The authors are very thankful for expert technical assistance by Annabella Pittl and the pilot method development by Haymo Pircher.

<ol>
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The authors have nothing to disclose and declare no competing financial interests. H. G. is CEOCSO at MoleculeCrafting.HuGs e.U. and provided acylpyruvates for this study via custom synthesis. Work in P. J. D.&#8217;s lab was supported by the Austrian Science Fund (FWF): project number P 31582-B26. Publication fees for this manuscript have partly been covered by the Austrian Science Fund (FWF) under project number P 31582-B26. A. N. and B. R. are supported by the Austrian Science Fund (FWF) under project P28395-B26.

Critical Steps
FAHD proteins are very sensitive to salt concentrations. At low NaCl concentrations, the proteins may precipitate upon thawing, but they can usually be fully reconstituted at higher salt concentrations. That is, if a FAHD protein precipitates for some reason, it may be recovered or refolded with higher salt concentrations (&#62;300 &#181;M). Some more hydrophobic proteins, however, may not be recovered (for example, human FAHD2), but detergents such as CHAPS (maximum 1%) or glycerol (10%) may be used to keep them in stable solution. In any case, shock-freezing using liquid nitrogen and storage at -80 &#176;C is recommended, as it is a gentle and slow process of thawing.
Some unexpected problems may occur during Ni-NTA purification in step 3.1.10. Of note, a higher OD in the second collected sample than in the first sample indicates a too high volume of the agarose resin (take a note and use less resin in the next experiment). Also, the agarose resin itself leads to an OD signal at 280 nm (i.e., disruption of the agarose resin bed will give artificial signals). In case of doubt, it is advised to use other methods like a Bradford or BSA assay to determine protein concentrations.
In enzymatic assays, there are three critical aspects to be considered. First, assessing the protein concentration is critical to obtain the correct specific activities. The level of purity of the protein is influencing the result and needs to be estimated. In case of tagged protein, the mass of the tag-part has to be computed, and the specific activity has to be correspondingly corrected. For simple assays described in section 7 of the protocol, Ni-NTA purity is sufficient to distinguish between active and inactive substrates, cofactors, etc. In the case of more complex Michaelis-Menten kinetics, all reactant and substrate concentrations must be correctly determined. Especially when using oxaloacetate (which auto-decarboxylates over time) the enzymatic part of the reaction must be corrected for auto-decarboxylation (under the assumption that both reactions occur simultaneously). Initial changes in the optical density signal addressed to keto-enol tautomerization of the substrate must be considered. Third, concentrations and volumes must be adjusted. A reaction with defined concentrations of enzyme and substrate may give different results dependent on the assay volume. If there is too much enzyme per well, adhesion of the liquid may in fact bias the result.
For assessing Michaelis-Menten kinetics it is recommended to perform initial experiments in 100 &#181;L, 200 &#181;L, and 300 &#181;L batches in order to find the optimal combination. Similar aspects apply to the ratio of enzyme-substrate concentrations for kinetic assays. Too much enzyme per substrate or too much substrate per enzyme put the system outside the linear steady-state Michaelis range. Initial experiments are required to optimize these conditions. Exemplary adjustment for human FAHD1 (wild-type) protein are provided in section 8, resulting in kinetic diagrams (as presented in Figure 5B, for example).
For crystallization a droplet of protein solution is pipetted in the center of a coverslip and mixed with a droplet of crystallization cocktail, which is usually composed of a buffer (e.g., Tris-HCl, HEPES) and a precipitant (e.g., polyethylene glycol, ammonium sulfate). A droplet of inhibitor solution for co-crystallization (such as oxalate in this protocol) may optionally be applied. The coverslip is then placed upside down above a well of reservoir containing crystallization cocktail, sealing the well air tight with the help of sealant oil (Figure 6B). Ideally, no precipitation occurs within the drop at the beginning of the experiment meaning the protein remains in solution. Since precipitant concentration in the reservoir is higher than in the drop, the drop starts to lose water by evaporation into the atmosphere of the well until equilibrium with the reservoir is reached. The diffusion of water into the reservoir causes a slow volume decrease of the drop which in turn causes an increase of both, protein and precipitant concentration in the drop. If the protein solution reaches the required state of super-saturation and thus meta-stability, spontaneous nucleation followed by crystal growth can occur. Reaching the supersaturated state is a necessary but not sufficient condition for crystallization. Crystallization of proteins needs both, favorable thermodynamic and kinetic conditions, and heavily depends on the unpredictable properties of the protein to be crystallized22.
Modifications and Troubleshooting
Expression of protein in E. coli may be inefficient. Varying IPTG concentrations, expression temperature, and amplification time, such as room temperature for several hours or in cold room overnight, may need to be tested for each new protein to find optimal conditions. Precipitation of protein in inclusion bodies is sometimes observed for more hydrophobic FAHD proteins. In such cases, protein expression in other model systems such as insect cells is recommended, as inclusion bodies are less likely to form26.
