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We thank all members of the Dingal laboratory for their advice, expertise, and discussions. We also thank the UTD Flow Cytometry Core for infrastructure and support. M.B.L. is supported by the UTD Eugene McDermott Graduate Fellowship. This research was supported by a UTD Startup Fund and National Institutes of Health-NIGMS awards to the laboratory of P.C.D.P.D. (R35GM150967).

<ol>
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	<li>Lim Suan MB et al. Identification and engineering of highly functional potyviral proteases in cells using co-evolutionary models. Nat Commun. 2026;17:3257. doi: 10.1038/s41467-026-69961-5.</li>
	<li>Kapust RB, Waugh DS. Controlled intracellular processing of fusion proteins by TEV protease. Protein Expr Purif. 2000;19:312&#8211;318.</li>
	<li>Nallamsetty S et al. Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro. Protein Expr Purif. 2004;38:108&#8211;115.</li>
	<li>Chen Z, Elowitz MB. Programmable protein circuit design. Cell. 2021;184:2284&#8211;2301.</li>
	<li>Sanchez MI, Ting AY. Directed evolution improves the catalytic efficiency of TEV protease. Nat Methods. 2020;17:167&#8211;174.</li>
	<li>Denard CA et al. YESS 2.0, a tunable platform for enzyme evolution, yields highly active TEV protease variants. ACS Synth Biol. 2021;10:63&#8211;71.</li>
	<li>Gietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007;2:31&#8211;34.</li>
	<li>Benatuil L, Perez JM, Belk J, Hsieh CM. An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel. 2010;23:155&#8211;159.</li>
	<li>Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997;15:553&#8211;557.</li>
	<li>Lau WL et al. Using yeast surface display to engineer a soluble and crystallizable construct of hematopoietic progenitor kinase 1 (HPK1). Acta Crystallogr Sect F Struct Biol Commun. 2021;77:22&#8211;28.</li>
	<li>Kieke MC et al. Selection of functional T cell receptor mutants from a yeast surface-display library. Proc Natl Acad Sci U S A. 1999;96:5651&#8211;5656.</li>
	<li>Ma H, Kunes S, Schatz PJ, Botstein D. Plasmid construction by homologous recombination in yeast. Gene. 1987;58:201&#8211;216.</li>
	<li>Varadarajan N, Cantor JR, Georgiou G, Iverson BL. Construction and flow cytometric screening of targeted enzyme libraries. Nat Protoc. 2009;4:893&#8211;901.</li>
	<li>Haas CM, Francino-Urdaniz IM, Steiner PJ, Whitehead TA. Identification of SARS-CoV-2 S RBD escape mutants using yeast screening and deep mutational scanning. STAR Protoc. 2021;2:100869. doi: 10.1016/j.xpro.2021.100869.</li>
	<li>Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983;153:163&#8211;168.</li>
	<li>Bloom JD, Arnold FH. In the light of directed evolution: pathways of adaptive protein evolution. Proc Natl Acad Sci U S A. 2009;106:9995&#8211;10000.</li>
	<li>Yi L et al. Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries. Proc Natl Acad Sci U S A. 2013;110:7229&#8211;7234.</li>
	<li>Martinusen SG et al. Modular and integrative activity reporters enhance biochemical studies in the yeast ER. Protein Eng Des Sel. 2024;37. doi: 10.1093/protein/gzae008.</li>
	<li>Li Q et al. Profiling protease specificity: combining yeast ER sequestration screening (YESS) with next generation sequencing. ACS Chem Biol. 2017;12:510&#8211;518.</li>
	<li>Packer MS, Rees HA, Liu DR. Phage-assisted continuous evolution of proteases with altered substrate specificity. Nat Commun. 2017;8:956. doi: 10.1038/s41467-017-01055-9.</li>
	<li>Yaghi RM, Andrews CL, Wylie DC, Iverson BL. High-resolution substrate specificity profiling of SARS-CoV-2 Mpro; comparison to SARS-CoV Mpro. ACS Chem Biol. 2024;19:1474&#8211;1483.</li>
</ol>

The authors declare no competing interests. The raw data supporting this protocol, including Nanopore sequencing FASTQ files, flow cytometry (FCS) files, and source data used to generate the figures and tables, and plasmids pDD1523 and pDD1524 have been deposited in Zenodo and are publicly available at 10.5281/zenodo.20746804

Directed evolution is a high-throughput method that uses iterative cycles of diversification and selection to identify proteins with enhanced or novel biochemical function17. In this protocol, we detail an optimized workflow to evolve protease-substrate recognition using a cell-surface display system in S. cerevisiae. The surface display system maps protease activity to a detectable fluorescence-based phenotype, enabling FACS-based enrichment of desired clones. We provide the detailed ste...

