This work was funded by Sanford Research startup funds to FB and the NIH grant R01CA233700 to MJS. The artwork was done by Felipe G. Serrano (www.illustrative-science.com).&#160;We thank Dr. Greg Findlay (University of&#160;Dundee) for the GST-USP27X plasmid.

The following protocol can be adapted for recombinant proteins using various affinity tags expressed in different strains of competent cells. Depending on the protein being expressed, the culturing and overnight expression conditions may require optimization of the OD600 at expression induction, expression time, expression temperature, and IPTG concentration. An overview of the protocol is illustrated in Figure 1. The details of the reagents and the equipment used in this study are listed in the Table of Materials.
1. Transformation of competent Rosetta 2 E. coli cells with the recombinant GST-USP27X expression plasmid
NOTE: To maintain the sterility of the bacterial culture, perform steps where media containers are open under a Bunsen burner flame. To allow optimal oxygen transfer, perform bacterial culture shaking in a benchtop temperature-controlled shaker with an orbit of 19 mm to 50 mm and a speed of 200 rpm29.
<ol>
	<li>Thaw 20 &#181;L of chemically competent Rosetta 2 E. coli cells on ice immediately before use. Add 1-10 ng of the pGEX6P1-USP27X expression plasmid (encoding for N-terminal Glutathione-S-transferase (GST)30 -tagged USP27X and containing Ampicillin resistance) and incubate 5 min on ice.</li>
	<li>Heat shock for 30 s at 42 &#176;C in a dry bath. Incubate on ice for 2 min.</li>
	<li>Add 80 &#181;L of room temperature (RT) SOC medium. Incubate for 60 min at 37 &#176;C with 200 rpm rotation in a 19 mm orbit benchtop temperature-controlled shaker.</li>
	<li>Plate 50 &#181;L of culture on an LB agar plate supplemented with 25 &#181;g/L Chloramphenicol and 50 &#181;g/L Ampicillin. Incubate for 20 h at 37 &#176;C lid-side down in a temperature-controlled incubator.</li>
</ol>
2. Overnight bacterial expression of recombinant protein from expression plasmid
NOTES: To maintain the sterility of the bacterial culture, perform steps where media containers are open under a Bunsen burner flame. To allow optimal oxygen transfer, perform bacterial culture shaking in a benchtop temperature-controlled shaker with an orbit of 19 mm to 50 mm and a speed of 200 rpm29. Measure culture OD600 using a spectrophotometer.
<ol>
	<li>Prepare 1 L of Terrific Broth (TB) medium by adding 47.6 g of TB Powder to 1 L of ultrapure water with 0.4% glycerol in a 2 L baffled culture flask. Autoclave medium for 30 min and cool to RT.</li>
	<li>Pick a single colony of transformed bacteria and add to 10 mL of sterile LB medium supplemented with 25 &#181;g/L Chloramphenicol and 50 &#181;g/L Ampicillin. Incubate for 20 h at 37 &#176;C with 200 rpm rotation in a 19 mm orbit benchtop temperature-controlled shaker.</li>
	<li>Add 10 mL overnight culture to 1 L of TB medium supplemented with 25 &#181;g/L Chloramphenicol and 50 &#181;g/L Ampicillin (1:100 ratio of starting culture to expression culture). Incubate at 37 &#176;C with 200 rpm rotation in a 19 mm orbit benchtop temperature-controlled shaker until the culture OD600 is between 0.5-0.6.</li>
	<li>Add 50 &#181;M of IPTG to the culture to induce expression, cool to 16 &#176;C, and incubate for 20 h at 16 &#176;C with 200 rpm rotation.</li>
	<li>Pellet cells from expression culture by centrifugation for 20 min at &#8805;3,000 x g and 4 &#176;C. Store the pellet at -80 &#176;C for at least 1 h. 
	NOTE: At this point, the experiment can be paused and restarted later (preferably the same week). The pellet can be stored for the long term at -80 &#176;C.</li>
</ol>
3. Protein purification by gravity-flow affinity column
NOTE: The resin, binding, wash, elution, and storage buffers appropriate for each purification will depend on the recombinant protein being purified. Collect samples from the cell pellet, supernatant, flow-through, wash fractions, and elution fractions in SDS-PAGE buffer. Perform SDS-PAGE and Coomassie staining for the samples to evaluate the success of the purification. Perform purification at 4 &#176;C and handle fractions on ice. Cleavage of protein tags can be performed on or off the column using the appropriate protease to target the relevant protease-specific cleavage site.
<ol>
	<li>Secure the empty gravity flow column in the retort stand and fill it with glutathione agarose resin. Use 2-3 mL of resin to purify a cell pellet collected from 1 L of expression culture.</li>
