Name: Video: Protein Purification by Gravity-Flow Affinity Column and In Vitro Ubiquitin Chain Cleavage Assay
Uploaded: 2024-09-27T14:55:00
Description: 22 vistas. Protein Purification by Gravity-Flow Affinity Column and In Vitro Ubiquitin Chain Cleavage Assay

protein purification by gravity-flow affinity column and in vitro ubiquitin chain cleavage assay

Assessing DUB Variants in Neurodevelopmental Disorders Using a Cleavage Assay

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.

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

We're interested in defining the molecular basis of neurodevelopmental disorders associated with gene variants in enzymes of the ubiquitin system. We use cutting edge techniques to dissect the signaling pathways controlled by those enzymes and to determine the impact of gene variants on protein biology. The advantage of this method is that it is a substrate-independent technique that does not require previous knowledge of the substrates that are regulated by a specific DUB.This way we can directly measure the impact of neurodevelopmental disorder-associated gene variance on DUB catalytic activity and suggest possible pathogenic mechanisms. Because the cellular functions of most DUBs are poorly understood, our lab focus will be on identifying the signaling pathways that these enzymes mediate and how gene variants associated with genetic disease affect these processes. To begin, thaw 20 microliters of chemically competent Rosetta 2 Escherichia coli cells on ice immediately before use.Add one to 10 nanograms of the pGEX6P1 USP27X expression plasmid and incubate for five minutes on ice. Heat shock the cells for 30 seconds at 42 degrees Celsius in a dry bath. Then incubate the cells on ice for two minutes.Add 80 microliters of room temperature SOC medium to the cells. Incubate for 60 minutes at 37 degrees Celsius with 200 RPM rotation in a 19-millimeter orbit benchtop temperature controlled shaker. Then plate 50 microliters of the culture on an LB agar plate, supplemented with chloramphenicol and ampicillin.Incubate for 20 hours at 37 degrees Celsius, lid side down, in a temperature controlled incubator. Pick a single colony of transformed bacteria and add it to 10 milliliters of sterile LB medium supplemented with chloramphenicol and ampicillin. Incubate for 20 hours as shown previously.Then add 10 milliliters of the overnight culture to one liter of TB medium supplemented with chloramphenicol and ampicillin. Incubate until the optical density at 600 nanometers reaches 0.5 to 0.6. Next, add 50-micromolar isopropyl beta-d-1-thiogalactopyranoside to the culture to induce expression.After cooling the sample to 16 degrees Celsius, incubate for 20 hours at 16 degrees Celsius with 200 RPM rotation. Then centrifuge the cells for 20 minutes at 3000 G or higher at four degrees Celsius to pellet the cells from the expression culture. Finally, store the pellet at minus 80 degrees Celsius for at least one hour.To begin, secure the empty gravity flow column in a retort stand. Fill the column with two to three milliliters of gluthathione agarose resin to purify a cell pellet collected from one liter of expression culture. Wash the column with one resin bed volume of 20%ethanol.After washing the column, thaw the cell pellet at four degrees Celsius. Add 30 milliliters of MS500 buffer to the thawed pellet and incubate with gentle end-over-end rotation. Now sonicate the lysed cells in a 50-milliliter centrifuge tube on ice until the lysate flows freely when dispensed from a pipette tip.Centrifuge for 30 minutes at four degrees Celsius and 20, 000 G or higher to clear the supernatant. Then decant the supernatant into a beaker and load the cleared lysate onto the column. Run the lysate through the column by gravity flow while collecting the flow-through.Afterward, wash the column with at least two resin bed volumes of MS500 wash buffer and collect the wash flow-through in five milliliter fractions. Next, add one microliter of each fraction to 100 microliters of Bradford reagent to check for protein presence. Recover the protein by running MS500 elusion buffer through the column and collecting five milliliter elution fractions.To precipitate the protein, add two volumes of four-molar ammonium sulfate to the eluate and gently invert the tube until it becomes cloudy. Centrifuge for 30 minutes at four degrees Celsius and 20, 000 G or higher. Remove the supernatant after each centrifugation without disturbing the protein pellet.Then re-dissolve the protein in MS500 buffer supplemented with 25%glycerol before storing it. Prepare 10 microliters of purified GST-tagged USP27X in deubiquitylating enzyme activation buffer for each time point for each deubiquitylating enzyme. Before adding ubiquitin chains, add seven microliters of SDS-PAGE loading buffer to the time zero sample to prevent the deubiquitylation reaction from starting.To each time point for each deubiquitylating enzyme, add 375 nanograms of K63-linked di-ubiquitin chains diluted in 10 microliters of enzyme activation buffer. Incubate the tubes at 30 degrees Celsius, stopping each time point by adding seven microliters of SDS-PAGE loading buffer. Perform SDS-PAGE using a four to 12%gradient gel.Wild type USP27X cleaved K63-linked di-ubiquitin chains into mono-ubiquitin after one hour of incubation. The XLID-105 associated variance F313V, Y381H, and S404N did not cleave K63-linked di-ubiquitin chains after one hour of incubation.

Protein Purification by Gravity-Flow Affinity Column and In Vitro Ubiquitin Chain Cleavage Assay

protein-purification-gravity-flow-affinity-column-vitro-ubiquitin

Research

JoVE Journal

Biochemistry

22 vistas. Protein Purification by Gravity-Flow Affinity Column and In Vitro Ubiquitin Chain Cleavage Assay