Name: using phage display to develop ubiquitin variant modulators for e3 ligases
Uploaded: 2021-08-27T14:45:00
Description: Watch this Scientific Journal Video about Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases at JoVE.com

The ubiquitin variant technology was devised in the laboratory of Dr. Sachdev Sidhu (University of Toronto). WZ is currently a CIFAR Azrieli Global Scholar in the Humans &#38; The Microbiome Program. This research was funded by NSERC Discovery Grants awarded to WZ (RGPIN-2019-05721).

1. Reagent preparation
<ol>
	<li>PBS (phosphate buffered saline): Mix 50 mL of 10x PBS solution with 450 mL of ultrapure H2O. Sterilize by filtration and store at 4 &#176;C or room temperature (~20-25 &#176;C).</li>
	<li>10% BSA (bovine serum albumin): Slowly add 1 g of BSA to 7 mL of ultrapure H2O and mix until fully dissolved (no clumps). Top up with ultrapure H2O until the final volume is 10 mL. Sterilize by filtration and store at 4 &#176;C.</li>
	<li>PB buffer (PBS supplemented wit...

<ol>
	<li>Karlsson, O. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+PDZbody,+a+bivalent+binder+of+the+E6+protein+from+human+papillomavirus.">Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus.</a> Scientific Reports. 5 (1), 9382 (2015).</li><li>Veggiani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+SH2+domains+with+tailored+specificities+and+enhanced+affinities+for+phosphoproteome+analysis.">Engineered SH2 domains with tailored specificities and enhanced affinities for phosphoproteome analysis.</a> Protein Science. 28 (2), 403-413 (2019).</li><li>Kaneko, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superbinder+SH2+domains+act+as+antagonists+of+cell+signaling.">Superbinder SH2 domains act as antagonists of cell signaling.</a> Science Signaling. 5 (243), (2012).</li><li>Wiechmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+inhibition+of+the+small+ubiquitin-like+modifier+(SUMO)-conjugating+enzyme+Ubc9+selectively+impairs+SUMO+chain+formation.">Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation.</a> Journal of Biological Chemistry. 292 (37), 15340-15351 (2017).</li><li>Ernst, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+strategy+for+modulation+of+enzymes+in+the+ubiquitin+system.">A strategy for modulation of enzymes in the ubiquitin system.</a> Science. 339 (6119), 590-595 (2013).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+selective+inhibition+of+pathogenic+viruses+by+engineered+ubiquitin+variants.">Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants.</a> PLOS Pathogens. 13 (5), 1006372 (2017).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=System-wide+modulation+of+HECT+E3+ligases+with+selective+ubiquitin+variant+probes.">System-wide modulation of HECT E3 ligases with selective ubiquitin variant probes.</a> Molecular Cell. 62 (1), 121-136 (2016).</li><li>Gabrielsen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+strategy+for+discovery+of+inhibitors+and+activators+of+RING+and+U-box+E3+ligases+with+ubiquitin+variants.">A general strategy for discovery of inhibitors and activators of RING and U-box E3 ligases with ubiquitin variants.</a> Molecular Cell. 68 (2), 456-470 (2017).</li><li>Brown, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+RING+E3+architectures+regulate+multiubiquitination+and+ubiquitin+chain+elongation+by+APC/C.">Dual RING E3 architectures regulate multiubiquitination and ubiquitin chain elongation by APC/C.</a> Cell. 165 (6), 1440-1453 (2016).</li><li>Gorelik, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+SCF+ubiquitin+ligases+by+engineered+ubiquitin+variants+that+target+the+Cul1+binding+site+on+the+Skp1-F-box+interface.">Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.</a> Proceedings of the National Academy of Sciences. 113 (13), 3527-3532 (2016).</li><li>Goldring, J. P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+quantification+methods+to+determine+protein+concentration+prior+to+electrophoresis.">Protein quantification methods to determine protein concentration prior to electrophoresis.</a> Protein Electrophoresis. 869, 29-35 (2012).</li><li>Zhang, W., Sidhu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+intracellular+modulators+of+E3+ligases+and+deubiquitinases+from+phage-displayed+ubiquitin+variant+libraries.">Generating intracellular modulators of E3 ligases and deubiquitinases from phage-displayed ubiquitin variant libraries.