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).

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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. 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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. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. 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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. Centrifugation and filtration after thawing is generally recommended to remove microprecipitation.
The initial protein lysate obtained from a tissue still contains many substances besides proteins. Ionic exchange chromatography columns can be used to clear the lysate. Even if the protein one wants to extract does not bind to the ionic exchange column, the flow-through may be much clearer than the input solution, and easier to be processed in subsequent steps. This is shown for the extraction of FAHD1 from mouse kidneys (see Figure 5A).
Limitations and modifications of the method 
This protocol describes a protocol for the extraction of FAHD1 from the tissue. Based on this strategy, a general approach for the extraction of FAHD proteins (FAHD1, FAHD2) from other tissue may be derived, while specific adaptions may be required for individual cases. The implicit limitation of this method is the relative expression levels of FAHD1 (or FAHD proteins) in a given tissue and the availability of this tissue in adequate quantities. The overall scheme described in this protocol requires a high expression of the protein in a suitable amount of tissue to start with. Examples for the extraction of FAHD1 from swine kidney and mouse liver have been provided, which are two organs that show high expression levels of the protein. At each step described in this protocol, there is an implicit loss of sample, i.e., the low initial amount of protein is even further lessened. Ultimately, only low amounts of protein can be achieved using this protocol. Because the swine kidney is a large organ, about a gram of protein (about 80% purity; estimated) could be extracted by this approach from two kidneys. Applying the same protocol for the extraction of FAHD1 from mouse liver required collecting liver samples from 20 mice, to obtain only a few &#181;g of protein in the end. Obtaining higher purity of the protein using the outlined methods may be tedious, and silver staining reveals that after size exclusion chromatography there might still be many other proteins in solution. One could augment this protocol via affinity chromatography (NHS activated columns), using a monoclonal antibody (e.g., CAB025530).
All the protocols listed here necessitate that the protein one wants to extract is soluble. Optimal conditions of ammonium sulfate precipitation and pH/salt adjustments for chromatography need to be tested and defined. In case the ionic exchange chromatography does not provide any good purification results, dialysis against a chromatography buffer before performing FPLC may be an option of improving the solubility of the protein and the resolution of the chromatography. It is not recommended, however, to dialyze the crude lysate, as this may lead to massive and uncontrolled protein precipitation inside the dialysis tube. Problems may occur if the protein tends to be hydrophobic, and the protocols may not be applicable if the protein is highly hydrophobic (e.g., membrane proteins). If FAHD proteins are partly insoluble as found for FAHD2 (data not shown) in a given tissue lysate, other types of chromatography, for example, hydrophobic interaction chromatography (HIC) may be explored as well12.
Two modifications of the standard protocol have been shown in the results section. For the purification of FAHD1 from a swine kidney, size exclusion chromatography was used before ionic exchange chromatography (see Supplementary Figure 2). A problem with this method is that size exclusion chromatography features larger running tracks, and the sample volume increases strongly during the run. The sample volume should not exceed 2% of the bed volume to achieve high resolution23, and the final dilution factor also depends on the collected elution volume. In the provided example using a 120 mL column, an initial volume of 2 mL is recommended. The initial sample may be collected from several mL of the lysate after ammonium sulfate precipitation, by concentration using centrifugal filter units, without aggregation or precipitation artifacts. An example for the purification of FAHD1 from a swine kidney is provided as a representative result (also see Supplementary Figure 2). However, the limitation to only small sample volumes may render this approach to be unpractical.
Applications or directions of the method 
Expression and purification of recombinant proteins from bacteria is a straightforward and well-established method, to obtain feasible amounts (in grams) of protein24,25. This has been presented for FAHD112. However, there are several possible drawbacks in using these methods, such as possible poor growth of the host, inclusion body formation, loss of or altered protein activity, and, therefore, the possibility of not obtaining any protein at all, or resulting in a biased form of the protein (misfolding, bad post-translational modifications, etc.). Developing methods that can extract well-folded and properly modified protein from tissue directly is, therefore, appealing. The major problem, however, is that most proteins are not expressed at significant levels, and compared with the implicit induction and subsequent extraction of protein from bacteria, the amount of protein to be obtained is typically several orders of magnitude lower. There is a tradeoff between the inexpensive and easy extraction of recombinant protein from bacteria and the more expensive and tedious extraction of protein from tissue. The major advance with the latter is that one may extract the protein in its physiological form that is present in the tissue, including all mutual effects that may impact its folding and/or catalytic activity.
Protein extracted and purified by this protocol will help to identify competent inhibitors of FAHD113, possible protein interaction partners9, and possible yet undiscovered roles of the protein9. The study of enzymatic inhibitors, the development of monoclonal antibodies, and the study of the protein structure in the first place are greatly supported when having protein at high purity extracted from tissue rather than extracted from bacteria.

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="Figure 1" class="xfigimg" src="/files/ftp_upload/63333/63333fig01.jpg" /> 
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.remotexs.ntu.edu.sg/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.

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

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4.6K Views.  University of Innsbruck. 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.

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

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

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 ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

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 ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a &#8220;one-size-fits-all&#8221; approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one&#160;day in a detergent-free environment.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

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 ...

Optimizing FPLC for Efficient Purification of His-Tagged FEN1 Protein

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...

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 ...

Medicine

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

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

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Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

Expression and Purification of Mammalian Bestrophin Ion Channels

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

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

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

Cell Type-specific Gene Expression Profiling in the Mouse Liver