Cysteine proteases of the Potyviridae family, specifically the nuclear inclusion A (NIa) proteases, represent a largely untapped source of sequence-specific endopeptidases. With more than 3,800 characterized family members, NIa proteases cleave a distinct seven-amino-acid substrate sequence. The potyviral family offers a diverse set of proteases for engineering programmable protein control systems, constructing orthogonal protease-based genetic circuits, and developing sequence-specific biotechnological tools1,2. Despite this natural diversity, only two NIa proteases, TEVp and tobacco vein mottling virus protease (TVMVp), have been thoroughly characterized and engineered for broad laboratory and biotechnological use3,4. This limited protease toolkit limits the design space for multicomponent circuits that require orthogonal protease activities5, thereby motivating efforts to further expand and characterize NIa proteases through directed evolution6,7.
Yeast surface display has emerged as one of the most powerful platforms for directed evolution, enabling high-throughput protein library screening via fluorescence-activated cell sorting (FACS)6,7. For example, the yeast endoplasmic reticulum sequestration screening (YESS) system couples protease activity to surface display through the secretory pathway of S. cerevisiae6,7. The YESS system has been applied to engineer novel protease-substrate specificities and has demonstrated utility for multi-round directed evolution campaigns.&#160;In this system, protease variants are co-expressed with a fusion protein containing a cleavage substrate cassette with epitope markers, and the Aga1p surface anchor is expressed in the EBY100 yeast strain. Differential labeling with fluorescent antibodies distinguishes cleaved from non-cleaved substrate cassettes displayed on the cell surface. This activity-dependent display mechanism is directly compatible with iterative FACS-based selection for libraries of 107 or more variants6,7.
A fundamental prerequisite for directed evolution is the ability to introduce variant libraries at a sufficient scale to achieve adequate coverage of the targeted sequence space. For site-saturation mutagenesis libraries spanning multiple amino acid positions, transformation yields of 108 to 109 unique transformants are needed to ensure that the diversity introduced into the library is well represented in the sorted population. Several methods exist for introducing exogenous DNA into S. cerevisiae, each with distinct throughput ceilings. The conventional chemical transformation method uses lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ssDNA/PEG) due to its simplicity and accessibility. However, it typically achieves transformation efficiencies of only 104&#8211;106 colony-forming units (CFU) per microgram of DNA, falling short of the sequence variation needed for large library campaigns8. By contrast, the electroporation method applies a high-voltage electric field to transiently permeabilize the cell membrane and facilitate DNA uptake. Electroporation consistently achieves the highest transformation efficiencies among available transformation methods, with optimized protocols reporting yields of 108&#8211;109 CFU9. It has been observed that electroporation efficiency in S. cerevisiae is critically sensitive to cell growth phase, DNA mass, and buffer composition, providing a framework that has since been widely adapted for library-scale directed evolution9.