As FAHD proteins are sensitive to salt and cofactor concentrations, as well as pH, purification strategies for different homologues, orthologues, and point mutation variants may differ in individual settings. The purification methods described are developed for the wild-type human and mouse FAHD1 protein. Concentrations of chemicals, such as NaCl and imidazole, as well as pH, may have to be adapted for individual proteins with a different isoelectric point (pI). Also of note, not every His-tagged protein may bind well to a Ni-NTA resin. If protein binding to the Ni-NTA column is inefficient, adapted concentrations of NaCl and imidazole, as well as varying pH conditions in the Ni-NTA running buffer may help to improve the quality of the outcome. If not, skipping the Ni-NTA step and proceeding to the step of ionic exchange chromatography may also lead to a successful purification strategy. If a protein binds to the Ni-NTA column but cannot be eluted from the column, addition of some mM EDTA may help disrupt the Ni2+ complex.
Concerning the process of crystallization, it needs to be understood that self-organization of large and complex protein molecules into a regular periodic lattice is an inherently unlikely process that depends heavily on difficult to control kinetic parameters. Even small changes in the set-up used for crystallization can dramatically alter the result and no crystals will form. Protein purity is generally of paramount importance. As a rule of thumb, a heavily overloaded SDS-PAGE gel should not show other bands. Also, the sequence in which steps are performed may affect the outcome. As an example, to ensure reproducibility, it is often necessary to keep the pipetting sequence the same, then first add the protein, and finally add precipitant to the crystallization droplet (or vice versa). Whichever method used, it should be kept the same when trying to reproduce or scale-up experiments. If no crystals are observed following this protocol, the chemical precipitant composition, pH, drop size, and protein-to-precipitate ratio can be varied in small increments. Patience and consistent observations of the drops are of virtue.
Remarks to Catalytic Mechanisms of FAHD1
The presented methods have been developed specifically to obtain FAHD1 proteins of high-quality. This enabled growth of FAHD1 crystals as well as engineering of crystals containing FAHD1 complexed to an inhibitor (oxalate, PDB:6FOG). The X-ray structures provide a 3D architecture of the enzyme&#8217;s catalytic cavity. These results establish a comprehensive description of residues potentially important for the catalytic mechanisms of this intriguing enzyme. FAHD1 was first described to be able to cleave acylpyruvates (acetylpyruvate, fumarylpyruvate)11. Later on, it was found that FAHD1 operates also as a decarboxylase of oxaloacetate12. Although the substrates acylpyruvate and oxaloacetate are different chemical moieties, the chemical transformations share mechanistically the strategic cleavage of a common single C3-C4 bond, energetically facilitated if the C3-C4 bond orbitals stay orthogonal to the &#960;-orbitals of the C2-carbonyl15. Such a conformation allows resonance stabilization of the C3-carbanion transiently formed during the cleavage process. The FAHD1 substrates (oxaloacetate and acylpyruvates) are flexible molecules and may exist in tautomeric (keto-enol) as well as C2-hydrated forms (Figure 9A). The equilibria between the different species are determined mainly by the nature of buffer composition used, pH and presence of metal ions. In the following we discuss hypothetic mechanistic scenarios inspired from analysis of X-ray crystal structures which disclosed the catalytic center of FAHD1.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/59729/59729fig09v2.jpg" /> 
Figure 9: Details on the proposed catalytic mechanism of human FAHD1. 
(A) Oxaloacetate exists in crystalline state as well as in neutral solution mainly in the Z-enol form24. However, under physiological pH-conditions the 2-keto form is the predominant representation25. (B) Chemical sketch of the hFAHD1 cavity15 with Mg-bound oxaloacetate (left) and acylpyruvate (right, with R1 as organic rest; the red arrow denotes a nucleophilic attack of the adjacent stabilized water molecule) (see discussion). (C) Comparison of favored conformations for C3-C4 cleavage in decarboxylase (b to c) and hydrolase (b&#8217; to c) mechanism of FAHD1: both processes result in Mg-complexed pyruvate-enolate (see discussion). Intermediates b and b&#8217; are expected to be stabilized by Q109, as sketched in panel B (see discussion). <a href="https://www.jove.com/files/ftp_upload/59729/59729fig09v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The Decarboxylase Activity of FAHD1
Oxaloacetate exists in crystalline state as well as in neutral solution mainly in the Z-enol form24. But it was shown that under physiological pH-conditions (buffer conditions at pH 7.4) the 2-keto form is the predominant representation of oxaloacetate25 (Figure 9A), and that enolization is not a prerequisite for decarboxylation27. Of note, Mg2+ ions have no influence on the ratio of the oxaloacetate species at a pH of 7.4 or below28. Transposition of the oxaloacetate keto form into the catalytic center of FAHD1 (guided by the bound oxalate in the complexed enzyme (PDB: 6FOG15)) revealed residue Q109 as a conformational regulator of the bound oxaloacetate15. As outlined in another article15, hydrogen bonding to the carbamoyl group of Q109 stabilizes an oxaloacetate-conformation resulting from rotation around the C2-C3 bond (Figure 9B, left panel). As a consequence of this rotation, the C3-C4 bond (to be cleaved) adopts a close to orthogonal disposition relative to the &#960;-orbitals of the C2-carbonyl (Figure 9C). Carbon dioxide can be released. The primary product of this process would be resonance stabilized Mg-enolate of pyruvate. It is known from investigations of oxaloacetate-Mg complexes that the enolate forms the most stable complex28,29. Assuming a comparable stability for a Mg-pyruvate enolate-complex the cofactor of FAHD1 could be blocked, but lysine residue K123 can protonate the pyruvate-enolate in an equilibrium to prohibit loss of the cofactor15.
