The authors are very thankful for the technical assistance by Ayse &#214;zt&#252;rk and Eva Albertini. Mice used for the generation of liver tissue were maintained under the supervision of Univ.-Doz. Dr. Pidder Jansen-D&#252;rr (Institute for Biomedical Aging Research at Innsbruck University, Rennweg 10, 6020 Innsbruck, Austria).

<ol>
	<li>Pircher, 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=Identification+of+FAH+domain-containing+protein+1+(FAHD1)+as+oxaloacetate+decarboxylase.">Identification of FAH domain-containing protein 1 (FAHD1) as oxaloacetate decarboxylase.</a> Journal of Biological Chemistry. 290 (11), 6755-6762 (2015).</li><li>Pircher, 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=Identification+of+human+Fumarylacetoacetate+Hydrolase+Domain-containing+Protein+1+(FAHD1)+as+a+novel+mitochondrial+acylpyruvase.">Identification of human Fumarylacetoacetate Hydrolase Domain-containing Protein 1 (FAHD1) as a novel mitochondrial acylpyruvase.</a> Journal of Biological Chemistry. 286 (42), 36500-36508 (2011).</li><li>Kang, T. -. 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=Senescence+surveillance+of+pre-malignant+hepatocytes+limits+liver+cancer+development.">Senescence surveillance of pre-malignant hepatocytes limits liver cancer development.</a> Nature. 479 (7374), 547-551 (2011).</li><li>Hong, H., Seo, H., Park, W., Kim, K. K. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence,+structure+and+function-based+classification+of+the+broadly+conserved+FAH+superfamily+reveals+two+distinct+fumarylpyruvate+hydrolase+subfamilies.">Sequence, structure and function-based classification of the broadly conserved FAH superfamily reveals two distinct fumarylpyruvate hydrolase subfamilies.</a> Environmental Microbiology. 22 (1), 270-285 (2020).</li><li>Timm, D. E., Mueller, H. A., Bhanumoorthy, P., Harp, J. M., Bunick, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+and+mechanism+of+a+carbon-carbon+bond+hydrolase.">Crystal structure and mechanism of a carbon-carbon bond hydrolase.</a> Structure. 7 (9), 1023-1033 (1999).</li><li>Bateman, R. L., 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=Mechanistic+inferences+from+the+crystal+structure+of+Fumarylacetoacetate+Hydrolase+with+a+bound+phosphorus-based+inhibitor.">Mechanistic inferences from the crystal structure of Fumarylacetoacetate Hydrolase with a bound phosphorus-based inhibitor.</a> Journal of Biological Chemistry. 276 (18), 15284-15291 (2001).</li><li>Weiss, A. K. 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=Structural+basis+for+the+bi-functionality+of+human+oxaloacetate+decarboxylase+FAHD1.">Structural basis for the bi-functionality of human oxaloacetate decarboxylase FAHD1.</a> Biochemical Journal. 475 (22), 3561-3576 (2018).</li><li>Etemad, 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=Oxaloacetate+decarboxylase+FAHD1+-+a+new+regulator+of+mitochondrial+function+and+senescence.">Oxaloacetate decarboxylase FAHD1 - a new regulator of mitochondrial function and senescence.</a> Mechanisms of Ageing and Development. 177, 22-29 (2019).</li><li>Weiss, A. K. 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=Regulation+of+cellular+senescence+by+eukaryotic+members+of+the+FAH+superfamily+-+A+role+in+calcium+homeostasis.">Regulation of cellular senescence by eukaryotic members of the FAH superfamily - A role in calcium homeostasis.</a> Mechanisms of Ageing and Development. 190, 111284 (2020).</li><li>Petit, M., Koziel, R., Etemad, S., Pircher, H., Jansen-D&#252;rr, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+oxaloacetate+decarboxylase+FAHD1+inhibits+mitochondrial+electron+transport+and+induces+cellular+senescence+in+human+endothelial+cells.">Depletion of oxaloacetate decarboxylase FAHD1 inhibits mitochondrial electron transport and induces cellular senescence in human endothelial cells.</a> Experimental Gerontology. 92, 7-12 (2017).</li><li>Wiley, C. D., 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=Mitochondrial+dysfunction+induces+senescence+with+a+distinct+secretory+phenotype.">Mitochondrial dysfunction induces senescence with a distinct secretory phenotype.</a> Cell Metabolism. 