	<li>Wash the column with one resin-bed volume of 20% ethanol. Wash column 3 times with one resin-bed volume of MS500 buffer (20 mM of Tris pH 7.5, 500 mM of NaCl, 0.5 mM of TCEP). Stop the column so the resin remains covered with buffer to prevent drying while preparing the cell pellet.</li>
	<li>Thaw the cell pellet at 4 &#176;C. Add 30 mL of MS500 buffer (20 mM of Tris pH 7.5, 500 mM of NaCl, 0.5 mM of TCEP, 60 mg of lysozyme, and one protease inhibitor tablet) to the thawed pellet. Lyse for 30 min at 4 &#176;C with gentle end-over-end rotation.</li>
	<li>Sonicate lysed cells in either a 50 mL centrifuge tube or a metal beaker on ice until the lysate flows freely when dispensed from a pipette tip. Centrifuge for 30 min at 4 &#176;C and &#8805;20,000 x g to clear the supernatant. 
	NOTE: Determine sonicator settings empirically. Set the sonicator such that 120 s total time is enough to reduce the viscous lysate to a free-flowing and transparent liquid.</li>
	<li>Decant the supernatant into a beaker and load cleared lysate onto the column. Run lysate through the column by gravity flow while collecting the flow through. Load the column with the collected flow through and run it through the column.</li>
	<li>Wash the column with at least two resin-bed volumes of MS500 wash buffer (20 mM of Tris pH 7.5, 500 mM of NaCl, 0.5 mM of TCEP) 5 times. Collect the wash flow through in 5 mL fractions. Add 1 &#181;L of each fraction to 100 &#181;L of Bradford reagent to visually check for protein presence. Wash until unbound protein is no longer present in the last wash, adding additional wash steps if necessary.</li>
	<li>Recover protein by running MS500 elution buffer (MS500 buffer supplemented with 10 mM of glutathione and 10 mM of NaOH) through the column, collecting 5 mL elution fractions. Check for protein presence by adding 1 &#181;L of eluate to 100 &#181;L of Bradford reagent. Stop collecting fractions when the Bradford reagent no longer indicates proteins are present.</li>
	<li>Perform buffer exchange by precipitation and centrifugation. Precipitate protein by adding 2 volumes of 4 M of ammonium sulfate to the eluate, gently inverting until cloudy, and then centrifuging for 30 min at 4 &#176;C and &#8805;20,000 x g and again for another 5 min. Remove the supernatant after each centrifugation without disturbing the protein pellet.</li>
	<li>Re-dissolve and store protein in MS500 supplemented with 25% glycerol. Store protein pellets or protein in storage buffer at -80 &#176;C.</li>
</ol>
4. In vitro ubiquitin chain cleavage assay
NOTE: Select ubiquitin chain length and linkage types based on DUB specificity described in previous reports31 or determined empirically. If necessary, this protocol could be used to test the activity of the wild-type DUB of interest on a panel of commercially available ubiquitin chains of defined length and linkage type. A di-ubiquitin chain amount of 375-750 ng and a DUB concentration of 1-2 &#181;M can be used as starting points for the assay27.
<ol>
	<li>Prepare 10x DUB activation buffer (500 mM of Tris-HCl pH 7.5, 500 mM of NaCl, and 100 mM of TCEP).</li>
	<li>For each time point for each DUB, prepare 10 &#181;L total of 2 &#181;M of purified GST-USP27X in 1x DUB activation buffer (DUB mix). Prepare master mixes and split them into time points.</li>
	<li>Incubate the DUB mix for 10 min at RT.</li>
	<li>Add 7 &#181;L of SDS-PAGE loading buffer to time 0 before adding ubiquitin chains to prevent the deubiquitylation reaction from starting.</li>
	<li>To each time point for each DUB, add 375 ng of K-63 di-ubiquitin chains diluted in 10 &#181;L 1x DUB activation buffer. The total volume is 20 &#181;L per reaction.</li>
	<li>Incubate the tubes at 30 &#176;C, stopping each time point with 7 &#181;L of SDS-PAGE loading buffer.</li>
	<li>Perform SDS-PAGE with a 4%-12% gradient gel and immunoblot7,32 for ubiquitin and USP27X to analyze the change in mono-ubiquitin presence across selected time points.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67367/67367fig01v2.jpg" /> 
Figure 1: Schematic of the study design. (A) Transformation of competent E. coli cells with recombinant protein expression plasmid. (B) Overnight bacterial expression of recombinant deubiquitylase protein. (C) Protein purification of recombinant deubiquitylase using a gravity-flow affinity column. (D) In vitro ubiquitin chain cleavage assay to evaluate deubiquitylating activity. <a href="https://www.jove.com/files/ftp_upload/67367/67367fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Assessing DUB Variants in Neurodevelopmental Disorders Using a Cleavage Assay