</a> The Ubiquitin Proteasome System: Methods and Protocols. 1844, 101-119 (2018).</li><li>Sheehan, J., Marasco, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+and+yeast+display.">Phage and yeast display.</a> Microbiology Spectrum. 3 (1), 1-17 (2015).</li><li>Lowman, H. B., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monovalent+phage+display:+A+method+for+selecting+variant+proteins+from+random+libraries.">Monovalent phage display: A method for selecting variant proteins from random libraries.</a> Methods. 3 (3), 205-216 (1991).</li><li>Lowman, H. B., Bass, S. H., Simpson, N., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+high-affinity+binding+proteins+by+monovalent+phage+display.">Selecting high-affinity binding proteins by monovalent phage display.</a> Biochemistry. 30 (45), 1-7 (1991).</li><li>Beaber, J. W., Tam, E. M., Lao, L. S., Rondon, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+helper+phage+for+improved+monovalent+display+of+Fab+molecules.">A new helper phage for improved monovalent display of Fab molecules.</a> Journal of Immunological Methods. 376 (1-2), 46-54 (2012).</li><li>Gal&#225;n, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Library-based+display+technologies:+where+do+we+stand.">Library-based display technologies: where do we stand.</a> Molecular BioSystems. 12 (8), 2342-2358 (2016).</li><li>Huang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+display+and+selection+of+single-chain+variable+fragments+effectively+inhibit+growth+and+progression+of+microspheres+in+vitro+and+in+vivo.">Ribosome display and selection of single-chain variable fragments effectively inhibit growth and progression of microspheres in vitro and in vivo.</a> Cancer Science. 109 (5), 1503-1512 (2018).</li><li>Yamaguchi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cDNA+display:+a+novel+screening+method+for+functional+disulfide-rich+peptides+by+solid-phase+synthesis+and+stabilization+of+mRNA-protein+fusions.">cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions.</a> Nucleic Acids Research. 37 (16), 1-13 (2009).</li><li>Mochizuki, Y., Kumachi, S., Nishigaki, K., Nemoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+library+size+in+cDNA+display+by+optimizing+purification+procedures.">Increasing the library size in cDNA display by optimizing purification procedures.</a> Biological Procedures Online. 15 (1), 1-5 (2013).</li><li>Odegrip, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CIS+display:+In+vitro+selection+of+peptides+from+libraries+of+protein-DNA+complexes.">CIS display: In vitro selection of peptides from libraries of protein-DNA complexes.</a> Proceedings of the National Academy of Sciences. 101 (9), 2806-2810 (2004).</li><li>Reiersen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+antibody+display--an+in+vitro+antibody-DNA+library+selection+system.">Covalent antibody display--an in vitro antibody-DNA library selection system.</a> Nucleic Acids Research. 33 (1), 1-9 (2005).</li><li>Houlihan, G., Gatti-Lafranconi, P., Lowe, D., Hollfelder, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+anti-HER2+DARPins+by+SNAP+display+reveals+stability/function+trade-offs+in+the+selection+process.">Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.</a> Protein Engineering Design and Selection. 28 (9), 269-279 (2015).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+validation+of+intracellular+ubiquitin+variant+inhibitors+for+USP7+and+USP10.">Generation and validation of intracellular ubiquitin variant inhibitors for USP7 and USP10.</a> Journal of Molecular Biology. 429 (22), 3546-3560 (2017).</li><li>Teyra, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+characterization+of+ubiquitin+variant+inhibitors+of+USP15.">Structural and functional characterization of ubiquitin variant inhibitors of USP15.</a> Structure. 27 (4), 590-605 (2019).</li></ol>

As mentioned in step 2.1 (protein preparation), a variety of methods can be used to assess the protein concentration, and each will have unique benefits and drawbacks based on the specific target protein used for phage display. A source of detailed descriptions and protocols for popular methods has been provided previously11.
Using the phage retained by a previous round of phage display as the input for a subsequent round enriches the good binders by gradually removing ...