The given interpretation suggests pyruvate enol as a distinct intermediate in the catalytic ODx function of FAHD1. At this step in the hypothesized model, experimental data does not provide any further indication as to why the closed lid should open to release the product. It may be deduced, however, that the proposed mechanism looks like an enzyme inhibition by the product: The crystal structure reveals a conserved water molecule held in directional orientation towards the FAHD1 catalytic center by residues H30 and E33 presented in a short helix15, which is induced upon ligand binding and lid closure. If the primary enol would stay in an equilibrium with the enolate, the resonance stabilized enolate could be quenched to pyruvate by the water molecule. The resulting hydroxyl would be capable to displace the pyruvate from the Mg-cofactor upon which the lid would open. Finally, the catalytic center would be restored in the mitochondrial environment. In this hypothetic scenario, the cavity water molecule would operate as an acid, respectively.
Hydrolase Activity of FAHD1
Hydrolase activity of an enzyme implicitly requires the intermediate formation of a hydroxyl nucleophile. This mechanism is usually found in combination with acid-base catalytic activity. The transitional state of the reaction has to be prepared via conformational control by critical amino acid side chains in the cavity. In analogy to the discussion of the decarboxylase function, enzyme-bound acylpyruvate in 2-keto form will be put under conformational control by hydrogen-bonding of the 4-carbonyl oxygen to Q109 (Figure 9B, right panel). The crystal structure of oxalate-bound FAHD1 (PDB:6FOG) reveals a conserved water molecule held in directional orientation towards the FAHD1 catalytic center by residues H30 and E33 presented in a short helix15. The E33-H30 dyad is competent to deprotonate the directional positioned water and the resulting hydroxyl is in ideal disposition to attack the 4-carbonyl of acylpyruvate presented under conformational control by Q10915.
Of note, a similar mechanism has been proposed for FAH18. Attack by the hydroxyl nucleophile is expected to result in an oxyanion species, that is stabilized upon orbital controlled C3-C4 bond cleavage (Figure 9C). In this model, the C3-C4 bond rotation (Figure 9C) happens after the nucleophilic attack by the formed hydroxyl indicated in Figure 9B (i.e., it prepares the acylpyruvate for the bond cleavage). The primary products would be acetic acid and Mg-pyruvate enolate. In this hypothetic scenario, the acetic acid could quench the enol to pyruvate and subsequently assist displacement of the product. Above a pH of 7.5 and in the presence of Mg ions, acylpyruvates exist in an equilibrium between keto- and enol-forms, the latter in slight preference30. Most probably both forms are capable to bind to the cofactor of FAHD1 under subsequent lid closure. Processing of enolic acylpyruvate substrates by the enzyme is hampered due to the flat structure of the enol-form. The C3-C4 cleavage would result in a vinylic carbanion without resonance stabilization.
Therefore, we propose a catalytic ketonization step to prepare for attack of the hydroxyl nucleophile on the acyl carbonyl. This process of ketonization, however, would require control over proton transpositions by FAHD1 residues, which would attribute an inherent isomerase activity to FAHD1. It is reported that the acidity of Mg-bound enol hydrogen reveals a ten-thousand-fold increase compared to the un-complexed form28. A deprotonation of the Mg bound enol-form would be feasible by un-protonated K123. Deprotonation of K123 may be assisted by the carboxylate of D102. A hydrogen bond network formed by residues D102-K47-K123 could operate as the necessary proton relay in the catalytic center of FAHD115. A such-formed intermediate enolate could then be quenched by a E33-H30-H20 triad under ketonization of the substrate15. The 2-keto form would come under conformational control of Q109, and the concomitantly formed hydroxyl would attack the acyl carbonyl. The summarized discussion implies a control of FAHD1 about a water molecule for switching between acid and base through interplay of cavity-forming residues.
Future Applications or Directions of the Method
Future applications of the methods described here are numerous. A plethora of prokaryotic members of the FAH superfamily still awaits functional characterization. Even the available information on the catalytic activities of known FAH superfamily members is scarce and, in most cases, based on theoretical assumptions rather than experimental data. Application of the methods described here for prokaryotic FAH superfamily members depends on the specific research interests in bacteriology. On the other hand, the recent demonstration that eukaryotic FAH superfamily members play essential roles in various cellular compartments (e.g., cytosol vs. mitochondria) highlights the need to better characterize these proteins (three of which have been identified so far), in particular because current data suggest that some uncharacterized proteins may carry out different functions in the context of mitochondrial biology, aging research, and cancer research. It is proposed that the full molecular and physiological characterization of these eukaryotic FAH superfamily members may provide important insight into major fields of contemporary research in the biomedical sector. More research on the mechanisms of FAHD1 (and related enzymes) are needed to better understand mechanisms underlying the bi-functionality of FAHD1, which is still not fully clarified. Additional studies with FAHD1 mutants, NMR-investigations, and structural studies on inhibitor complexes may help resolve the true mechanistic scenarios for which FAHD1 seems to be competent. Furthermore, computer-aided design of enol mimics capable to bind to the Mg-cofactor will eventually lead to potent inhibitors of FAHD1.