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The authors declare no conflicts of interest.

This article presents a protocol for the expression and purification of recombinant USP27X DUBs and an in vitro ubiquitin chain cleavage assay to compare the deubiquitylating activity of wild-type USP27X and NDD-associated variant proteins. This assay determined that XLID105-associated variants disrupt USP27X catalytic activity7. This mechanistic insight helped us define XLID105 as a USP27X functional disruption disorder.
This protocol can be adapted to other D...

Neurodevelopmental disorders (NDDs) arise from diverse etiologies with environmental or genetic determinants that drive atypical nervous system development1. Next-generation sequencing genetic testing has linked an increasing number of variants in ubiquitin system-related genes with genetic NDDs2. The ubiquitin system catalyzes the ligation of the small protein modifier ubiquitin to primarily lysine residues in protein substrates to drive changes in cellular behavior, including localization, stability, protein-protein interactions, or activity3. Ubiquitylation is mediated by E1 activating, E2 conjugating, and E3 ligase enzymes4 and is reversible by the activity of deubiquitylating enzymes (DUBs) that catalyze the cleavage and removal of ubiquitin from protein substrates5. Ubiquitin can be ligated to the substrate as a monomer (monoubiquitylation) or polymeric chains (polyubiquitylation) that are formed on any of the seven lysine residues (K6, K11, K27, K29, K33, K48, K63) or the M1 residue of ubiquitin. These different ubiquitin chain topologies and their combinations create a cellular code that is key for signal transduction6.
DUBs such as USP27X, USP7, USP9X, USP48, STAMBP, OTUD4, OTUD6B, and OTUD5 have been associated with NDDs2,7,8,9,10,11. For most NDDs, the molecular mechanisms that drive pathogenesis remain experimentally undefined. Some of the DUBs driving recently described disorders are poorly understood and lack known cellular readouts that can be used to assess the impact of genetic variation on protein function. In vitro, ubiquitin chain cleavage assays overcome this limitation as substrate-independent DUB activity readouts that can measure the impact of gene variants on enzymatic activity12.
In vitro ubiquitin cleavage assays have been used since the 1980s. These assays using radiolabeled substrates allowed for the discovery of the first DUBs, including isopeptidase, identified for its capacity to deubiquitylate Histone H2A13, and ubiquitin carboxyl-terminal hydrolase (UCH), identified by its ability to hydrolyze ubiquitin from a variety of chemical conjugates14,15,16. Further, radiolabeled polyubiquitylated full-length proteins or peptides were used to identify isopeptidase T and several UCHs and ubiquitin-specific proteases (UBPs) from erythrocytes and skeletal muscle, respectively17,18,19,20. Ubiquitin chains of a defined length and linkage type (K48-linked tetra-ubiquitin) were first used to measure the DUB activity of Isopeptidase T21. Since then, this assay has become the gold standard to measure DUB activity in mutational analyses22,23. The refinement of this assay currently allows visualization of ubiquitin cleavage via electrophoresis and conventional gel stains such as Coomassie blue, SYPRO orange, ruby, and silver stain or fluorescent or immunoblotting-based detection12,24. Molecular aspects of DUB activity, such as minimum chain length and linkage specificity25,26,27,28, can be clarified by using ubiquitin chains of different lengths (e.g., di-, tri-, tetra-ubiquitin) and linkages (K6, K11, K27, K29, K33, K48, K63, linear) in functional assays. NDD-associated variants can drive DUB activity defects that are ubiquitin chain linkage type specific11.
A di-ubiquitin cleavage assay using purified recombinant DUB proteins can directly measure the impact of NDD variants on DUB activity. USP27X, which is mutated in the NDD X-linked intellectual disability disorder 105 (XLID105)7,28 models the process presented here. This approach allows for the determination of how DUB activity is disrupted by gene variants in existing and unknown DUB-associated NDDs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amersham Protran 0.45 NC 200 mm &#215; 200 mm 25/PK</td>
			<td>Cytiva</td>
			<td>10600041</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Fisher Scientific</td>
			<td>AC205872500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760 25</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti- Ubiquitin (Mouse monoclonal)</td>
			<td>Biolegend</td>
			<td>Cat# 646302, RRID:AB_1659269</td>
			<td>(WB: 1:1000)</td>
		</tr>
		<tr>
			<td>Anti-GST (Sheep polyclonal)</td>
			<td>MRC-PPU Reagents and Services</td>
			<td>Cat# S902A Third bleed</td>
			<td>(WB: 1:1000) https://mrcppureagents.dundee.ac.uk/</td>
		</tr>
		<tr>
			<td>Baffled Culture Flasks 2 L</td>
			<td>Fisher Scientific</td>
			<td>10-042-5N</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Reagent</td>
			<td>Millipore Sigma</td>
			<td>B6916-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Gold Biotechnology</td>