Understanding the molecular details of protein-protein interactions is critical for delineating the signal transduction mechanisms of biological processes, particularly those that contribute to clinically important diseases. In recent years, phage display has been utilized as a practical and accessible method to isolate proteins/peptides with much improved binding to a desired target protein1,2,3,4, which in turn can be used as intracellular probes of protein-protein interactions.
Ubiquitination is a cascade of enzymatic activities (E1 activating enzyme &#8594; E2 conjugating enzyme &#8594; E3 ligases) that covalently conjugate ubiquitin (Ub) to protein substrates to target them for degradation or to mediate cell signaling changes. In addition, deubiquitinases catalyze the removal of ubiquitin from proteins. Therefore, in cells, there are thousands of Ub-dependent protein-protein interactions, the vast majority of which recognize a common surface with low affinity but high specificity to allow weak interactions through large and diverse surfaces.
Ernst et al. introduced mutations into known binding regions of Ub in order to see if they could enhance binding affinity for a protein of interest while still maintaining high selectivity5. A combinatorial library of over 10 billion (7.5 x 1010) Ub variants (UbVs) with mutations at positions across the Ub surface that mediate the known Ub-protein interactions was developed. This library consisted of phagemids that express the M13 bacteriophage pIII coat protein fused to diversified UbVs. Therefore, individual UbVs can be displayed on the phage surface via the coat protein upon expression. During the selection process, phage that display UbVs with considerable binding interactions with the target protein will be retained and enriched in subsequent rounds of phage display, whereas phage displaying UbVs that bind poorly to the target protein are washed away and removed from the phage pool. The retained phage particles contain the phagemid corresponding to their displayed UbV, allowing them to be sequenced and further characterized once isolated.
Using this protein engineering strategy, UbV inhibitors were developed for human deubiquitinases5 and viral proteases6. Importantly, we have generated inhibitory UbVs for human HECT-family E3 ligases through hijacking the E2-binding site and activating UbVs that occupy a Ub-binding exosite on the HECT domain7. We can also inhibit monomeric RING-family E3s by targeting the E2 binding site and induce UbV dimerization to activate homodimeric RING E3s8. For multi-subunit RING E3s, UbVs can achieve inhibition by targeting the RING subunit (e.g., for APC/C complex9) or disrupting complex formation (e.g., for SCF E3s10). Collectively, UbVs can be leveraged to systematically interrogate protein-protein interactions in the Ub-proteasome system (UPS) so that we can better decipher biochemical mechanisms of UPS enzymes and to identify and validate functional sites for therapeutic intervention.
The following protocol describes how to employ a previously generated phage displayed UbV library to target a protein of interest and how to enrich the UbV binders that interact with the target protein through successive rounds of phage display.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Axygen Mini Tube System (0.65 mL, sterile, 96/Rack, 10 Racks/pack)</td>
			<td>Fisher Scientific</td>
			<td>14-222-198</td>
			<td>Culturing phage outputs after phage display.</td>
		</tr>
		<tr>
			<td>BD Difco Dehydrated Culture Media: LB Broth, Miller (Luria-Bertani)</td>
			<td>Fisher Scientific</td>
			<td>DF0446-17-3</td>
			<td>Preparing plates for titering.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA), Fraction V</td>
			<td>BioShop Canada</td>
			<td>ALB001</td>
			<td>Buffer component.</td>
		</tr>
		<tr>
			<td>Carbenicillin disodium salt 89.0-100.5% anhydrous</td>
			<td>Millipore-Sigma</td>
			<td>C1389-5G</td>
			<td>Culturing phagemid-infected cells.</td>
		</tr>
		<tr>
			<td>Compact Digital Microplate Shaker</td>
			<td>Fisher Scientific</td>
			<td>11-676-337</td>
			<td>Shaking plates during incubation with the phage library.</td>
		</tr>
		<tr>
			<td>Corning Microplate Aluminum Sealing Tape</td>
			<td>Fisher Scientific</td>
			<td>07-200-684</td>
			<td>Sealing phage glycerol stocks.</td>
		</tr>
		<tr>
			<td>Dehydrated Agar</td>
			<td>Fisher Scientific</td>
			<td>DF0140-01-0</td>
			<td>Preparing plates for titering.</td>
		</tr>
		<tr>
			<td>DS-11 Spectrophotometer/Fluorometer</td>
			<td>DeNovix</td>
			<td>DS-11 FX+</td>
			<td>Protein concentration measurement.</td>
		</tr>
		<tr>
			<td>Greiner Bio-One CellStar 96-Well, Non-Treated, U-Shaped-Bottom Microplate</td>
			<td>Fisher Scientific</td>
			<td>7000133</td>
			<td>Storing phage glycerol stocks.</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>Phage elution.</td>
		</tr>
		<tr>
			<td>Invitrogen One Shot OmniMAX 2 T1R Chemically Competent E. coli</td>
			<td>Fisher Scientific</td>
			<td>C854003</td>
			<td>Bacterial strain for phage infection.</td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>AAJ1792406</td>
			<td>Culturing M13K07 helper phage-infected cells.</td>
		</tr>
		<tr>
			<td>M13KO7 Helper Phage</td>
			<td>New England Biolabs</td>
			<td>&#160;N0315S</td>
			<td>Permit phagemid packing and secretion.</td>
		</tr>
		<tr>
			<td>MaxQ 4000 Benchtop Orbital Shaker</td>
			<td>Fisher Scientific</td>
			<td>11-676-076</td>
			<td>Bacterial cell culture.</td>
		</tr>
		<tr>
			<td>Nunc MaxiSorp 96 well microplate, flat bottom</td>
			<td>Life Technologies</td>
			<td>44-2404-21</td>
			<td>Immobilizing proteins.</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) 10X Solution</td>
			<td>Fisher Scientific</td>
			<td>BP3994</td>
			<td>Buffer component/phage resuspension medium.</td>
		</tr>
		<tr>
			<td>Polyester Films for ELISA and Incubation</td>
			<td>VWR</td>
			<td>60941-120</td>
			<td>Covering the microplates during incubation.</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol 8000 (PEG)</td>
			<td>Fisher Scientific</td>
			<td>BP233-1</td>
			<td>Phage precipitation.</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Millipore-Sigma</td>
			<td>S3014</td>
			<td>Phage precipitation.</td>
		</tr>
		<tr>
			<td>Sterile Plastic Culture Tubes: Translucent Polypropylene</td>
			<td>Fisher Scientific</td>
			<td>14-956-1D</td>
			<td>Culturing phage inputs.</td>
		</tr>
		<tr>
			<td>Tetracycline Hydrochloride</td>
			<td>Fisher Scientific</td>
			<td>BP912-100</td>
			<td>Culturing E. coli OmniMax cells.</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP1525</td>
			<td>Neutralizing eluted phage solution.</td>
		</tr>
		<tr>
			<td>Tryptone Powder</td>
			<td>Fisher Scientific</td>
			<td>BP1421-2</td>
			<td>Cell growth media component.</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337500</td>
			<td>Buffer component.</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Fisher Scientific</td>
			<td>BP1422-2</td>
			<td>Cell growth media component.</td>
		</tr>
	</tbody>
</table>