23 (2), 303-314 (2016).</li><li>Weiss, A. K. 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=Expression,+purification,+crystallization,+and+enzyme+assays+of+Fumarylacetoacetate+Hydrolase+Domain-containing+proteins.">Expression, purification, crystallization, and enzyme assays of Fumarylacetoacetate Hydrolase Domain-containing proteins.</a> Journal of Visualized Experiments: JoVE. (148), e59729 (2019).</li><li>Weiss, A. K. 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=Inhibitors+of+Fumarylacetoacetate+Hydrolase+Domain+Containing+Protein+1+(FAHD1).">Inhibitors of Fumarylacetoacetate Hydrolase Domain Containing Protein 1 (FAHD1).</a> Molcules. 26 (16), 5009 (2021).</li><li>Mizutani, H., Kunishima, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+crystallization+and+preliminary+X-ray+analysis+of+the+fumarylacetoacetase+family+member+TTHA0809+from+Thermus+thermophilus+HB8.">Purification, crystallization and preliminary X-ray analysis of the fumarylacetoacetase family member TTHA0809 from Thermus thermophilus HB8.</a> Acta Crystallographica Section F Structural Biology and Crystallization Communications. 63 (9), 792-794 (2007).</li><li>Lee, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+outline+of+methods+for+protein+isolation+and+purification.">A simple outline of methods for protein isolation and purification.</a> Endocrinology and Metabolism. 32 (1), 18-22 (2017).</li><li>Amer, H. E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+proteins:+Between+meaning+and+different+methods).">Purification of proteins: Between meaning and different methods).</a> Proteomics Technologies and Applications. , (2019).</li><li>Niu, L., Yuan, H., Gong, F., Wu, X., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+extraction+methods+shape+much+of+the+extracted+proteomes.">Protein extraction methods shape much of the extracted proteomes.</a> Frontiers in Plant Science. 9, 802 (2018).</li><li>Gordon, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+vanadate+as+protein-phosphotyrosine+phosphatase+inhibitor.">Use of vanadate as protein-phosphotyrosine phosphatase inhibitor.</a> Methods in Enzymology. 201, 477-482 (1991).</li><li>Gallagher, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDS-polyacrylamide+gel+electrophoresis+(SDS-PAGE).">SDS-polyacrylamide gel electrophoresis (SDS-PAGE).</a> Current Protocols in Essential Laboratory Techniques. , (2012).</li><li>. Effect of pH on Protein Size Exclusion Chromatography Available from: <a target="_blank" href="https://www.agilent.com/cs/library/applications/5990-8138EN.pdf">https://www.agilent.com/cs/library/applications/5990-8138EN.pdf</a> (2011)</li><li>S&#248;rensen, B. K., 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=Silver+staining+of+proteins+on+electroblotting+membranes+and+intensification+of+silver+staining+of+proteins+separated+by+polyacrylamide+gel+electrophoresis.">Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis.</a> Analytical Biochemistry. 304 (1), 33-41 (2002).</li><li>Fagerberg, L., 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=Analysis+of+the+human+tissue-specific+expression+by+genome-wide+integration+of+transcriptomics+and+antibody-based+proteomics.">Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics.</a> Molecular &amp; Cellular Proteomics. 13 (2), 397-406 (2014).</li><li>. Cytiva Life Fundamentals of size exclusion chromatography Available from: <a target="_blank" href="https://www.cytivalifesciences.com/en/us/solutions/protein-research/knowledge-center/protein-purification-methods/size-exclusion-chromatography">https://www.cytivalifesciences.com/en/us/solutions/protein-research/knowledge-center/protein-purification-methods/size-exclusion-chromatography</a> (2022)</li><li>Rosano, G. L., Ceccarelli, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+expression+in+Escherichia+coli:+advances+and+challenges.">Recombinant protein expression in Escherichia coli: advances and challenges.</a> Frontiers in Microbiology. 5, 172 (2014).</li><li>Rosano, G. L., Morales, E. S., Ceccarelli, E. 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The authors have no competing financial interests.