			<td>C-105-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete, Protease Inhibitor tablets</td>
			<td>Millipore Sigma</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Econo-Column 1.5 &#215; 5 cm</td>
			<td>Bio-Rad</td>
			<td>7371507</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf ThermoMixer F1.5&#160;</td>
			<td>Eppendorf</td>
			<td>5384000020</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>https://www.microsoft.com/en-us/microsoft-365/excel</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Genesee Scientific</td>
			<td>18-205</td>
			<td></td>
		</tr>
		<tr>
			<td>Illustrator</td>
			<td>Adobe</td>
			<td></td>
			<td>https://www.adobe.com/products/illustrator.html</td>
		</tr>
		<tr>
			<td>Image Studio</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td>https://www.licor.com/bio/image-studio/</td>
		</tr>
		<tr>
			<td>Inkscape</td>
			<td>Inkscape</td>
			<td></td>
			<td>https://inkscape.org/</td>
		</tr>
		<tr>
			<td>Invitrogen 4-12% NuPAGE 1mm 12 well gel</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0322BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG (Isopropyl-b-D-Thiogalactopyranoside)</td>
			<td>Genesee Scientific</td>
			<td>20-109</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 800CW Donkey anti-Goat IgG Secondary Antibody</td>
			<td>LI-COR Biosciences</td>
			<td>Cat# 926-32214</td>
			<td>(WB: 1:10000)</td>
		</tr>
		<tr>
			<td>IRDye 800CW Donkey anti-Mouse IgG Secondary Antibody</td>
			<td>LI-COR Biosciences</td>
			<td>Cat# 926-32212</td>
			<td>(WB: 1:10000)</td>
		</tr>
		<tr>
			<td>Isotemp Digital Dry Bath</td>
			<td>Fisher Scientific</td>
			<td>88860022</td>
			<td></td>
		</tr>
		<tr>
			<td>K63 Di-Ubiquitin</td>
			<td>South Bay Bio LLC</td>
			<td>SBB-UP0072</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Genesee Scientific</td>
			<td>11-119</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Genesee Scientific</td>
			<td>11-118</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Gold Biotechnology</td>
			<td>L-040-100</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQ 4000 Benchtop Orbital Shaker</td>
			<td>Thermo Fisher Scientific</td>
			<td>SHKE4000-7</td>
			<td></td>
		</tr>
		<tr>
			<td>MES-SDS Running Buffer</td>
			<td>Boston Bioproducts Inc</td>
			<td>BP-177</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Tube Rotator</td>
			<td>Fisher Scientific</td>
			<td>88-861-051</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPage LDS Sample buffer 4x</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey&#160;Fc Imager</td>
			<td>LI-COR Biosciences</td>
			<td>43214</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Plus Ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>26620</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEX6P1 human USP27X</td>
			<td>MRC-PPU Reagents and Services</td>
			<td>DU21193</td>
			<td>https://mrcppureagents.dundee.ac.uk/</td>
		</tr>
		<tr>
			<td>pGEX6P1 human USP27X F313V</td>
			<td>Addgene</td>
			<td>225715</td>
			<td>Koch et at 2024 (PMID: 38182161)</td>
		</tr>
		<tr>
			<td>pGEX6P1 human USP27X S404N</td>
			<td>Addgene</td>
			<td>225717</td>
			<td>Koch et at 2024 (PMID: 38182161)</td>
		</tr>
		<tr>
			<td>pGEX6P1 human USP27X Y381H</td>
			<td>Addgene</td>
			<td>225716</td>
			<td>Koch et at 2024 (PMID: 38182161)</td>
		</tr>
		<tr>
			<td>Pierce Glutathione Agarose</td>
			<td>Thermo Fisher Scientific</td>
			<td>16100</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF (Phenylmethylsulfonyl fluoride)</td>
			<td>Gold Biotechnology</td>
			<td>P-470-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20 (Tween 20)</td>
			<td>Fisher Scientific</td>
			<td>AC233360010</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosetta 2 Competent Cells</td>
			<td>Millipore Sigma</td>
			<td>71402-M</td>
			<td></td>
		</tr>
		<tr>
			<td>SimplyBlue SafeStain</td>
			<td>Thermo Fisher Scientific</td>
			<td>LC6060</td>
			<td></td>
		</tr>
		<tr>
			<td>SmartSpec 3000</td>
			<td>Bio-Rad</td>
			<td>170-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>15544034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Genesee Scientific</td>
			<td>18-216</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonifier 250</td>
			<td>Branson</td>
			<td>100-132-135</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall RC 6 Plus Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>46910</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP (Tris-(carboxyethyl) phosphine hydrochloride)</td>
			<td>Gold Biotechnology</td>
			<td>TCEP10</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth Powder</td>
			<td>Genesee Scientific</td>
			<td>18-225</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Genesee Scientific</td>
			<td>18-146</td>
			<td></td>
		</tr>
		<tr>
			<td>XCell SureLock Mini-Cell and XCell II Blot Module&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>EI0002</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring enzymatic activity of neurodevelopmental disorder-associated deubiquitylating enzymes via an in vitro ubiquitin chain cleavage assay