The protocol described here uses S. cerevisiae strain EBY100, a widely adopted host strain for yeast surface display. EBY100 was engineered by Boder and Wittrup by integrating an AGA1 expression cassette under the control of the galactose-inducible GAL1 promoter into the chromosomal AGA1 locus of the protease-deficient parent strain BJ546510. The strain is commercially available from the ATCC (catalog no. MYA-4941; depositor K.D. Wittrup), carrying the genotype MATa AGA1::GAL1-AGA1::URA3 ura3-52 trp1 leu2-&#916;1 his3-&#916;200 pep4::HIS3 prb1&#916;1.6R can1 GAL (URA+, leu&#8722;, trp&#8722;). As a result, both Aga1p and the Aga2p-substrate cassette fusion are expressed from the same galactose-inducible promoter, ensuring that the two subunits are co-induced and assemble into the disulfide-linked Aga1p&#8211;Aga2p complex that anchors the displayed fusion to the cell wall.
Despite the widespread use of EBY100 in yeast display applications, no published protocol has been systematically optimized for electroporating this strain at library-scale yields. EBY100-specific parameters, such as the optimal growth phase at harvest, the medium composition during subculture, and the quantitative relationship between initial optical density at 600 nm (OD600) and transformation efficiency, remain to be rigorously characterized and optimized. Here, we report that transformation efficiency in EBY100 increases monotonically with the culture&#39;s OD600 at harvest, with cells in late log phase yielding substantially higher transformation rates than those harvested at early log phase. We evaluated critical protocol parameters, including initial OD600 values at inoculation, the use of 2 &#215; Yeast Extract Peptone Dextrose (YPD) medium for subcultures, electroporation buffer formulations, and post-electroporation outgrowth conditions. The protocol presented here provides an optimized, step-by-step workflow (Figure 1A&#8211;C, Figure 2, Figure 3A&#8211;D, Figure 4A&#8211;E) for preparing electrocompetent EBY100 cells and performing high-efficiency DNA library electroporation, yielding transformation rates consistently above 109 CFU. To validate this protocol, we conducted a comprehensive directed evolution campaign to engineer TEVp with altered cleavage specificity across four rounds of FACS-based selection and enrichment. Sequencing of enriched protease variants confirmed substantial diversification at targeted residues within the protease&#39;s substrate-binding pocket, demonstrating that the protocol provides sufficient library coverage of sequence space to support productive evolution campaigns. This protocol will be of broad utility to researchers using EBY100 for yeast surface display-based directed evolution and FACS-compatible, high-throughput screening of proteases, antibodies9,&#160;kinases11, and receptors12, among other proteins.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 mM dNTP Mix</td><td>NEB</td><td>N0447S</td><td>PCR reactions (Tables 1, 3)</td></tr><tr><td>10X PBS, pH 7.4</td><td>Fisher</td><td>BP3994</td><td>Staining buffer base (Steps 3.2&amp;ndash;3.3)</td></tr><tr><td>5X Q5 Reaction Buffer</td><td>NEB</td><td>B9027S</td><td>Sold separately; also included with Q5 polymerase</td></tr><tr><td>Agar, Bacto</td><td>BD Difco</td><td>281230-500g</td><td>SDCAA agar plates (Table 5); 15 g/L</td></tr><tr><td>Agarose (Low-EEO/Multi-Purpose/Molecular Biology Grade)</td><td>Fisher BioReagents</td><td>BP160-500</td><td>Gel Electrophoresis</td></tr><tr><td>Anti-FLAG PE Antibody</td><td>BioLegend</td><td>637309</td><td>FACS staining; PE-conjugated anti-FLAG</td></tr><tr><td>Anti-HA Tag Antibody [FITC]</td><td>GenScript</td><td>A01621</td><td>FACS staining; FITC-conjugated anti-HA</td></tr><tr><td>Bacto Casamino Acids</td><td>BD Difco</td><td>223120</td><td>SDCAA+, SGCAA+, SCAA media (Table 5); 5 g/L</td></tr><tr><td>Bacto Peptone / Tryptone</td><td>RPI</td><td>T60060-10000.0</td><td>LB/SOB media preparation; 20 g/L in YPD (Table 5)</td></tr><tr><td>BSA, Bovine Serum Albumin, Fraction V</td><td>Sigma-Aldrich</td><td>126575-10GM</td><td>0.5% in PBS; staining buffer (Steps 3.2&amp;ndash;3.3)</td></tr><tr><td>BsmBI-v2 (200 U)</td><td>NEB</td><td>R0739S</td><td>Golden Gate assembly of substrate cassette (Table 8)</td></tr><tr><td>Calcium chloride solution (1M)</td><td>Sigma-Aldrich</td><td>21115-100mL</td><td>Electroporation buffer: 1 mM final (Table 5)</td></tr><tr><td>ColiRollers&amp;reg; Plating Beads</td><td>Sigma-Aldrich</td><td>71013-3</td><td>Sterilize by putting in reagent bottles and autoclaving at 121&amp;ordm;C, 15 psi, for 15 mins.