using phage display to develop ubiquitin variant modulators for e3 ligases

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins with high specificity but low affinity. Through phage display, ubiquitin variants (UbVs) can be engineered such that they exhibit improved affinity over wildtype ubiquitin and maintain binding specificity to target proteins. Phage display utilizes a phagemid library, whereby the pIII coat protein of a filamentous M13 bacteriophage (chosen because it is displayed externally on the phage surface) is fused with UbVs. Specific residues of human wildtype ubiquitin are soft and randomized (i.e., there is a bias towards to native wildtype sequence) to generate UbVs so that deleterious changes in protein conformation are avoided while introducing the diversity necessary for promoting novel interactions with the target protein. During the phage display process, these UbVs are expressed and displayed on phage coat proteins and panned against a protein of interest. UbVs that exhibit favorable binding interactions with the target protein are retained, whereas poor binders are washed away and removed from the library pool. The retained UbVs, which are attached to the phage particle containing the UbV's corresponding phagemid, are eluted, amplified, and concentrated so that they can be panned against the same target protein in another round of phage display. Typically, up to five rounds of phage display are performed, during which a strong selection pressure is imposed against UbVs that bind weakly and/or promiscuously so that those with higher affinities are concentrated and enriched. Ultimately, UbVs that demonstrate higher specificity and/or affinity for the target protein than their wildtype counterparts are isolated and can be characterized through further experiments.

Binders produced from phage display can be verified and analyzed in many ways. It is recommended to first proceed with sequencing the phage with primers that flank the diversified insert in the phagemid library. An ideal phage display experiment will show a clear bias towards several sequences (Figure 1). Other sequences will also be present but with a lower count, appearing more as background noise. In the example provided, where phage display was performed between ubiquitin variants (UbVs)...

Watch this Scientific Journal Video about Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases at JoVE.com

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins ...