The fumarylacetoacetate hydrolase (FAH)1,2 superfamily of enzymes describes a group of enzymes that share the highly conserved catalytic FAH domain3,4,5,6,7,8,9,10. Despite their common catalytic center, these enzymes are multi-functional, and most are found in prokaryotes, where they are used to break down compounds retrieved from complex carbon sources3. Only three members of this family were identified in eukaryotes so far: the name giving FAH2, as well as FAH domain-containing protein 1 (FAHD1)11,12,13,14,15 and FAH domain-containing protein 2 (FAHD2). Depletion of FAHD1 has been associated with impaired mitochondrial respiration13,16 and associated with a reversible type of cellular senescence phenotype14 that is linked to intermediate potential shortcomings in the electron transport system. Human FAHD1 and its orthologues in model systems (mouse, nematode, cancer cell lines, plants, etc.), as well as selected point mutation variants, have become druggable targets of potential interest. For this research, recombinant protein at high levels of purity, as well as information on catalytic mechanisms guided by crystal structures and selective antibodies are vital.
This manuscript describes methods for FAHD protein expression in E. coli, affinity chromatography, ion exchange chromatography, ammonium sulfate precipitation, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. The purpose of the methods and protocols described here is to provide guidance for scientists working in diverse fields such as bacteriology, plant biology, as well as animal and human studies, to characterize members of the FAH superfamily, including uncharacterized superfamily members should they become relevant in a particular field. The protocols described here may provide valuable support for projects aiming to characterize other prokaryotic or eukaryotic FAH superfamily members.
The rationale behind the methods described here is the fact that for characterization of poorly described proteins (in particular, metabolic enzymes of unknown physiological relevance), the approach to start with purified recombinant proteins allows the development of invaluable, high-quality research tools such as in vitro active enzyme preparations, high-quality antibodies, and potent and specific pharmacological inhibitors for selected enzymes. The described methods require fast protein liquid chromatography (FPLC) and X-ray crystallography. Alternative methods (e.g., to express protein without chemical induction, or to display protein purification by centrifugation after heat treatment followed by desalting and size exclusion chromatography), may be found elsewhere17. While a broader spectrum of methods is available for the expression and purification of FAH superfamily enzymes2,7,9,17,18, this work focuses on the expression and purification of FAHD proteins in particular.
In the discussion section of this manuscript, the catalytic mechanisms identified for the FAHD1 protein (hydrolase, decarboxylase)15 are described in more detail, in order to demonstrate the chemical character of the catalyzed reactions. The data obtained based on previous work7,15,18 (PDB: 6FOG, PDB:6FOH) imply a third activity of the enzyme as keto-enol isomerase.