Critical steps in the protocol 
Following common guidelines for the handling of proteins is essential, such as working on ice and at moderate pH and salt conditions. The use of protease inhibitors is beneficial to the method, while the use of proteasome inhibitors is highly recommended. Freezing and thawing the sample may always result in protein precipitation (at least partially), so any thawed aliquot of initial protein lysate (step 2) should be processed continuously without a break. Centrifugati...

The eukaryotic FAH domain-containing protein 1 (FAHD1) acts as bi-functional oxaloacetate (OAA) decarboxylase (ODx)1 and acylpyruvate hydrolase (ApH)2. It is localized in mitochondria2 and belongs to the broad FAH superfamily of enzymes1,2,3,4,5,6. While its ApH activity is only of minor relevance, the ODx activity of FAHD1 is involved in the regulation of the TCA cycle flux1,7,8,9. OAA is not only required for the central citrate synthase reaction in the tricarboxylic acid cycle but also acts as a competitive inhibitor of succinate dehydrogenase as part of the electron transport system and as a cataplerotic metabolite. Downregulation of FAHD1 gene expression in human umbilical vein endothelial cells (HUVEC) resulted in a significant reduction in the rate of cell proliferation10, and significant inhibition of mitochondrial membrane potential, associated with a concomitant switch to glycolysis. The working model refers to mitochondrial dysfunction associated senescence (MiDAS)11-like phenotype8, where mitochondrial OAA levels are tightly regulated by FAHD1 activity1,8,9.
Recombinant protein is easier to obtain via expression and purification from bacteria12 rather than from tissue. However, a protein expressed in bacteria may be biased by possible lack of post-translational modifications, or may simply be problematic (i.e., due to plasmid loss, bacterial stress responses, distorted/unformed disulfide bonds, none or poor secretion, protein aggregation, proteolytic cleavage, etc.). For certain applications, protein needs to be obtained from cell lysate or tissue, in order to include such modifications and/or to exclude possible artifacts. Purified protein from tissue supports the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors for selected enzymes, such as for FAHD113.
This manuscript presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. The described methods require fast protein liquid chromatography (FPLC) but otherwise use common laboratory equipment. Alternative methods may be found elsewhere14,15,16,17. After total protein extraction, the proposed protocol involves a testing phase, in which sub-protocols for ammonium sulfate precipitation and ionic exchange chromatography are discussed (Figure 1). After defining these sub-protocols, the protein of interest is extracted via a sequential strategy using ionic exchange and size-exclusion chromatography with FPLC. Based on these guidelines, the final protocol may be adapted individually for other proteins of interest.
<img alt="Tissue processing to Western Blot: centrifugation, precipitation, chromatography; diagram." class="xfigimg" src="/files/ftp_upload/63333/63333fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: The overall strategy of this protocol. From top to bottom: Protein is extracted from tissues. Tissue homogenate is prepared, centrifuged, and filtrated. For each pair of supernatant and pellet-derived samples, tests for ammonium sulfate precipitation and ionic exchange chromatography (FPLC) need to be performed to probe for optimal conditions. After establishing these sub-protocols, the protein may be extracted via a sequential procedure of ammonium sulfate precipitation, ionic exchange chromatography, and repetitive size exclusion chromatography (FPLC) at varying pH and salt concentrations. All steps need to be controlled by western blot. <a href="https://www.jove.com/files/ftp_upload/63333/63333fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>0.22 &amp;micro;m filter units</td><td>MERCK</td><td>SLGP033RS</td><td>Millex-HP, 0.22 &amp;micro;m, PES 33 mm, not steril</td></tr><tr><td>0.45 &amp;micro;m filter units</td><td>MERCK</td><td>SLHP033NS</td><td>Millex-HP, 0.45 &amp;micro;m, PES 33 mm, not steril</td></tr><tr><td>15 mL Falcon tubes</td><td>VWR</td><td>734-0451</td><td>centrifugal tubes</td></tr><tr><td>50 mL Falcon tubes</td><td>VWR</td><td>734-0448</td><td>centrifugal tubes</td></tr><tr><td>96-Well UV Microplate</td><td>Thermo-Fischer</td><td>8404</td><td>UV/VIS transparent flat-bottom 96 well plates</td></tr><tr><td>Acrylamide/Bis Solution (40%, 29:1 