Phage display not only helps us investigate the binding characteristics of clinically important enzymes, but also develop modulators for those enzymes and the corresponding diseases. Phage display can be used to develop novel binders for a wide array of target proteins and is also easier to perform than other conventional display methods. This procedure involved a lot of repetitive action, so the key is to not autopilot and to remain focused throughout.Begin by inoculating 30 milliliters of 2YT tetracycline broth with 200 microliters of the seed culture. Incubate it for approximately three hours at 37 degrees Celsius with 200 RPM orbital shaking until the bacteria are in the mid-log phase. Discard the coating solution from the plate into a sink.Dry it gently with paper towels. Then add 200 microliters of PB buffer to each coated well. Discard the PB buffer from the plate and dry it on paper towels.Add another 200 microliters of PB buffer to each coated well of the plate. Incubate it at room temperature for an hour with 300 RPM orbital shaking. Thaw the phage library on ice, then dilute it to 100X the library diversity in PBS.Add the polyethylene glycol sodium chloride solution at one-fifth of the diluted library volume and incubate the solution on ice for 30 minutes. Next, centrifuge the solution at 11, 000 times G for 30 minutes at four degrees Celsius. Discard the supernatant and centrifuge it for another two minutes to pull down the remaining supernatant and concentrate the phage pellet.Gently resuspend the phage pellet in one milliliter of PBT buffer per target protein to be analyzed, typically four in total. Add 100 microliters of phage library to each coated well in the control plate. Incubate it at room temperature for an hour with 300 RPM orbital shaking.Discard the PB buffer from the target plate into the sink and dry the plate on paper towels. Transfer all 100 microliters of the phage library in the control plate to each coated well of the target plate. Incubate it at room temperature for an hour with 300 RPM orbital shaking.Next, remove the phage library from the plate and wash the coated wells four times with PT buffer. Invert the plate and tap on a paper towel to remove the last drops of buffer. Add 100 microliters of 0.1 molar hydrochloric acid to each coated well to elute the phage.Incubate the plate at room temperature for five minutes with 300 RPM orbital shaking. After that, neutralize the pH by adding 12.5 microliters of pH 11 one molar tris hydrochloride to each coated well. Transfer the eluted phage from all eight wells into a single 1.5 milliliter microcentrifuge tube.Pipette up and down during the transfer to make solutions homogenous and aspirate all liquid from the wells. Add 10%BSA to the eluted phage to make the final concentration 1%Store the microcentrifuge tube at four degrees Celsius. This is the round one output.Prepare a seed culture for culturing the phage input for the next round of selection by inoculating five milliliters of 2YT tetracycline seed culture with an isolated E.coli colony from an agar plate. Incubate it overnight at 37 degrees Celsius with 200 RPM orbital shaking. Dilute the target protein in the rounds two and three tube with an appropriate amount of PBS.Take half of the contents of rounds two and three tube and coat four wells per target protein in a 96-well binding plate for the next round. Next, use half of the round one output to inoculate three milliliters of mid-log phase cells. Incubate it at 37 degree Celsius for 30 minutes with 200 RPM orbital shaking.Add M13KO7 helper phage to a final concentration of 10 billion PFU per milliliter. Incubate it again at 37 degrees Celsius for an hour with 200 RPM orbital shaking. After an hour, transfer the entire three milliliters of culture to 30 milliliters of 2YT carbenicillin kanamycin solution.Grow overnight at 37 degrees Celsius with 200 RPM orbital shaking. The representative results for a ubiquitin variant selection against UBE4B are shown here. UBVs were ordered from the highest to lowest frequency.Sequences represent the diversified ubiquitin region in the UBV with all the randomized residues. The relative binding affinity of the binders to the wild type target protein, mutated target protein, as well as off-target proteins is measured by enzyme-linked immunosorbent assays or ELISA. All ELISA absorbances were normalized against 96 averaged BSA ELISA scores and 96 averaged GST ELISA scores.Darker green represents the stronger relative binding. GST was included as a control for nonspecific binding because the target protein is GST tagged. The ELISA results are graphically presented here.The x-axis represents the UBVs and the y-axis represents the corresponding normalized absorbance. When attempting this procedure, it's important that you don't discard the solution in the plate after you add the hydrochloric acid. The phages are now suspended in solution and you want to keep these so that you can perform subsequent enrichment final analyses or both.Following this procedure, sequencing should be done to determine UBV frequencies. ELISAs and IC50 assays can be done to determine binding efficacies and inhibitory efficacies respectively and also to help determine which UBVs to further characterize.

Research

JoVE Journal

Biochemistry

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 ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

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. ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...