</td></tr><tr><td>D-Galactose</td><td>Thermo Fisher</td><td>A12813.30</td><td>250 g; 20% w/v stock for SGCAA+ induction (Table 5)</td></tr><tr><td>D-Sorbitol</td><td>Thermo Fisher</td><td>1327300.10</td><td>Electroporation buffer (1 M) and outgrowth media (Table 5)</td></tr><tr><td>Dextrose (D-Glucose), anhydrous</td><td>Sigma-Aldrich</td><td>G8270-1KG</td><td>YPD, SDCAA+, 20% w/v dextrose stock (Table 5)</td></tr><tr><td>Difco Yeast Nitrogen Base w/o Amino Acids</td><td>Sigma</td><td>Y0626-250G</td><td>6.7 g/L in SDCAA/SGCAA (Table 5)</td></tr><tr><td>Difco YPD Broth (pre-mixed)</td><td>BD</td><td>242820-500g</td><td>Pre-made YPD alternative; 500 g</td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>D0632-1G</td><td>Conditioning buffer: 10 mM final; aliquot 250 &amp;mu;L, store &amp;minus;20&amp;deg;C</td></tr><tr><td>E.Z.N.A. CyclePure PCR Purification Kit</td><td>Omega Bio-tek</td><td>D6492-02</td><td>PCR product cleanup</td></tr><tr><td>E.Z.N.A. Gel Extraction Kit</td><td>Omega Bio-tek</td><td>D2500-01</td><td>Gel purification post-PCR and restriction digest (Steps 1.1.8, 1.3.2)</td></tr><tr><td>E.Z.N.A. Plasmid DNA Mini Kit I</td><td>Omega Bio-tek</td><td>D6942-02</td><td>Plasmid purification (Step 4.2)</td></tr><tr><td>Glycerol (&amp;ge;99%)</td><td>Fisher</td><td>BP229-4</td><td>50% v/v stock for glycerol stocks (Step 4.1.1)</td></tr><tr><td>Kanamycin Sulfate&amp;nbsp;</td><td>Fisher</td><td>BP906-5</td><td>SDCAA+ and SGCAA+: 50 &amp;mu;g/mL final; replaces Sigma K0879-5G</td></tr><tr><td>KLD Enzyme Mix</td><td>NEB</td><td>M0554S</td><td>Kinase-Ligase-DpnI; site-directed mutagenesis</td></tr><tr><td>KLD Reaction Buffer</td><td>NEB</td><td>B0554A</td><td>Used with KLD Enzyme Mix</td></tr><tr><td>LB Broth (Lennox)</td><td>Sigma-Aldrich</td><td>L3022-1KG</td><td>E. coli liquid culture</td></tr><tr><td>Lithium Acetate Dihydrate (LiOAc&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Thermo Fisher</td><td>A17921.30</td><td>Conditioning buffer: 0.1 M final; add before DTT (Table 5)</td></tr><tr><td>Luria Agar (Luria-Bertani Agar)</td><td>RPI</td><td>L24020-2000.0</td><td>E. coli plating; replaces Sigma LB Agar Lennox L2897-1KG</td></tr><tr><td>OmniPur Casamino Acids</td><td>Sigma</td><td>2240-500GM</td><td>Alternative casamino acids source</td></tr><tr><td>pDD1523</td><td>Zenodo</td><td>10.5281/zenodo. 20746804</td><td>pCTCon2-TRP-CEN/ARS-Gal10-SPAga2-BsaI; Gal1-Aga2-sfGFP</td></tr><tr><td>pDD1524</td><td>Zenodo</td><td>10.5281/zenodo. 20746804</td><td>pCTCon2-Aga2-6xHis-ENLYFQS-FLAG-ENLYFES-HA-ERS</td></tr><tr><td>Penicillin-Streptomycin Solution, 100X</td><td>Corning</td><td>30-002-CI</td><td>SDCAA+, SGCAA+: 1X final</td></tr><tr><td>PstI-HF (5000 U)</td><td>NEB</td><td>R3140S</td><td>Vector linearization (Table 10); use HF version</td></tr><tr><td>Q5 HF DNA Polymerase</td><td>NEB</td><td>M0492S</td><td>High-fidelity polymerase; alternative to Q5 Hot Start</td></tr><tr><td>Q5 High GC Enhancer</td><td>NEB</td><td>B9028A</td><td>For high GC-content PCR reactions</td></tr><tr><td>Q5 Hot Start DNA Polymerase</td><td>NEB</td><td>M0491L</td><td>PCR assembly and amplification (Tables 1, 3)</td></tr><tr><td>rCutSmart Buffer (10X)</td><td>NEB</td><td>B6004S</td><td>Supplied with PstI-HF and SphI-HF (Table 10)</td></tr><tr><td>SalI-HF</td><td>NEB</td><td>R3138L</td><td>Additional restriction enzyme</td></tr><tr><td>SOC Medium</td><td>NEB</td><td>B9020S</td><td>Post-transformation recovery</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher</td><td>S271-1</td><td>LB / SOB media preparation</td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Fisher</td><td>S318-500</td><td>Media