><td>8986.3</td><td></td></tr><tr><td>Q5 High-Fidelity DNA Polymerase&amp;nbsp;</td><td>NEB</td><td>M0491S</td><td></td></tr><tr><td>SaII-HF</td><td>NEB</td><td>R3138S</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S8750-1KG</td><td></td></tr><tr><td>Sodium chloride</td><td>Carl Roth</td><td>3957.2</td><td></td></tr><tr><td>Sodium dihydrogen phosphate monohydrate</td><td>Carl Roth</td><td>K300.2</td><td></td></tr><tr><td>Steritop threaded bottle top filter</td><td>MERCK</td><td>S2GPT01RE</td><td></td></tr><tr><td>Streptavidin, AF488</td><td>Invitrogen</td><td>S32354 (Thermo Fisher)</td><td></td></tr><tr><td>Streptavidin, AF647</td><td>Invitrogen</td><td>S32357 (Thermo Fisher)</td><td></td></tr><tr><td>Streptomycin sulfate</td><td>Sigma-Aldrich</td><td>S6501</td><td></td></tr><tr><td>Tri-Sodium citrate dihydrate</td><td>Carl Roth</td><td>4088.1</td><td></td></tr><tr><td>UV-Vis spectrophotometer</td><td>Agilent</td><td>8453</td><td></td></tr><tr><td>Yeast extract, micro-granulated</td><td>Carl Roth</td><td>2904.4</td><td></td></tr><tr><td>Zymoprep Yeast Plasmid Miniprep II</td><td>Zymo Research</td><td>D2004&amp;nbsp;</td><td></td></tr></tbody></table>

protein engineering by yeast surface display

Protein Engineering by Yeast Surface Display

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and versatile tools in the protein engineering field is yeast surface display, where a pool of randomized proteins is expressed on the surface of yeast. The linkage of phenotype (e.g., binding of the yeast-displayed protein to the antigen of interest) and genotype (the plasmid encoding for the protein variant) enables selection of this library for desired properties and subsequent sequencing of enriched variants. By combining magnetic bead selection with flow cytometric sorting, protein variants with enhanced binding to a target antigen can be selected and enriched. Notably, in addition to affinity maturation, binding to a target can also be achieved without any initial binding affinity. Here, we provide a step-by-step protocol that covers all essential parts of a yeast surface display selection campaign and gives examples of typical yeast surface display results. We demonstrate that yeast surface display is a broadly applicable and robust method that can be established in any molecular biology laboratory with access to flow cytometry.

The G4 library was selected against the antigen hRBP4 bound to the small molecule drug A1120. The staining of the libraries for flow cytometric sorting was performed as described in Method 6, and the applied gating strategy is shown in Figure 2A. An initial gate included all cells based on cell morphology, and the second gate (histogram of FSC-Width) showed a stringent gating strategy that was applied to select single cells and remove cell aggregates. The third and final gate showed the display of protein variants (x-axis) versus antigen binding (y-axis). Yeast cells showing both display and binding signals were sorted. Importantly, the sorting gate was set in a stringent way to enrich binding domains with high binding signal and thus high affinity. This stringent selection yielded an enrichment of displaying yeast cells that specifically bind to the target antigen throughout the selection campaign (Figure 2B). In later flow cytometric sorting rounds, the antigen concentration was decreased 10-fold (from 100 nM to 10 nM). Therefore, the overall binding signal was reduced, and only binders with a high affinity were still detectable and sorted (Figure 2C).
<img alt="Flow cytometry gating diagram for cell sorting; displays FSC, SSC, and antigen binding levels." class="xfigimg" src="/files/ftp_upload/66994/66994fig2v5.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Representative results from a yeast surface display selection of the Fn3-based G4 library for binding to the antigen (hRBP4 in the presence of A1120). (A) The general gating strategy for sorting of yeast libraries. The first gate (FSC vs. SSC) is to select all yeast cells and exclude scatter events; the second gate (histogram of FSC-W) aims to remove cell aggregates and only select single yeast cells. The third gate plots the surface display level (detection of the HA- or c-myc-tag) vs. binding to the antigen (here hRBP4 in the presence of 5 µM A1120, detected by anti-His antibody). The library was additionally stained with secondary antibodies only (without antigen), where no antigen binding is expected. Sorted cells are highlighted in blue. (B) Evolution of the G4 library throughout 3 rounds of flow cytometric sorting. Enrichment of the binding population can be observed with each selection round. (C) The use of lower antigen concentrations enables the selection of protein variants with a higher affinity towards the target antigen. Upon reduction of the antigen concentration (here hRBP4) by 10-fold, different diagonals appear, indicating the presence of clones with higher (sorted cells, blue) or lower affinity. <a href="https://www.jove.com/files/ftp_upload/66994/66994fig2largev5.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol describes the essential steps for conducting yeast surface display selection campaigns to enrich protein variants binding to an antigen of interest.

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and ...

protein-engineering-by-yeast-surface-display

Research

JoVE Journal

Bioengineering

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Here, we introduce a protocol for using yeast surface-displayed substrates for enzymatic modification assays. The platform was demonstrated using the analysis of the dephosphorylation activity of tyrosine phosphatase SHP-2 against one of its substrates as a representative enzymatic modification assay.

Enzymatic Modification and Flow Cytometry Assessment of Yeast Surface Displayed Proteins

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Procedure for Lung Engineering

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment.  This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Plasma Lithography Surface Patterning for Creation of Cell Networks

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Self-reporting Scaffolds for 3-Dimensional Cell Culture

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Learn yeast surface display for protein engineering using flow cytometric sorting and controlled selection conditions. This method enables enrichment of antigen-binding variants for therapeutic development.

Protein Engineering by Yeast Surface Display

Explore More Videos