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. Discrete disorders caused by single genes provide models to investigate mechanisms driving atypical neurodevelopment. Variants of genes encoding deubiquitylating enzyme (DUB) family proteins are associated with several NDDs, but there is a need to determine the pathogenic mechanisms of disorders driven by these gene variants. The impact of gene variants on DUB activity can be experimentally determined using a substrate-independent in vitro ubiquitin cleavage assay. This assay does not require knowledge of downstream substrates to directly measure catalytic activity. Here, the protocol for determining the impact of gene variants on enzymatic activity is modeled using the DUB Ubiquitin Specific Protease 27, X-linked (USP27X), which is mutated in X-linked intellectual disability 105 (XLID105). This experimental pipeline can be used to clarify the mechanisms underlying neurodevelopmental disorders driven by variants in DUB genes.

To determine the impact of XLID105-associated variants on USP27X catalytic activity, GST-tagged wild-type USP27X and the XLID105-associated variant F313V, Y381H, and S404N USP27X proteins were purified from bacteria. These variants are located within the USP catalytic domain of USP27X (Figure 2A). Because USP27X was previously reported to cleave K63 ubiquitin chains31, wild-type USP27X and the XLID105-associated variant F313V, Y381H, and S404N USP27X proteins were inc...

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

Neurodevelopmental disorders (NDDs) are associated with impairments in nervous system function but often remain poorly understood at the molecular level. ...

measuring-enzymatic-activity-neurodevelopmental-disorder-associated

Research

JoVE Journal

Biochemistry

376 Views.  Sanford Research. This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

Video: Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

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 ...