<table>
	<tbody>
		<tr>
			<td>BL21(DE3) pLysS competent E. coli</td>
			<td>Promega</td>
			<td>L1195</td>
			<td>High-efficiency protein expression from gene with T7 promoter and ribosome binding site</td>
		</tr>
		<tr>
			<td>pET E. coli T7 Expression Vectors</td>
			<td>MERCK</td>
			<td>-</td>
			<td>http://www.merckmillipore.com/AT/de/life-science-research/genomic-analysis/dna-preparation-cloning/pet-expression-vectors/qFSb.qB.mLQAAAFA6.VkiQ0G,nav</td>
		</tr>
		<tr>
			<td>0.45 &#181;m filter units</td>
			<td>MERCK</td>
			<td>SLHP033NS</td>
			<td>Millex-HP, 0.45 &#181;m, PES 33 mm, not steril</td>
		</tr>
		<tr>
			<td>0.22 &#181;m filter units</td>
			<td>MERCK</td>
			<td>SLGP033RS</td>
			<td>Millex-HP, 0.22 &#181;m, PES 33 mm, not steril</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>15 mL Falcon</td>
			<td>VWR</td>
			<td>734-0451</td>
			<td>centrifugal tubes</td>
		</tr>
		<tr>
			<td>50 mL Falcon</td>
			<td>VWR</td>
			<td>734-0448</td>
			<td>centrifugal tubes</td>
		</tr>
		<tr>
			<td>PS Cuvettes Spectrophotometer Semi-Micro</td>
			<td>VWR</td>
			<td>30622-758</td>
			<td>VIS transparent cuvettes</td>
		</tr>
		<tr>
			<td>UV Cuvettes Spectrophotometer Semi-Micro</td>
			<td>VWR</td>
			<td>47727-024</td>
			<td>UV/VIS transparent cuvettes</td>
		</tr>
		<tr>
			<td>isopropyl-&#946;-D-thiogalactopyranosid (IPTG)</td>
			<td>ROTH</td>
			<td>2316</td>
			<td>chemical used for induction of protein expression with the DE3/pET system</td>
		</tr>
		<tr>
			<td>imidazole</td>
			<td>ROTH</td>
			<td>X998</td>
			<td>chemical used for elution of polyhistidine (6xHis) sequences from a nickel-charged affinity resin</td>
		</tr>
		<tr>
			<td>Glass Econo-Column Columns</td>
			<td>Bio-Rad</td>
			<td>-</td>
			<td>http://www.bio-rad.com/de-at/product/glass-econo-column-columns?ID=2cfb1c6e-32e8-4c72-b532-dd39013d707d&#38;pcp_loc=catprod</td>
		</tr>
		<tr>
			<td>chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378</td>
			<td>antibiotic for bacterial growth selection; resistance endi&#243;ded in pLysS plasmid of BL21(DE3) E. coli; 25 &#181;g/mL final concentration</td>
		</tr>
		<tr>
			<td>kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td align="right">60615</td>
			<td>antibiotic for bacterial growth selection; to be used if this resistance is encoded in the employed pET vector; 50 &#181;g/mL final concentration</td>
		</tr>
		<tr>
			<td>ampicillin</td>
			<td>Sigma-Aldrich</td>
			<td>A1593</td>
			<td>antibiotic for bacterial growth selection; to be used if this resistance is encoded in the employed pET vector; 100 &#181;g/mL final concentration</td>
		</tr>
		<tr>
			<td>Ultra-15, MWCO 10 kDa</td>
			<td>Sigma-Aldrich</td>
			<td>Z706345</td>
			<td>centrifigal filters for protein enrichment; https://www.sigmaaldrich.com/catalog/product/sigma/z706345?lang=de&#174;ion=AT</td>
		</tr>
		<tr>
			<td>Ultra-0.5 Centrifugal Filter Units</td>
			<td>Sigma-Aldrich</td>
			<td>Z677108</td>
			<td>centrifigal filters for protein enrichment; https://www.sigmaaldrich.com/catalog/product/ALDRICH/Z677108?lang=de&#38;region=AT&#38;cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold5-2</td>
		</tr>
		<tr>
			<td>oxaloacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>O4126</td>
			<td>TCA metabolite</td>
		</tr>
		<tr>
			<td>sodium oxlalate</td>
			<td>Sigma-Aldrich</td>
			<td>71800</td>
			<td>a competitive inhibitor of FAH superfamily enzymes</td>
		</tr>
		<tr>
			<td>Dialysis tubing cellulose membrane</td>
			<td>Sigma-Aldrich</td>
			<td>D9277</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sigma/d9277; or comparable</td>
		</tr>
		<tr>
			<td>Ni-NTA agarose</td>
			<td>Thermo-Fischer</td>
			<td>R90101</td>
			<td>a nickel-charged affinity resin that can be used to purify recombinant proteins containing a polyhistidine (6xHis) sequence</td>
		</tr>
		<tr>
			<td>96-Well UV Microplate</td>
			<td>Thermo-Fischer</td>
			<td align="right">8404</td>
			<td>UV/VIS transparent flat-bottom 96 well plates</td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder, 10 to 180 kDa</td>
			<td>Thermo-Fischer</td>
			<td>26616</td>
			<td>https://www.thermofisher.com/order/catalog/product/26616?SID=srch-hj-26616</td>
		</tr>
		<tr>
			<td>&#196;KTA FPLC system</td>
			<td>GE Healthcare Life Sciences</td>
			<td>-</td>
			<td>using the FPLC system by GE Healthcare; different custom versions exist; this work used the &#34;&#196;KTA pure&#34; system</td>
		</tr>
		<tr>
			<td>HiTrap Phenyl HP column</td>
			<td>GE Healthcare Life Sciences</td>
			<td>-</td>
			<td>https://www.gelifesciences.com/en/it/shop/chromatography/prepacked-columns/hydrophobic-interaction/hitrap-phenyl-hp-p-05630</td>
		</tr>
		<tr>
			<td>Mono S 10/100 GL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>-</td>
