The research presented here was supported by the Pennsylvania Department of Health, The Ellison Medical, The V and The Emerald Foundations.

1. Expression of recombinant TcTERT
<ol>
<li>Inoculate 6 L of 2YT media (6 x 2 L Baffled Erlenmeyer Flasks containing 1 L of 2YT broth in each) from six fresh plates of transformed Rosetta(DE3)pLysS cells containing the synthetic TcTERT plasmid with a TEV cleavable N-terminal hexahistidine-tag.</li>
<li>Grow cells at 37&deg;C while shaking at 220 rpm to an OD600 of 0.5-0.6.</li>
<li>Induce protein expression by adding 1 mM IPTG.</li>
<li>Reduce temperature to 30&deg;C, slow shaking to 150 r...

<ol>
	<li>Gillis, A. J., Schuller, A. P., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+Tribolium+castaneum+telomerase+catalytic+subunit+TERT.">Structure of the Tribolium castaneum telomerase catalytic subunit TERT.</a> Nature. 455, 633-637 (2008).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+specific+telomere+terminal+transferase+activity+in+Tetrahymena+extracts.">Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.</a> Cell. 43, 405-413 (1985).</li><li>Harley, C. B., Futcher, A. B., Greider, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres+shorten+during+ageing+of+human+fibroblasts.">Telomeres shorten during ageing of human fibroblasts.</a> Nature. 345, 458-460 (1990).</li><li>Hayflick, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Limited+in+vitro+Lifetime+of+Human+Diploid+Cell+Strains.">The Limited in vitro Lifetime of Human Diploid Cell Strains.</a> Exp Cell Res. 37, 614-636 (1965).</li><li>Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeres:+no+end+in+sight.">Telomeres: no end in sight.</a> Cell. 77, 621-623 (1994).</li><li>Kim, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+association+of+human+telomerase+activity+with+immortal+cells+and+cancer.">Specific association of human telomerase activity with immortal cells and cancer.</a> Science. 266, 2011-2015 (1994).</li><li>Bodnar, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+of+life-span+by+introduction+of+telomerase+into+normal+human+cells.">Extension of life-span by introduction of telomerase into normal human cells.</a> Science. 279, 349-352 (1998).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telomere+terminal+transferase+of+Tetrahymena+is+a+ribonucleoprotein+enzyme+with+two+kinds+of+primer+specificity.">The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity.</a> Cell. 51, 887-898 (1987).</li><li>Greider, C. W., Blackburn, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+telomeric+sequence+in+the+RNA+of+Tetrahymena+telomerase+required+for+telomere+repeat+synthesis.">A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis.</a> Nature. 337, 331-337 (1989).</li><li>Jacobs, S. A., Podell, E. R., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+essential+N-terminal+domain+of+telomerase+reverse+transcriptase.">Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase.</a> Nat Struct Mol Biol. 13, 218-225 (2006).</li><li>Rouda, S., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+RNA-binding+domain+of+telomerase:+implications+for+RNA+recognition+and+binding.">Structure of the RNA-binding domain of telomerase: implications for RNA recognition and binding.</a> Structure. 15, 1403-1412 (2007).</li><li>Mitchell, M., Gillis, A., Futahashi, M., Fujiwara, H., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+telomerase+catalytic+subunit+TERT+binding+to+RNA+template+and+telomeric+DNA.">Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA.</a> Nat Struct Mol Biol. 17, 513-518 (2010).</li></ol>

The method presented here allows for the production of large amounts of the catalytic subunit of T. castaneum telomerase TERT in soluble, active form for structural and biochemical studies. The method of TcTERT over-expression is sensitive to subtle changes in temperature or cell density from those stated above and can dramatically affect the levels of protein expression. Specifically, we have found that inducing the cells for protein expression before the optical density reaches 0.5 at 600 nm, or lowe...

<table border="1">
 <tbody>
 <tr>
 <th>Name of Reagent</th>
 <th>Company</th>
 <th>Catalog Number</th>
 </tr>
 <tr>
 <td>Rosetta(DE3)plysS Cells</td>
 <td>Novagen</td>
 <td>70956</td>
 </tr>
 <tr>
 <td>2YT Broth</td>
 <td>Teknova</td>
 <td>Y0215</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Gold Biotechnology</td>
 <td>I2481C</td>
 </tr>
 <tr>
 <td>MISONIX Sonicator 3000</td>
 <td>Qsonica, LLC.</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&Auml;KTA Purifier FPLC</td>
 <td>GE Life Sciences </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ni-NTA Superflow Resin</td>
 <td>Qiagen</td>
 <td>30410</td>
 </tr>
 <tr>
 <td>Amicon Ultra-15 Centrifugal Filter Device</td>
 <td>Millipore</td>
 <td>UFC903008</td>
 </tr>
 <tr>
 <td>POROS 50 HS Strong Cation Exchange Packing</td>
 <td>Applied Biosystems</td>
 <td>1-3359-06</td>
 </tr>
 <tr>
 <td>POROS 50 HQ Strong Cation Exchange Packing</td>
 <td>Applied Biosystems</td>
 <td>1-2559-06</td>
 </tr>
 <tr>
 <td>Superdex 200 10/300 Size-Exclusion Column</td>
 <td>GE Life Sciences</td>
 <td>17-5175-01</td>
 </tr>
 <tr>
 <td>Phenol: Chloroform: Isoamyl 25:24:1 with 10mM Tris, pH 8, 1mM EDTA</td>
 <td>Sigma</td>
 <td>P3803-100mL</td>
 </tr>
 <tr>
 <td>RNaseZap</td>
 <td>Ambion</td>
 <td>AM9780</td>
 </tr>
 <tr>
 <td>Recombinant Rnasin Ribonuclease Inhibitor</td>
 <td>Promega</td>
 <td>N251B</td>
 </tr>
 <tr>
 <td>RNeasy Mini Kit</td>
 <td>Qiagen</td>
 <td>74104</td>
 </tr>
 <tr>
 <td>DNA oligonucleotides</td>
 <td>Integrated DNA Technologies</td>
 <td>&nbsp;</td>
 </tr>
 
 </tbody>
</table>

in vitro reconstitution of the active t. castaneum telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

Watch this Scientific Journal Video about In vitro Reconstitution of the Active T. castaneum Telomerase at JoVE.com

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

in-vitro-reconstitution-of-the-active-t-castaneum-telomerase

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Biology

11.6K visualizações.  University of Pennsylvania. Esforços para isolar a subunidade catalítica da telomerase, TERT, em quantidades suficientes para estudos estruturais, foram cumpridos com sucesso limitado por mais de uma década. Aqui, apresentamos métodos para o isolamento do recombinante Tribolium castaneum TERT ( Tc TERT) ea reconstituição do ativo T. castaneum Ribonucleoproteína telomerase (RNP) complexo In vitro.