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 with 1% BSA): Slowly add 5 g of BSA to 400 mL of ultrapure H2O and 50 mL of PBS and mix until fully dissolved (no clumps). Top up with ultrapure H2O until the final volume is 500 mL. Sterilize by filtration and store at 4 &#176;C.</li>
	<li>PBT buffer (PBS supplemented with 1% BSA and 0.05% Tween 20): Slowly add 5 g of BSA to 400 mL of ultrapure H2O and 50 mL of PBS and mix until fully dissolved (no clumps). Add 250 &#181;L of Tween 20. Top up with ultrapure H2O until the final volume is 500 mL. Sterilize by filtration and store at 4 &#176;C.</li>
	<li>PT buffer (PBS supplemented with 0.05% Tween 20): Mix 1 mL of Tween 20 with 400 mL of PBS. Top up with ultrapure H2O until the final volume is 2 L. Sterilize by filtration and store at 4 &#176;C or room temperature (~20-25 &#176;C).</li>
	<li>2YT broth: Add 16 g of tryptone, 10 g of yeast extract, and 5 g of NaCl to 800 mL of ultrapure H2O and mix until fully dissolved (no clumps). Top up with ultrapure H2O until the final volume is 1 L. Sterilize by autoclaving and store at room temperature (~20-25 &#176;C).</li>
	<li>LB/carb plates: Add 12.5 g of pre-made LB mixture and 7.5 g of agar to 400 mL of H2O. Top up with ultrapure H2O until the final volume is 500 mL and sterilize by autoclaving. Make sure agar is fully dissolved and wait for it to cool to below 60 &#176;C. Add 500 &#181;L of 100 mg mL carbenicillin, mix well, and pour into plate. Store at 4 &#176;C.</li>
	<li>LB/tet plates: Add 12.5 g of pre-made LB mixture and 7.5 g of agar to 400 mL of ultrapure H2O. Top up with ultrapure H2O until the final volume is 500 mL and sterilize by autoclaving. Ensure agar is fully dissolved and wait for it to cool to below 60 &#176;C. Add 500 &#181;L of 10 mg mL tetracycline, mix well, and pour into plate. Store at 4 &#176;C.</li>
	<li>20% PEG (polyethylene glycol)/2.5 M NaCl: Add 50 g of PEG-8000 and 36.5 g of NaCl to 200 mL of ultrapure H2O. Mix until fully dissolved. 
	NOTE: This may take a while; heating can help. Top up with ultrapure H2O until the final volume is 250 mL. Sterilize by filtration or autoclaving.</li>
	<li>0.1 M HCl: Mix 20 mL of 1 M HCl with 180 mL of ultrapure H2O. Sterilize by filtration and store at room temperature (~20-25 &#176;C).</li>
	<li>1 M Tris (pH 11.0): Add 6.1 g of Tris base to 40 mL of ultrapure H2O. Adjust pH to 11.0 with HCl and mix until Tris is fully dissolved. Top up with ultrapure H2O until the final volume is 50 mL. Sterilize by filtration and store at room temperature (~20-25 &#176;C).</li>
	<li>100 mg/mL (1000x) carbenicillin: Add 2 g of the carbenicillin disodium salt to 20 mL of ultrapure H2O. Mix until fully dissolved. Sterilize by filtration and store at -20 &#176;C.</li>
	<li>50 mg/mL (1000x) kanamycin: Add 1 g of kanamycin sulfate to 20 mL of ultrapure H2O. Mix until fully dissolved. Sterilize by filtration and store at -20 &#176;C.</li>
	<li>10 mg/mL (1000x) tetracycline: Add 0.2 g of tetracycline hydrochloride to 20 mL of 70% ethanol. Mix until fully dissolved. Sterilize by filtration and store at -20 &#176;C.</li>
</ol>
2. Protein preparation
<ol>
	<li>Determine the concentration of the target protein stock in &#181;M. The most convenient way to assess protein concentration is to measure the absorbance of the protein stock at 280 nm. If the concentration is known in mg/mL, convert it to &#181;M using the molecular weight of the target protein. A range of methods can be used depending on the protein of interest; see the Discussion section for details. 
	NOTE: If the protein is truncated (specific protein domain/motif) or tagged, remember to account for these changes in molecular weight when converting from mg/mL to &#181;M.</li>
	<li>Prepare three microcentrifuge tubes labeled &#34;round 1&#34;, &#34;round 2/3&#34;, and &#34;round 4/5&#34;.
	<ol>
		<li>Calculate the volume of protein necessary to dilute it to 1 &#181;M in 800 &#181;L PBS and aliquot this volume into the tubes without adding PBS. 
