Sample Preparation of Telomerase Subunits for Crystallization
用于结晶化的端粒酶亚基样品的制备
发布: 2015年08月20日第5卷第16期 DOI: 10.21769/BioProtoc.1565 浏览次数: 8342
评审: Arsalan DaudiAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2014
Abstract
Telomerase is a large ribonucleoprotein complex that replicates the linear chromosome ends in most eukaryotes. Large-scale preparation of the telomerase core components in vitro has long been a big challenge in this field, hindering the understanding of the catalytic mechanism of telomerase, as well as slowing down the development of telomerase inhibitors for cancer therapy. We have successfully developed a protocol for large-scale preparation of the TRBD-CR4/5 complex of the medaka telomerase in vitro, and used this method to study the high-resolution structure of the TRBD-CR4/5 complex by X-ray crystallography. This procedure may be also adapted to purify other protein-RNA complexes for structural studies.
Materials and Reagents
- pMAL-C2X vector (New England Biolabs, catalog number: N8076S ) with an insertion of the protease 3C recognition site between MBP and the fusion protein
- Escherichia coli (E. coli) strain ScarabXpress T7lac cells (Scarab Genomics, catalog number: C-1709-05K )
- LB (Luria-Bertani) medium (BD Bioscience, catalog number: 214906 ) and LB agar (BD Bioscience, catalog number: 244520 )
- Ampicillin (AMRESCO, catalog number: 0339 )
- Glucose (AMRESCO, catalog number: 0188 )
- Isopropyl β-D-1-thiogalactopyranoside (IPTG) (AMRESCO, catalog number: 0487 )
- Phenylmethylsulfonyl fluoride (PMSF) (Roche Diagnostics, catalog number: 11359061001 )
- Tris(2-carboxyethyl)phosphine (TCEP) (AMRESCO, catalog number: K831 )
- Benzamidine (Sigma-Aldrich, catalog number: 12072 )
- Leupeptin (Sigma-Aldrich, catalog number: 62070 )
- Pepstatin (Sigma-Aldrich, catalog number: 77170 )
- Amylose resin (New England Biolabs, catalog number: E8021S )
- Ammonium sulfate (Sigma-Aldrich, catalog number: A4418 )
- Sodium chloride (Sigma-Aldrich, catalog number: S3014 )
- Magnesium Chloride (Sigma-Aldrich, catalog number: 63068 )
- Tris-HCl (Amresco, catalog number: 0 497 )
- Glycerol (Sigma-Aldrich, catalog number: G5516 )
- Maltose (Sigma-Aldrich, catalog number: 1B1184 )
- HEPES-KOH (Sigma-Aldrich, catalog number: 54457 )
- Spermidine (Sigma-Aldrich, catalog number: 85558 )
- Dithiothreitol (DTT) (Roche Diagnostics, catalog number: 10708984001 )
- Recombinant RNasin Ribonuclease Inhibitor (Promega Corporation, catalog number: N2511 )
- Nuclease-Free Water (Promega Corporation, catalog number: P1193 )
- T7 RNA polymerase (New England Biolabs, catalog number: M0251S )
- Pyrophosphatase (New England Biolabs, catalog number: M0361S )
- Glucosamine-6-phosphoate (Sigma-Aldrich, catalog number: G5509 )
- Lysis buffer (see Recipes)
- Column buffer (see Recipes)
- RNA buffer (see Recipes)
- 5x transcription buffer (see Recipes)
Equipment
- Incubator shaker (Eppendorf, model: New BrunswickTM Innova 44 )
- Centrifuge (Eppendorf, model: 5810R )
- Ultracentrifuge (Beckman Coulter, model: Optima TM)
- 45Ti rotor (Beckman Coulter)
- Sonicator (Branson Sonifier)
- AKTA purifier (GE)
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Huang, J., Bley, C. J., Rand, D. P., Chen, J. J. and Lei, M. (2015). Sample Preparation of Telomerase Subunits for Crystallization. Bio-protocol 5(16): e1565. DOI: 10.21769/BioProtoc.1565.
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分类
生物化学 > RNA > RNA-蛋白质相互作用
生物化学 > 蛋白质 > 结构
生物化学 > 蛋白质 > 分离和纯化
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