ratio)</td><td>BIO-RAD</td><td>#1610147</td><td>40% acrylamide/bis-acrylamide, 29:1 (3.3% crosslinker) solution for casting polyacrylamide gels</td></tr><tr><td>&amp;Auml;KTA FPLC system</td><td>GE Healthcare Life Sciences / Cytiva</td><td>-</td><td>using the FPLC system by GE Healthcare; different custom versions exist; this work used the &quot;&amp;Auml;KTA pure&quot; system</td></tr><tr><td>Amicon Ultra-15, PLGC Ultracel-PL Membran, 10 kDa</td><td>MERCK</td><td>UFC901024</td><td>centrifigal filters for protein enrichment; 10 kDa molecular mass filter; 15 mL</td></tr><tr><td>Amicon Ultra-4, PLGC Ultracel-PL Membran, 10 kDa</td><td>MERCK</td><td>UFC801024</td><td>centrifigal filters for protein enrichment; 10 kDa molecular mass filter; 4 mL</td></tr><tr><td>Ammonium sulfate powder</td><td>MERCK</td><td>A4418</td><td>ammonium sulphate for molecular biology, &amp;ge;99.0%</td></tr><tr><td>Ammoniumpersulfat reagent grade, 98%</td><td>MERCK</td><td>215589</td><td>Catalyst for acrylamide gel polymerization.</td></tr><tr><td>Coomassie Brilliant blue R 250</td><td>MERCK</td><td>1125530025</td><td>Coomassie Brilliant blue R 250 (C.I. 42660) for electrophoresis Trademark of Imperial Chemical Industries PLC. CAS 6104-59-2, pH 6.2 (10 g/l, H&lt;sub&gt;2&lt;/sub&gt;O, 25 &amp;deg;C)</td></tr><tr><td>Dialysis tubing cellulose membrane</td><td>MERCK</td><td>D9277</td><td>Cellulose membranes for the exchange of buffers via dialysis.</td></tr><tr><td>Eppendof tubes 1.5 mL</td><td>VWR</td><td>525-1042</td><td>microcentrifugal tubes; autoclaved</td></tr><tr><td>HiLoad 26/600 Superdex 75 pg</td><td>GE Healthcare Life Sciences / Cytiva</td><td>28989334</td><td>HiLoad Superdex 75 pg prepacked columns are for high-resolution size exclusion chromatography of recombinant proteins</td></tr><tr><td>Immun-Blot PVDF Membrane</td><td>BIO-RAD</td><td>#1620177</td><td>PVDF membranes are protein blotting membranes optimized for fluorescent and multiplex fluorescent applications.</td></tr><tr><td>Mini Trans-Blot Electrophoretic Transfer Cell</td><td>BIO-RAD</td><td>#1703930</td><td>Use the Mini Trans-Blot Cell for rapid blotting of Mini-PROTEAN precast and handcast gels.</td></tr><tr><td>Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels</td><td>BIO-RAD</td><td>#1658004</td><td>4-gel vertical electrophoresis system, includes electrode assembly, companion running module, tank, lid with power cables, mini cell buffer dam.</td></tr><tr><td>Mono Q 10/100 GL</td><td>GE Healthcare Life Sciences / Cytiva</td><td>17516701</td><td>Mono Q columns are strong anion exchange chromatography columns for protein analysis or small scale, high resolution polishing of proteins.</td></tr><tr><td>Mono S 10/100 GL</td><td>GE Healthcare Life Sciences / Cytiva</td><td>17516901</td><td>Mono S columns are strong cation exchange chromatography columns for protein analysis or small scale high resolution polishing of proteins.</td></tr><tr><td>PageRuler Prestained Protein Ladder, 10 to 180 kDa</td><td>Thermo-Fischer</td><td>26616</td><td>A mixture of 10 blue-, orange-, and green-stained proteins (10 to 180 kDa) for use as size standards in protein electrophoresis (SDS-PAGE) and western blotting.</td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo-Fischer</td><td>23225</td><td>A two-component, high-precision, detergent-compatible protein assay for determination of protein concentration.</td></tr><tr><td>Sonifier 250; Ultrasonic Cell Disruptor w/ Converter</td><td>Branson</td><td>-</td><td>New models at https://www.emerson.com/documents/automation/brochure-sonifier-sfx250-sfx550-cell-disruptors-homogenizers-branson-en-us-168180.pdf</td></tr><tr><td>Swine Anti-Rabbit Immunoglobulins/HRP (affinity isolated)</td><td>Agilent Dako</td><td>P0399</td><td>The antibody used for horseradish peroxidase conjugation reacts with rabbit immunoglobulins of all classes.</td></tr><tr><td>TEMED, 1,2-Bis(dimethylamino)ethane, TMEDA</td><td>MERCK</td><td>T9281</td><td>TEMED (N,N,N&amp;prime;,N&amp;prime;-Tetramethylethylenediamine) is molecule which allows rapid polymerization of polyacrylamide gels.</td></tr><tr><td>Tube Roller</td><td>-</td><td>-</td><td>A general tube rotator roller; e.g. a new model at https://labstac.com/de/Mixer/Roller/c/71</td></tr><tr><td>Tube Rotator</td><td>-</td><td>-</td><td>A general tube rotator wheel; e.g. a new model at https://labstac.com/de/Tube-Roller/p/MT123</td></tr><tr><td>ULTRA-TURRAX; T 25 digital</td><td>IKA</td><td>0003725000</td><td>New models at https://www.ika.com/de/Produkte-Lab-Eq/Dispergierer-Dipergiergeraet-Homogenisierer-Homogenisator-csp-177/T-25-digital-ULTRA-TURRAX-cpdt-3725000/</td></tr></tbody></table>