pH adjustment</td></tr><tr><td>Sodium Phosphate Dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S9763-500G</td><td>SDCAA plates, SCAA media: 38 mM (Table 5)</td></tr><tr><td>Sodium Phosphate Monobasic, anhydrous</td><td>VWR (BDH)</td><td>BDH4542-1KGP</td><td>SDCAA plates, SCAA media: 62 mM; replaces NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O monohydrate</td></tr><tr><td>SphI-HF (1000 U)</td><td>NEB</td><td>R3182S</td><td>Vector linearization in tandem with PstI-HF (Table 10)</td></tr><tr><td>Streptomycin</td><td>Life Technologies / Sigma</td><td>S6501-5g</td><td>Antibiotic</td></tr><tr><td>T4 DNA Ligase Buffer (10X)</td><td>NEB</td><td>B0202S</td><td>Table 6</td></tr><tr><td>T4 Polynucleotide Kinase (500 U)</td><td>NEB</td><td>M0201S</td><td>Table 6</td></tr><tr><td>T7 DNA Ligase (3000 U/mL)</td><td>NEB</td><td>M0318S</td><td>Table 8</td></tr><tr><td>Yeast Extract</td><td>RPI</td><td>Y20020-5000.0</td><td>YPD (10 g/L) and LB/SOB media</td></tr><tr><td>Zymolyase (1000 U)</td><td>Zymo Research</td><td>E1004-A</td><td>Yeast cell wall digestion for plasmid extraction (Step 4.2.2)</td></tr><tr><td>Zymoprep Yeast Plasmid Miniprep II</td><td>Zymo Research</td><td>D2004</td><td>Yeast plasmid extraction; includes Solutions 1, 2, 3 (Step 4.2)</td></tr><tr><td>ZymoPURE II Plasmid Midi Kit</td><td>Zymo Research</td><td>D4201-B</td><td>Mid-scale plasmid prep; for larger-volume preparations</td></tr><tr><td>&lt;strong&gt;Equipment and Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Disposable Cuvettes, 1.5 mL</td><td>Fisher</td><td>14-955-127</td><td>For measuring OD600 of liquid cultures</td></tr><tr><td>Erlenmeyer Flask, 125 mL</td><td>VWR</td><td>10536-912</td><td>Pre-culture and conditioning incubation</td></tr><tr><td>Erlenmeyer Flask, 250 mL</td><td>VWR</td><td>10536-914</td><td>Yeast subculture</td></tr><tr><td>Erlenmeyer Flask, 250 mL</td><td>VWR</td><td>75809-646</td><td>Wide-mouth variant</td></tr><tr><td>Erlenmeyer Flask, 500 mL</td><td>VWR</td><td>10536-926</td><td>Main pre-culture flask (Step 2.3.1, 2.4.3)</td></tr><tr><td>Falcon 50 mL Conical Tubes</td><td>Falcon</td><td>352070</td><td>Cell washes, centrifugation throughout protocol</td></tr><tr><td>Flow Cytometry Sorter: BD Fusion</td><td>BD Biosciences</td><td>FACSAria Fusion Flow Cytometer (catalog number not applicable)</td><td>For Fluorescence-Activated Cell Sorting (FACS)</td></tr><tr><td>Gene Pulser/MicroPulser Electroporation Cuvettes, 0.2 cm gap</td><td>Bio-Rad</td><td>1652086</td><td>For electroporation of electrocompetent EBY100</td></tr><tr><td>GenePulser Xcell Electroporation System</td><td>Bio-Rad</td><td>1652660</td><td>INSTRUMENT; 2.5 kV, 25 &amp;mu;F, 200 &amp;Omega; (Step 2.7.6)</td></tr><tr><td>New Brunswick Innova 40R - Benchtop Orbital Shaker</td><td>Eppendorf</td><td>M12990084</td><td>For yeast liquid culture incubation</td></tr><tr><td>Petri Dishes, 100 mm</td><td>Fisher</td><td>FB0875712</td><td>YPD and SDCAA agar plates</td></tr><tr><td>SpectraMax QuickDrop UV-Vis Spectrophotometer</td><td>Molecular Devices</td><td>SpectraMax QuickDrop</td><td>For measuring OD600 of liquid cultures and DNA concentration</td></tr><tr><td>Vacufuge plus</td><td>Eppendorf</td><td>2231001204</td><td>For concentrating DNA</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BD FACSDiva (Version: 8.0)</td><td>BD Biosciences</td><td>https://www.bdbiosciences.com/en-us/products/software/instrument-software/bd-facsdiva-software</td><td>Collection of tools for flow cytometer and application setup, data acquisition, and data analysis</td></tr><tr><td>Benchling (Version: Web-based platform, continuous release)</td><td>Benchling, Inc.</td><td>https://www.benchling.com/</td><td>Sequence design software</td></tr><tr><td>SnapGene (Version: 8.0.1)</td><td>SnapGene, Inc.</td><td>https://www.snapgene.com/</td><td>Sequence design software</td></tr></tbody></table>