			<td>https://www.gelifesciences.com/en/ch/shop/chromatography/prepacked-columns/ion-exchange/mono-s-cation-exchange-chromatography-column-p-00723</td>
		</tr>
		<tr>
			<td>Mono Q 10/100 GL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>-</td>
			<td>https://www.gelifesciences.com/en/ch/shop/chromatography/prepacked-columns/ion-exchange/mono-q-anion-exchange-chromatography-column-p-00608</td>
		</tr>
		<tr>
			<td>HiLoad Superdex column 75 pg (G75)</td>
			<td>GE Healthcare Life Sciences</td>
			<td>-</td>
			<td>https://www.gelifesciences.com/en/ch/shop/chromatography/prepacked-columns/size-exclusion/hiload-superdex-75-pg-preparative-size-exclusion-chromatography-columns-p-05800</td>
		</tr>
		<tr>
			<td>HiLoad Superdex column 200 pg (G200)</td>
			<td>GE Healthcare Life Sciences</td>
			<td>-</td>
			<td>https://www.gelifesciences.com/en/ch/shop/chromatography/prepacked-columns/size-exclusion/hiload-superdex-200-pg-preparative-size-exclusion-chromatography-columns-p-06283</td>
		</tr>
		<tr>
			<td>TECAN microplate reader</td>
			<td>TECAN Life Sciences</td>
			<td>-</td>
			<td>https://lifesciences.tecan.com/microplate-readers</td>
		</tr>
		<tr>
			<td>acetylpyruvate</td>
			<td>MoleculeCrafting.HuGs e.U.</td>
			<td>-</td>
			<td>custom synthesis</td>
		</tr>
		<tr>
			<td>benzoylpyruvate</td>
			<td>MoleculeCrafting.HuGs e.U.</td>
			<td>-</td>
			<td>custom synthesis</td>
		</tr>
		<tr>
			<td>VDX&#8482; plate (24 wells)</td>
			<td>Hampton</td>
			<td>HR3-142</td>
			<td>24 well plates used for crystallization via Hanging Drop Vapor Diffusion</td>
		</tr>
		<tr>
			<td>paraffin oil</td>
			<td>Hampton</td>
			<td>HR3-411</td>
			<td>used for crystallization via Hanging Drop Vapor Diffusion</td>
		</tr>
		<tr>
			<td>coverslips (22 mm)</td>
			<td>Karl Hecht KG</td>
			<td>14043</td>
			<td>coverslips used for crystallization via Hanging Drop Vapor Diffusion</td>
		</tr>
		<tr>
			<td>Luria broth (LB) medium</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>a general growth medium for E. coli: 5 g/L yeast extract; 10 g/L peptone from casein; 10 g/L sodium chloride; 12 g/L agar-agar</td>
		</tr>
		<tr>
			<td>NZCYM medium</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>a better growth medium for E. coli, used for amplification: 10 g/L NZ amine; 5 g/L NaCl; 5 g/L yeast extract; 1 g/L casamino acids; 2 g/L MgSO4; adjust pH to 7.4</td>
		</tr>
		<tr>
			<td>Luria broth (LB) agarose plates</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>autoclaved agarose plates containing LB-medium and antibiotics for bacterial groth selection; https://www.addgene.org/protocols/pouring-lb-agar-plates/</td>
		</tr>
		<tr>
			<td>Ni-NTA running buffer</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>20 mM Tris-HCl pH 7,4; 50-300 mM NaCl; 10-200 mM imidazole; ranges: optimal value varies among FAHD proteins</td>
		</tr>
		<tr>
			<td>Ni-NTA elution buffer</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>20 mM Tris-HCl pH 7,4; 50-300 mM NaCl; 200-500 mM imidazole; ranges: optimal value varies among FAHD proteins</td>
		</tr>
		<tr>
			<td>HIC running buffer</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>44 mM NaH2PO4; 6 mM Na2HPO4; 100 mM NaCl; 20 mM DTT; adjust to pH 7</td>
		</tr>
		<tr>
			<td>HIC running buffer AS</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>HIC running buffer saturated with ammonium sulfate (AS); adjust to pH 7: 70 g ammonium sulfate + 90 mL buffer, stirred overnight in the cold room; adjust to pH 7.0</td>
		</tr>
		<tr>
			<td>Mono S low salt buffer</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>44 mM NaH2PO4; 6 mM Na2HPO4; 10-300 mM NaCl; ranges: optimal value varies among FAHD proteins</td>
		</tr>
		<tr>
			<td>Mono S high salt buffer</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>44 mM NaH2PO4; 6 mM Na2HPO4; 1-2 M NaCl; ranges: optimal value varies among FAHD proteins</td>
		</tr>
		<tr>
			<td>Mono Q low salt buffer</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>20 mM Tris-HCl; 15 mM NaCl; adjust to pH 8.0</td>
		</tr>
		<tr>
			<td>Mono Q high salt buffer</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>20 mM Tris-HCl; 1 M NaCl; 10 % glycerol; adjust to pH 8.0</td>
		</tr>
		<tr>
			<td>G75 / G200 running buffer</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>15 mM Tris-HCl; 300 mM NaCl; adjust to pH 7.4</td>
		</tr>
		<tr>
			<td>enzyme assay buffer</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>50 mM Tris-HCl pH7.4; 100 mM KCl; 1 mM MgCl2</td>
		</tr>
		<tr>
			<td>protein crystallization buffer</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>G75 / G200 running buffer with 1 mM DTT</td>
		</tr>
		<tr>
			<td>reservoir solution for crystallization</td>
			<td>self-prepared</td>
			<td>-</td>
			<td>100 mM Na-HEPES pH 7.5; 5-20 % (w/v) PEG4k; 10 mM-200 mM MgCl2</td>
		</tr>
	</tbody>
</table>

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.

Starting with a prepared cloning vector and purchased BL21(DE3) pLysS E. coli, the plasmid is inserted into the bacteria via heat-shock or any appropriate alternative method (Figure 1). After a short period of amplification, the transformed bacteria are plated on LB agar plates, in order to grow overnight. Plates at this point may look different, depending on a variety of potential error sources. Plates may be empty (i.e., no colonies), completely overgrown by bacteria, or something in between, respectively. Two examples of LB agar plates after optimal and non-optimal transformation are depicted in Figure 7A. Too many bacterial colonies indicate either that too many bacteria were plated (likely) or that the antibiotics in use may be expired (unlikely). Too few bacterial colonies may indicate that either not enough plasmid was used for the transformation (use more next time) or that too much antibiotics were used to select the bacteria. In any case, if colonies are present, they should be fine, as using two selective antibiotics implies a rather insignificant chance of untransformed bacteria to grow. No colonies at all, however, indicates that either the bacteria lost their transformation competence (because of wrong storage or storage over longer periods, repetitive freeze and thaw, etc.), the heat-shock was not successful (no plasmid uptake or bacterial death by too much heat), the cloning vector is corrupted, or by mistake a wrong set of selective antibiotics was used (verify the resistance gene on the plasmid vector).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59729/59729fig07v2.jpg" /> 
Figure 7: Representative results for bacteria transformation and IMAC. 
(A) Representative LB agar plates with transformed BL21(DE3) E. coli, obtained by following protocol step 1.1. Left: A plate with well distributed colonies (positive example). Right: A plate with only one single colony (negative example). White circles mark good colonies. The red circle marks colonies that are growing too close to each other and should not be picked as long as isolated colonies are available. (B) A 12.5% acrylamide SDS-PAGE analysis of a series of induction controls (&#8220;-&#8220; indicates before IPTG induction; &#8220;+&#8221; indicates after IPTG induction, before pellet harvest), adjusted to equal amounts of total protein. This is described in step 1.2. (C) An exemplary 12.5% acrylamide SDS-PAGE analysis of Ni-NTA purification of His-tagged FAHD1 protein. This is described in section 3 of the protocol. The affinity chromatography yields protein of high purity (&#62;70%, black arrow), however, several small contaminations are also observed (red arrows). These contaminations consist of non-FAHD proteins that bind to the column, and from proteins that bind to the FAHD protein. <a href="https://www.jove.com/files/ftp_upload/59729/59729fig07v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Validated colonies are selected and picked. After amplification in nourishing medium, protein expression is triggered by application of the chemical IPTG. The bacterial pellet containing the expressed protein in milligram quantities is harvested, and expression is verified via SDS-PAGE (see for example Figure 7B). Some problems may occur during this otherwise simple process. First, some proteins form inclusion bodies, because they apparently somehow interfere with the natural metabolism of the host bacteria. This was observed for some point mutations of human FAHD1 and FAHD2. In such cases, other expression systems like insect cells may be more appropriate and should be considered. After harvesting a pellet from insect cells, for example, purification of the proteins follows the same steps as described in this protocol. Second, the DE3-pET system is sometimes found to be &#8220;leaky&#8221; (i.e., protein is already expressed to some extent before IPTG induction). The potential reason for this is not well-understood, but it may help to express the protein slowly overnight in a cold room incubator. Third, no protein is expressed. This is probably the worst-case scenario, as it likely indicates a corrupted plasmid vector and thus advisable to sequence the plasmid.
If a His-tag was used to tag the protein, affinity chromatography with Ni-NTA agarose is an easy and cheap capture method eliminating the majority of contaminations (Figure 7C). Similar methods exist for other tag systems (e.g., STREP-II). If no tag was used, a combination of ammonium sulfate precipitation and consecutive hydrophobic exchange chromatography may also separate the protein from the majority of other proteins (Figure 8A). However, comparing the two methods (Figure 7C vs. Figure 8A), the superiority of the Ni-NTA methods can be demonstrated by SDS-PAGE analysis. Using His-tagged protein is therefore advised.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59729/59729fig08v2.jpg" /> 
Figure 8: Representative results for FPLC experiments (HIC, ion exchange, SEC). 
(A) A typical chromatogram and 12.5% acrylamide SDS-PAGE analysis of HIC-phenyl chromatography after ammonium sulfate (AS) precipitation of untagged FAHD1 protein, as described in section 4 of the protocol. The green line reflects the gradient of buffer B that does not contain AS. During the process AS is gradually washed out from the system. Comparing this panel to Figure 7C displays the power of Ni-NTA affinity chromatography compared to the HIC-phenyl method, and the advantage of using a His-tag system for protein purification. (B) An exemplary chromatogram and 12.5% acrylamide SDS-PAGE analysis of cationic exchange chromatography of His-tagged FAHD following Ni-NTA purification. Using a salt gradient, the applied sample is separated into individual proteins. (C) An exemplary chromatogram and 12.5% acrylamide SDS-PAGE analysis of G75 size exclusion chromatography of His-tagged FAHD following cationic exchange chromatography. <a href="https://www.jove.com/files/ftp_upload/59729/59729fig08v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Consecutively, the protein is further separated from leftover contaminations by cation/anion exchange chromatography (for example, see Figure 8B), followed by size-exclusion chromatography (for example, see Figure 8C). It is advised to set up an initial purification strategy in this order; however, these columns should be used in combination, subsequently and in variation, until the protein is sufficiently pure.
Simple activity assays, in order to test for &#8220;yes or no&#8221; decisions on active substrates and/or cofactors, may be performed with His-tagged proteins after Ni-NTA purification, or untagged proteins after the ionic exchange column. Specific activities and kinetic constants must be determined with protein of highest purity. Crystallization may be attempted with proteins after the ionic exchange column, but the quality of crystals almost always correlates with protein purity. Polyclonal antibodies may be raised against proteins at any stage of the purification protocol; however, here the quality also correlates with the protein purity.

Watch this Scientific Journal Video about Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins at JoVE.com

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

Fumarylacetotate 하이드로 아세테이트 도메인 함유 단백질의 발현, 정제, 결정화 및 효소 어세포

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 ...

expression-purification-crystallization-enzyme-assays

Research

JoVE Journal

Biochemistry

23.9K 조회수.  University of Innsbruck Austria. 당사의 프로토콜은 단백질뿐만 아니라 일반적으로 단백질을 함유한 FAH 도메인의 효율적인 발현 및 정제 방법을 설명합니다. FHD 단백질-1은 미토콘드리아의 TCA 주기 및 에너지 대사에 있는 규제 역할을 합니다. 우리는 세포 노화에 FHD-1 다운 조절을 연결할 수 있었고 효소 활성 및 단백질 구조에 대한 정보는 일반적으로 관찰 된 표현형 뒤에 분자 메커니즘을 이해하기 위해 요?...

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

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

항온, 발현, 정제, 인간 세로토닌 전달체 결정화 바운드<em&gt; S</em&gt; 시탈 로프 람

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

연구

생화학

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

습식 및 건식 연구소의 기술을 결합하면 대형 코일 코일 포함하는 단백질의 결정화를 안내합니다

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis is an obligate pathogen that causes fatal inhalational anthrax. Bacillus cereus is considered a useful model for studying B. anthracis due to its close evolutionary relationship. The gene cluster ba1554 - ba1558 of B. anthracis is highly conserved with the bc1531- bc1535 cluster in B. cereus, as well as with the bt1364-bt1368 cluster in Bacillus thuringiensis, indicating the critical role of the associated genes in the Bacillus genus. This manuscript describes methods to prepare and characterize a protein product of the first gene (ba1554) from the gene cluster in B. anthracis using a recombinant protein of its ortholog in B. cereus, bc1531.

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Bacillus anthracis is the obligate pathogen of fatal inhalational anthrax, so studies of its genes and proteins are strictly regulated. An alternative approach is to study orthologous genes. We describe biophysicochemical studies of a B. anthracis ortholog in B. cereus, bc1531, requiring minimal experimental equipment and lacking serious safety concerns.

재조합 단백질 발현, 결정화 및 생물 물리학 연구<em&gt; 바실러스</em&gt; 보존 된 뉴클레오티드 피로 인산화 효소, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

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.

표현과 포유류 Bestrophin 이온 채널의 정화

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, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

표현, 가용 화, 그리고 진 핵 Borate 전송기의 정화

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

뉴클레아제 없는 산소 제거제 프로토카테의 발현 및 정제 3,4-디옥시게나아제

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

PCR 돌연변이 발생, 복제, 발현, 빠른 단백질 정화 프로토콜 및 트립토판 신타제의 야생 유형 및 돌연변이 형태의 결정화

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

박테리아 발현 및 친화성 크로마토그래피를 이용한 인간 매트릭스 메탈로프로테이나제-3의 정제

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

포유류 세포에서 유비퀴틴화된 p53 단백질의 정제

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...