extraction and purification of fahd1 protein from swine kidney and mouse liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

FAHD1 protein was extracted from swine kidney and mouse liver using the presented protocol. For mouse tissue, multiple organs are required to obtain several µg after the final purification step. For this reason, this article focuses on the extraction of FAHD1 from swine kidneys, which is a much more exemplary experiment. The extraction of FAHD1 from the mouse liver is performed to present the difficulties and possible pitfalls of this protocol. It is generally recommended to use organs that show a high expression level ...

Watch this Scientific Journal Video about Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver at JoVE.com

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as ...

extraction-purification-fahd1-protein-from-swine-kidney-mouse

Research

JoVE Journal

Biochemistry

4.7K Views.  University of Innsbruck. Zuivering van recombinant eiwit uit bacteriën is een gevestigde methode. Er kunnen echter verschillende problemen optreden bij het gebruik van deze technieken. Het hebben van methoden om eiwit uit weefsel te extraheren is zeker aantrekkelijk.Het belangrijkste voordeel van deze methode is dat men het eiwit in zijn fysiologische vorm kan extraheren, inclusief alle wederzijdse effecten die van invloed kunnen zijn op de vouw- en katalytische activiteit. Deze benadering kan worden aangepas...

Video: Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.
The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (&#8805;42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. 
This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (&gt;10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. 
Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

We provide scalable protocols covering construct design, transient transfection, and expression and purification of full-length human huntingtin protein variants in HEK293 cells.

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...

Purifying a Fibrinolytic Enzyme from Sipunculus nudus via Affinity Chromatography

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin directly, showing great advantages over traditional thrombolytic agents. However, due to the lack of structural information, all the purification programs for sFE are based on multistep chromatography purifications, which are too complicated and costly. Here, an affinity purification protocol of sFE is developed for the first time based on a crystal structure of sFE; it includes preparation of the crude sample and the lysine/arginine-agarose matrix affinity chromatography column, affinity purification, and characterization of the purified sFE. Following this protocol, a batch of sFE can be purified within 1 day. Moreover, the purity and activity of the purified sFE increases to 92% and&#160;19,200 U/mL,&#160;respectively. Thus, this is a simple, inexpensive, and efficient approach for sFE purification. The development of this protocol is of great significance for the further utilization of sFE and other similar agents.

Author Spotlight: Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus  

Here, we present an affinity purification method of a fibrinolytic enzyme from Sipunculus nudus that is simple, inexpensive, and efficient.

The fibrinolytic enzyme from Sipunculus nudus (sFE) is a novel fibrinolytic agent that can both activate plasminogen into plasmin and degrade fibrin ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Immunology and Infection

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

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