optimized yeast surface display workflow for directed evolution of proteases

Optimized Yeast Surface Display Workflow for Directed Evolution of Proteases

Directed evolution mimics the natural evolutionary process in a fast, controlled laboratory environment to evolve proteins with desirable functions or traits. Screening a large pool of mutant protein-coding genes allows functional selection of desired variants for applications in biotechnology, medicine, and synthetic biology. Using this protocol, we perform directed evolution in Saccharomyces cerevisiae to engineer protease variants with high specificity for a non-native substrate sequence. We display these protease variants on the yeast cell surface to facilitate the selection of desired variants via fluorescence-activated cell sorting (FACS) over multiple rounds of enrichment. Four key elements of the substrate cassette are co-expressed with the protease library: a fusion protein composed of (i) the yeast adhesion receptor subunit Aga2, (ii) selection and (iii) counter-selection substrate sequences, (iv) epitope tag sequences, and an optional endoplasmic reticulum (ER) retrieval signal sequence. Yeast cells in which only the selection substrate sequence is cleaved are isolated using multicolor FACS via fluorescent (phycoerythrin/fluorescein isothiocyanate&#8211;tagged) anti-epitope antibodies. Here we provide details of the optimized, step-by-step protocol to conduct a protease evolution campaign, including DNA insert library design and substrate cassette design, yeast cell electroporation with high transformation efficiency (up to 109 transformants per microgram of DNA), FACS-based selection, enrichment, and sequencing of evolved variants. We demonstrate our directed evolution protocol by evolving the tobacco etch virus nuclear inclusion A protease (TEVp) from cleaving its natural substrate sequence, ENLYFQ&#8595;S, to cleaving a new sequence, ENLYFE&#8595;S.

Efficient library construction is critical to ensure sufficient coverage of the mutational space. To avoid loss of rare functional variants, the number of transformants should reach at least 10&#215; to 100&#215; the theoretical library size. Because transformation efficiency in S. cerevisiae is influenced by cell concentration and is highest in early- to mid-log-phase cells16, we optimized the initial cell density prior to electroporation. Yeast cultures were subcultured at initial OD<su...

This protocol provides an optimized workflow for the directed evolution of viral proteases in Saccharomyces cerevisiae using a surface-display-based selection system. It outlines DNA insert library and substrate cassette design, electroporation optimized for high transformation efficiency, fluorescence-activated cell sorting-based selection for protease cleavage, and sequencing of enriched variants.

Directed evolution mimics the natural evolutionary process in a fast, controlled laboratory environment to evolve proteins with desirable functions or ...

optimized-yeast-surface-display-workflow-for-directed-evolution

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

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.

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

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 by Yeast Surface Display

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

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

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Polymer Microarrays for High Throughput Discovery of Biomaterials

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

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

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

The University of Texas at Dallas.  This protocol provides an optimized workflow for the directed evolution of viral proteases in Saccharomyces cerevisiae using a surface-display-based selection system. It outlines DNA insert library and substrate cassette design, electroporation optimized for high transformation efficiency, fluorescence-activated cell sorting-based selection for protease cleavage, and sequencing of enriched variants.

Optimized Yeast Surface Display Workflow for Directed Evolution of Proteases

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