		&#8203;NOTE: If the amount of target protein available is low, the concentration can be lowered to as little as 0.25 &#181;M.</li>
	</ol>
	</li>
</ol>
3. Preparation for round one of selection
<ol>
	<li>Prepare a seed culture for culturing the phage input for round two of selection.
	<ol>
		<li>Inoculate 5 mL of 2YT/tet with a well-isolated Escherichia coli colony and incubate overnight at 37 &#176;C with 200 rpm orbital shaking.</li>
	</ol>
	</li>
	<li>Coat the plate for the first round of selection.
	<ol>
		<li>Dilute the target protein in the &#34;round 1&#34; tube with the appropriate amount of PBS and aliquot 100 &#181;L into eight wells of a 96-well binding plate (i.e., the target plate). 
		&#8203;NOTE: Phage display can be done for four different proteins simultaneously on a single plate by coating the wells in the corners of the plate.</li>
		<li>Optional control: If the target protein is tagged, such as with GST or MBP (maltose binding protein), coat eight wells in another 96-well binding plate with 100 &#181;L of a 1 &#181;M solution containing the appropriate epitope tag. This will be used to remove undesired phage that bind to the tag non-specifically.</li>
		<li>Shake plate(s) overnight at 4 &#176;C with 200 rpm orbital shaking.</li>
	</ol>
	</li>
</ol>
4. Round one of selection
<ol>
	<li>Prepare the round two input.
	<ol>
		<li>Inoculate 30 mL of 2YT/tet with 200 &#181;L of the seed culture from step 3.1.</li>
		<li>Incubate at 37 &#176;C with 200 rpm orbital shaking until the bacteria are in mid-log phase (OD600 <img alt="Equation 1" src="/files/ftp_upload/62950/62950eq01v2.jpg" /> 0.6 - 0.8). This takes approximately three h.</li>
	</ol>
	</li>
	<li>Block target plate.
	<ol>
		<li>Remove the coating solution from the plate, or both plates if a control plate has been made, by inverting and shaking over a sink. Pat dry on paper towels.</li>
		<li>Add 300 &#181;L of PB buffer to each coated well.</li>
		<li>Remove the PB buffer as in step 4.2.1 and add another 200 &#181;L of PB buffer.</li>
		<li>Incubate at room temperature (~20-25 &#176;C) for 1 h with 300 rpm orbital shaking.</li>
	</ol>
	</li>
	<li>Prepare phage library.
	<ol>
		<li>Thaw the phage library on ice and dilute it to 100x the library diversity in PBS. For example, if the library diversity is 1 x 1010 and the library concentration is 1 x 1013, dilute the library so that the concentration is 1 x 1012. For the libraries used here, dilute them 10-fold in PBS by combining 1 mL of the library with 9 mL of PBS. 
		NOTE: Library diversity should have been determined during library creation and cannot be easily assessed otherwise. Library concentration can be determined by inoculating E. coli cells in mid-log phase (OD600 <img alt="Equation 1" src="/files/ftp_upload/62950/62950eq01v2.jpg" /> 0.6 - 0.8) and plating on LB/carb.</li>
		<li>Add 1/5 volume of PEG/NaCl. For example, for 10 mL of the previously diluted library, add 2 mL of PEG/NaCl.</li>
		<li>Incubate on ice for 30 min.</li>
		<li>Centrifuge at 11,000 x g for 30 min at 4 &#176;C, discard supernatant, and recentrifuge for 2 min to pull down the remaining supernatant. 
		NOTE: Put the tube in the rotor in the same orientation the second time as it was the first to keep the pellet in the same place, therefore, making it easier to see.</li>
		<li>Gently resuspend the phage pellet in 1 mL of PBT per protein. For four proteins, resuspend the pellet in 4 mL of PBT. 
		NOTE: Try not to touch the pellet and do not introduce air bubbles.</li>
	</ol>
	</li>
	<li>Display phage to the target protein(s).
	<ol>
		<li>Optional control: Add 100 &#181;L of phage library to each coated well in the control plate. Incubate at room temperature (~20-25 &#176;C) for 1 h with 300 rpm orbital shaking and transfer the library from the control plate to the target plate. Skip step 4.4.2.</li>
		<li>Remove the PB buffer from the target plate as in step 4.2.1 and add 100 &#181;L of the phage library to each coated well.</li>
