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="Figure 1" class="xfigimg" src="/files/ftp_upload/66994/66994fig1v5.jpg" /> 
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 &#34;RS3&#34; 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 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="Figure 2" class="xfigimg" src="/files/ftp_upload/66994/66994fig2v5.jpg" /> 
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 &#181;M 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>

Protein Engineering by Yeast Surface Display

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

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Bioengineering

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

Video: Protein Engineering by Yeast Surface Display

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Biochemistry

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Here, we present a workflow for the expression, purification and liposome binding of SNX-BAR heterodimers in yeast.

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and ...

Genetics

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic biotinylation by the E. coli biotin-protein ligase BirA is highly specific and allows for the biotinylation of target proteins in their native environment; however, the current usage of BirA mediated biotinylation requires the presence of a synthetic acceptor peptide (AP) in the target protein. Therefore, its application is limited to proteins that have been engineered to contain the AP. The purpose of the present protocol is to use the bacterial display of a peptide derived from an unmodified target protein to select for BirA variants that biotinylates the peptide. The system is based on a single plasmid that allows for the co-expression of BirA variants along with a scaffold for the peptide display on the bacterial surface. The protocol describes a detailed procedure for the incorporation of the target peptide into the display scaffold, creation of the BirA library, selection of active BirA variants and initial characterization of the isolated BirA variants. The method provides a highly effective selection system for the isolation of novel BirA variants that can be used for the further directed evolution of biotin-protein ligases that biotinylate a native protein in complex solutions.

Here we present a method to select for novel variants of the E. coli biotin-protein ligase BirA that biotinylates a specific target peptide. The protocol describes the construction of a plasmid for the bacterial display of the target peptide, generation of a BirA library, selection and characterization of BirA variants.

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic ...

Immunology and Infection

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Biology

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

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

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

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

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

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Protein Engineering by Yeast Surface Display

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Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

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

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry