This research was funded by the US Department of Defense in the form of an Idea Award (W81XWH-13-1-0174), Aplastic Anemia &amp; MDS Foundation Grant, and UMRB grant to M.M. Authors thank Mohit Jaiswal and Subhradeep Bhar for initial cloning of TET2 in pDEST14 vector.

1. Cloning and Purification of Untagged Human TET2 Dioxygenase
<ol>
	<li>Clone human TET2 dioxygenase (TET2 1129-1936, &#916;1481-1843) into the pDEST14 destination vector using the site-specific recombination technique as previously described22. 
	NOTE: Previous studies have demonstrated that the C-terminal TET2 dioxygenase (TET2 1129-1936, &#916;1481-1843) domain is the minimal catalytically active domain21,23. In order to express the untagged TET2 dioxygenase domain using the pDONR221 vector, Shine Dalgarno and Kozak sequences were incorporated in the forward primer during PCR (Table 1).</li>
	<li>For bacterial transformation, add 1 &#181;L of recombinant pDEST14 expression vector containing untagged human TET2 dioxygenase (TET2 1129-1936, &#916;1481-1843) to 100 &#181;L of chemically competent E. coli BL21 (DE3) cells in a 1.7 mL tube. Keep the mixture on ice for at least 15 minutes followed by heat shock at 42 &#176;C for 30 s in a water bath.
	<ol>
		<li>Immediately after heat shock, keep the cells back on ice for minimum of 2 min. Following this, add 250 &#181;L of super optimal broth with catabolite repression (S.O.C media) to cells. Incubate the bacterial cells for 1 h at 37 &#176;C in a shaker.</li>
		<li>After incubation, spin down the cells by centrifuging the tube at 9,000 x g for 1 min. Discard 70% supernatant by pipetting and dissolve the pellet in left over media.</li>
		<li>Spread the cell suspension on a Luria broth (LB) agar plate containing 100 &#181;g/mL ampicillin. Incubate the plate for 16 h at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Select one isolated colony and inoculate it into 10 mL of LB-ampicillin media in a 50 mL tube. Incubate the tube at 37 &#176;C in a shaker for overnight. Following this, use 100 &#181;L&#160; of culture from the 50 mL tube to inoculate 100 mL of LB-ampicillin media. Incubate the flask at 37 &#176;C on a shaker at 180 rpm and use it as primary culture. Next day, use 6 mL each of primary culture to inoculate 15 flasks, each containing 600 mL LB-ampicillin media. Now, incubate 15 flasks at 37 &#176;C on a shaker at 180 rpm. 
	NOTE: For verifying transformed clones, perform DNA sequencing or restriction digestion with the isolated plasmid DNA.</li>
	<li>To check the density of bacterial culture, measure its OD600 using a spectrophotometer. After the culture reaches a density of 0.8 at OD600, induce the expression of TET2 protein with 300 &#181;L of 1 M (final concentration of 0.5 mM in 600 mL) IPTG in each flask and further grow the culture for 16 h at 17 &#176;C.</li>
	<li>After 16 h, transfer the bacterial culture to centrifuge bottles. Centrifuge the bacterial culture expressing the TET2 enzyme at 5,250 x g for 45 min. Use the bacterial pellet for TET2 purification. 
	NOTE: Perform all remaining protein purification steps either on the ice or at 4 &#176;C.</li>
	<li>Resuspend the bacterial pellet in 100 mL of 50 mM MES (2-(N-morpholino)ethanesulfonic acid) buffer, pH 6 and sonicate for 5 &#215; 30 s at power 20 with 60 s cooling intervals.</li>
	<li>Spin the lysate at 5,250 x g for 45 min. Collect the supernatant containing the soluble TET2 enzyme and pass through 0.45-&#181;m filters before loading on an FPLC system. 
	NOTE: In these experiments, the TET2 enzyme was very stable, but if needed a protease inhibitor cocktail containing 1 mM benzamidine-HCl, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 0.5 mM 1,10-o-phenanthroline can be added to the cell lysate to prevent degradation of TET2 enzyme. Avoid using EDTA or EGTA in the inhibitor cocktail as these may interfere with the following cation-exchange chromatography.</li>
	<li>Pack 30 mL of a strong cation exchange resin into a FPLC column. Equilibrate the column with 10 bed volumes of wash buffer (50 mM MES buffer, pH 6) at the constant flow rate of 0.3 mL/min using a FPLC system.</li>
	<li>Load the clarified lysate onto the pre-equilibrated column and wash with &#8764;10 bed volumes of wash buffer until the flow through becomes clear.</li>
	<li>Elute TET2 using a 0-100% gradient from the wash buffer to the elution buffer (50 mM MES buffer, pH 6, 1 M NaCl) in 15 bed volumes followed by holding at 100% elution buffer for two-bed volumes. 
	NOTE: Collect samples (100 &#181;L each) of cell lysate before and after column loading along with all the elution fractions and analyze on 10% resolving SDS-PAGE.</li>
	<li>Pool the fractions containing TET2 protein, freeze dry, dissolve enzyme pallet in 10 mL water and store at -80 &#176;C.</li>
</ol>
2. 5mC Oxidation by TET2 Dioxygenase
<ol>
	<li>Perform all demethylation reactions in triplicate with 3 &#181;g of substrate (25-mer double stranded DNA, Table 2).
	<ol>
		<li>Add 100 &#181;g of purified TET2 enzyme in 50 &#181;L of total reaction buffer containing 50 mM HEPES (pH 8.0), 200 &#181;M FeSO4, 2 mM 2OG (2-oxoglutarate/&#945;-ketoglutarate), and 2 mM ascorbate and incubate at 37 &#176;C for 1 h21.</li>
		<li>Quench TET2 catalyzed oxidation reactions with 5 &#181;L of 500 mM EDTA.</li>
	</ol>
	</li>
	<li>After quenching TET2 reactions, prepare samples for LC-MS/MS analysis by separating the DNA from TET2 reaction mixture using oligo purification columns.
	<ol>
		<li>Add 100 &#181;L of oligo binding buffer to 55 &#181;L of quenched reaction.</li>
		<li>Following this, add 400 &#181;L of 100% ethanol to the mixture. Pass this mixture through an oligo binding column.</li>
		<li>Wash bound DNA with 750 &#181;L wash buffer and elute in 20 &#181;L water.</li>
	</ol>
	</li>
	<li>Digest the isolated DNA with 2 units of DNase I and 60 units of S1 nuclease at 37 &#176;C for 12 h to produce individual nucleoside-monophosphates.</li>
	<li>Following digestion, add 2 units of calf intestinal alkaline phosphatase (CIAP) in the samples and incubate for addition 12 h at 37 &#176;C to remove the terminal phosphate groups from nucleoside-monophosphates to obtain nucleosides.</li>
	<li>Quantify all nucleosides, especially modified cytosines, using the LC-MS/MS method described below.</li>
</ol>
3. Quantitative LC-MS/MS-based Assay Development
<ol>
	<li>Prepare 100 &#181;M stock solution of all modified cytosine nucleosides [5-methyl-2&#8242;-deoxycytidine (5mdC), 5-hydroxymethyl-2&#8242;-deoxycytidine (5hmdC), 5-formyl-2&#8242;-deoxycytidine (5fdC), and 5-carboxy-2&#8242;-deoxycytidine (5cadC)] and normal DNA bases (adenine, thymine, cytosine, and guanine) in HPLC-grade water for the development of the LC-MS/MS method.</li>
	<li>Optimize nucleoside-dependent MS/MS parameters by infusing stock solutions, one at a time, in mass spectrometer at a flow rate of 10 &#181;L/min in EMS scan mode. Optimize following parameters: Declustering Potential (DP), Entrance Potential (EP), Collision Cell Entrance Potential (CEP), Collision Energy (CE), and Collision Cell Exit Potential (CXP) for each DNA nucleoside using automated quantitative optimization feature of the software.</li>
	<li>Optimize source-dependent MS/MS parameters by injecting 10 &#181;L of stock solution using a gradient with 25% solvent B at a flow rate of 0.3 mL/min where solvent A is 10 mM ammonium acetate (pH 4.0) and solvent B is 20% acetonitrile with 10 mM ammonium acetate (pH 4.0). Optimize following parameters: Curtain Gas (CUR): 10-50, Temperature: 0-600 &#176;C, Gas Flow 1 (GS1): 0-50, Gas Flow 2 (GS2): 0-50, Collisionally Activated Dissociation (CAD): Low-Medium-High, Ion spray Voltage (IS): 4000-5500 for each DNA nucleoside using manual quantitative optimization feature of the software in FIA (Flow Injection Analysis) mode.</li>
	<li>To separate all eight DNA nucleosides, perform liquid chromatography using the following gradient: 0% solvent B (0-2 min), 0-20% solvent B (2-5 min), 20-60% solvent B (5-9 min), 60-0% solvent B (9-10 min) and then equilibrate with solvent A for 5 min at a flow rate of 0.3 mL/min on a C18 column (Particle Size: 5 &#181;m, Pore Size: 120 &#197;).</li>
	<li>Using the optimum MS/MS parameters (step 3.2 and 3.3) coupled with the above-mentioned liquid chromatography gradient (step 3.4), determine the response linearity, limit of detection (LOD), and lower limit of quantification (LLOQ) using a two-fold serial dilution of a 100 &#181;M standard mixture containing all eight nucleosides. Draw standard curves for all eight DNA nucleosides.</li>
	<li>Detect and quantify all nucleosides, especially modified cytosines, produced in step 2.4 using the LC-MS/MS method and standard curves.</li>
</ol>

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The authors have no financial interests to declare.

Mutations in TET2 gene are some of the most frequently detected genetic changes in patients with diverse hematopoietic malignancies. To date hundreds of different TET2 mutations, which include nonsense, frame-shift, and missense mutations, have been identified in patients12. Patients with TET2 mutations show low levels of genomic 5hmC in the bone marrow compared to those with wt-TET214. Mutant TET2 knock-in experiments have recapitulated the effects of the...

The C5 position of cytosine bases within CpG dinucleotides is the predominant methylation site (5mCpG) in mammalian genomes1. In addition, a number of recent studies have uncovered extensive C5 cytosine methylation (5mC) in non-CpG sites (5mCpH, where H = A, T, or C)2,3. 5mC modification serves as a transcriptional silencer at endogenous retrotransposons and gene promoters3,4,5. DNA methylation at 5mC also plays important roles in X chromosome inactivation, gene imprinting, nuclear reprogramming and tissue-specific gene expression5,6,7. Methylation of cytosine at the C5 position is carried out by DNA methyltransferases, and mutations in these enzymes cause significant developmental defects8. The removal of 5mC marks are initiated by TET1-3 5mC oxidases9,10. These TET-family dioxygenases convert 5mC into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by sequential oxidation steps11,12,13. Finally, thymine-DNA glycosylase replaces 5fC or 5caC to unmodified cytosine using the base excision repair pathway11.
The human TET2 gene was identified as a frequently mutated gene in diverse hematopoietic malignancies including myelodysplastic syndromes (MDS)14,15,16, MDS-myeloproliferative neoplasms (MDS-MPN), and acute myeloid leukemia (AML) originating from MDS and MDS-MPN16. The levels of 5hmC modification in the bone marrow DNA are lower in patients with TET2 mutations compared to those with wild type (wt)-TET214. A number of groups have developed TET2-knockout mouse models to elucidate its role in normal hematopoiesis and myeloid transformation17,18,19,20. These mice with mutations in the TET2 gene were initially normal and viable, but manifested diverse hematopoietic malignancies as they aged causing their early death. These studies showed the important roles played by the wt-TET2 in normal hematopoietic differentiation. In these mouse models, the heterozygous hematopoietic stem cells (TET2+/- HSCs) and homozygous TET2-/-&#160;HSCs had a competitive advantage over homozygous wt-TET2 HSCs in repopulating hematopoietic lineages as both TET2+/- and TET2-/-&#160;HSCs developed diverse hematopoietic malignancies17,18. These studies demonstrate that haploinsufficiency of TET2 dioxygenase alters the development of HSCs and results in hematopoietic malignancies.
Similar to mice with mutations in the TET2 gene, most leukemia patients manifest haploinsufficiency of TET2 dioxygenase activity. These mostly heterozygous somatic mutations include frame-shift and nonsense mutations dispersed throughout the TET2 gene body while missense mutations that are most clustered in the dioxygenase domain12. To date, little characterization of wt- and mutant-TET2 is reported in the literature mainly due to difficulties with the production of TET2 dioxygenase and its assay21. Here, we report a simple single-step purification of native TET2 dioxygenase using ion exchange chromatography. Further, a quantitative LC-MS/MS assay was optimized and used to measure the enzymatic activity of native TET2 dioxygenase.

<table border="0" cellpadding="0" cellspacing="0" style="width:739px;" width="739">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">HEPES</td>
			<td style="width:201px;">Carbosynth</td>
			<td style="width:119px;">FH31182</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Iron(II) sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>F8633</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">&#945;-Ketoglutaric acid (2-Oxoglutaric acid)</td>
			<td>Sigma-Aldrich</td>
			<td>K1750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">L-Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
			<td style="width:201px;">Sigma-Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Ammonium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>A1542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">HPLC grade water</td>
			<td>Fisher Scientific</td>
			<td>7732-18-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Oligo clean and concentrator</td>
			<td>Zymo Research</td>
			<td>D4061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">DNAse I</td>
			<td>New England Biolabs</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">S1 Nuclease</td>
			<td>Thermo Scientific</td>
			<td>ENO321</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">CIAP (Calf intestinal alkaline phosphatase)</td>
			<td>New England Biolabs</td>
			<td>M0290S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">LB Media</td>
			<td>Affymetrix</td>
			<td>J75852</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">IPTG</td>
			<td>Carbosynth</td>
			<td>EI05931</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">MES [2-(N-Morpholino)ethanesulfonic acid monohydrate]</td>
			<td style="width:201px;">Carbosynth</td>
			<td>FM37015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G7893</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">SP Sepharose</td>
			<td>Fisher Scientific</td>
			<td>45-002-934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxy-5-methylcytidine</td>
			<td>TCI</td>
			<td>D3610</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxy-5-hydroxymethyalcytidine</td>
			<td>TCI</td>
			<td>D4220</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:252px;">2&#39;-Deoxycytidine-5-carboxylic acid, sodium salt</td>
			<td>Berry &#38; Associates</td>
			<td>PY 7593</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">5-Formyl-2&#39;-deoxycytidine</td>
			<td>Berry &#38; Associates</td>
			<td>PY 7589</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxycytidine</td>
			<td>Berry &#38; Associates</td>
			<td>PY 7216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxyadenosine</td>
			<td>Carbosynth</td>
			<td>ND04011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxyguanosine</td>
			<td>Carbosynth</td>
			<td>ND06306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">2&#39;-Deoxythymidine</td>
			<td>VWR Life Science</td>
			<td>97061-764</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Gateway technology</td>
			<td style="width:201px;">Thermo Fisher</td>
			<td style="width:119px;">11801016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:252px;">Beckman Allegra X-15R centrifuge&#160;</td>
			<td style="width:201px;">Beckman Coulter</td>
			<td style="width:119px;">392932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Sonic Dismembrator 550</td>
			<td style="width:201px;">Fisher Scientific</td>
			<td style="width:119px;">XL2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">&#196;KTA FPLC system</td>
			<td style="width:201px;">Pharmacia (GE Healthcare)</td>
			<td style="width:119px;">18116468</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">FreeZone 4.5 freeze dry system</td>
			<td style="width:201px;">Labconco</td>
			<td style="width:119px;">7750020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Zymo Oligo purification columns&#160;</td>
			<td style="width:201px;">Zymo Research</td>
			<td style="width:119px;">D4061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">BDS Hypersil C18 column</td>
			<td style="width:201px;">Keystone Scientific, INC</td>
			<td style="width:119px;">105-46-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">3200 Q-Trap mass spectrometer</td>
			<td style="width:201px;">AB Sciex</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">HPLC&#160;</td>
			<td style="width:201px;">Shimadzu HPLC&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">XK16/20 FPLC column</td>
			<td style="width:201px;">Pharmacia (GE Healthcare)</td>
			<td style="width:119px;">28988937</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient purification and lc-ms/ms-based assay development for ten-eleven translocation-2 5-methylcytosine dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Dynamic modification of 5mC in DNA by TET-family dioxygenases plays important roles in epigenetic transcriptional regulations. TET2 dioxygenase is frequently mutated in diverse hematopoietic malignancies12. To investigate the role of the TET2 enzyme in normal development and disease, we have cloned its minimal catalytically active domain without any affinity tag into the pDEST14 vector22. The untagged TET2 dioxygenase was produced at &#8764;...

Watch this Scientific Journal Video about Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase at JoVE.com

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

efficient-purification-lc-msms-based-assay-development-for-ten-eleven

Research

JoVE Journal

Biochemistry

8.1K Views.  University of Missouri-Kansas City. Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

Video: Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

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

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

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

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

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

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.

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

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 ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...