		<li>Incubate at room temperature (~20-25 &#176;C) for 1 h with 300 rpm orbital shaking.</li>
	</ol>
	</li>
	<li>Elute round one phage.
	<ol>
		<li>Remove phage library and wash the coated wells four times with PT buffer. Invert the plate and tap on a paper towel to remove the last drops. 
		NOTE: If multiple target proteins are coated on one plate, try to minimize the flow of all subsequent solutions between the wells.</li>
		<li>Add 100 &#181;L of 0.1 M HCl to each coated well and incubate at room temperature for 5 min with 300 rpm orbital shaking.</li>
		<li>Neutralize the pH by adding 12.5 &#181;L of 1 M Tris-HCl (pH 11) to each coated well.</li>
		<li>Pool the eluted phage from all 8 wells into a single 1.5 mL microcentrifuge tube. Pipette up and down during the transfer to make solutions homogenous and aspirate all liquid from the wells.</li>
		<li>Add 10% BSA to the pooled eluted phage to a final concentration of 1%. For a typical round one elution volume of 950 &#181;L, add 95 &#181;L of 10% BSA. Store at 4 &#176;C. This is the round one output.</li>
	</ol>
	</li>
</ol>
5. Preparation for subsequent rounds of selection
<ol>
	<li>Prepare a seed culture for culturing the phage input for the next round of selection as in step 3.1.</li>
	<li>Coat the plate for the third round of selection as in step 3.2 with the changes noted below.
	<ol>
		<li>Only coat four wells per protein in a 96-well binding plate. Use half of the contents of the &#34;round 2/3&#34; or &#34;round 4/5&#34; tubes for the appropriate round. Dilute the contents of these tubes as needed, in order to avoid proteins settling out of the solution.</li>
	</ol>
	</li>
	<li>Prepare the phage input for the next round of selection.
	<ol>
		<li>Use half of the round one output from step 4.5.5 to inoculate 3 mL of mid-log phase cells from step 4.1. For a typical round one output, inoculate with 500 &#181;L of the output.</li>
		<li>Incubate at 37 &#176;C for 30 min with 200 rpm orbital shaking.</li>
		<li>Add M13K07 helper phage to a final concentration of 1 x 1010 PFU/mL.</li>
		<li>Incubate at 37 &#176;C for 1 h with 200 rpm orbital shaking.</li>
		<li>Transfer the entire 3 mL of culture to 30 mL of 2YT/carb/kan. Grow overnight at 37 &#176;C with 200 rpm orbital shaking.</li>
		<li>The next day, transfer the culture into a 50 mL centrifuge tube and centrifuge at 11,000 x g for 10 min at 4 &#176;C to precipitate cells.</li>
		<li>Decant the supernatant into a new 50 mL centrifuge tube and mix with 8 mL of PEG/NaCl and incubate on ice for 10 min.</li>
		<li>Centrifuge at 11,000 x g for 10 min at 4 &#176;C, discard the supernatant, and centrifuge again for 2 min. 
		NOTE: Put the tube in the rotor in the same orientation the second time as it was the first to keep the pellet in the same place, therefore, making it easier to see.</li>
		<li>Resuspend the phage pellet in 800 &#181;L of PBT so that it is fully homogenous and no clumps are visible.</li>
		<li>Transfer the phage solution to a 1.5 mL microcentrifuge tube and centrifuge at 16,200 x g for 4 min at 4 &#176;C to pellet debris.</li>
		<li>Transfer the supernatant to a new microcentrifuge tube. This is the input for the next round of selection.</li>
	</ol>
	</li>
	<li>Optional: Titer the input and output solutions.
	<ol>
		<li>Use 500 &#181;L of an E. coli seed culture to inoculate 5 mL of 2YT/tet and incubate at 37 &#176;C with 200 rpm orbital shaking until bacteria is in mid-log phase (OD600 <img alt="Equation 1" src="/files/ftp_upload/62950/62950eq01v2.jpg" /> 0.6 - 0.8). This takes approximately 1 h.</li>
		<li>Dilute phage input/output solutions to 10-3&#160;&#160;-&#160;10-5 in PBS and add 10 &#181;L of each dilution to 90 &#181;L of cultured cells. 
		NOTE: Use diluted phage solutions immediately because phage will adsorb to the tube over time, and transformations may produce false negatives.</li>
		<li>Incubate at 37 &#176;C for 20 min with 200 rpm orbital shaking.</li>
		<li>Plate 5 &#181;L on separate LB/carb plates. 
		&#8203;NOTE: Amount plated is variable depends on previous results/personal preference.</li>
	</ol>
	</li>
</ol>
6. Subsequent rounds of selection
<ol>
	<li>Perform all subsequent rounds along with the preparation as done in steps 3 and 4, respectively, with differences noted below.
	<ol>
		<li>Use the input produced in the previous round as the phage source instead of a library. Coat 4 wells per target protein with 100 &#181;L of the phage input acquired from the previous round. Store remaining phage inputs at 4 &#176;C.</li>
		<li>Make the washing step in 4.5.1 more stringent with each round of selection. Round one requires washing 4 times, round two requires 6 times, round three requires 8 times, and rounds four and five both require washing 10 times.</li>
		<li>Reduce some volumes compared to those used in round one because subsequent rounds only coat four wells instead of eight. For example, in step 4.5.5 only 45 &#181;L of 10% BSA is added to the phage output, and in step 5.3.1 only 250 &#181;L of the phage output is used to inoculate cells.</li>
	</ol>
	</li>
</ol>
7. Post selection processing and phage Isolation
<ol>
	<li>Titer the input and output solutions for rounds four and five.
	<ol>
		<li>If not already done in step 5.4, follow the same steps to titer rounds four and five phage outputs, ideally producing a range of 30-300 colonies across a few plates.</li>
	</ol>
	</li>
	<li>Culture and isolate phage.
	<ol>
		<li>Aliquot 450 &#181;L of 2YT/carb/M13K07 into every tube of a 96 mini tube culture box.</li>
		<li>Pick well-isolated colonies from any of the plates from step 7.1 to inoculate each tube. 
		NOTE: Use the top half of the box for round four outputs and the bottom half for round five outputs.</li>
		<li>Incubate overnight at 37 &#176;C with 200 rpm orbital shaking.</li>
		<li>Centrifuge at 1,200 x g for 10 min at 4 &#176;C.</li>
		<li>Transfer as much supernatant as possible to a new 96 mini tube culture box without disturbing the cell pellet and discard the old one. Store at 4 &#176;C.</li>
		<li>From the new box, transfer 100 &#181;L from each tube to a 96 well non-binding plate containing 100 &#181;L of 50% glycerol in all wells. Mix well and store at -80 &#176;C as a backup stock.</li>
	</ol>
	</li>
</ol>

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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>

The authors declare no conflict of interests.

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 ...

using-phage-display-to-develop-ubiquitin-variant-modulators-for-e3

Research

JoVE Journal

Biochemistry

3.0K Views.  University of Guelph. 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.

Video: Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

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 ...

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 these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

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

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

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. 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. ...

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 validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

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 purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

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 cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

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 attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

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 protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

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

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

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Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates