用于结晶化的端粒酶亚基样品的制备

Jing Huang; Christopher J. Bley; Dustin P. Rand; Julian  J.-L. Chen; Ming Lei

doi:10.21769/BioProtoc.1565

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Sample Preparation of Telomerase Subunits for Crystallization

用于结晶化的端粒酶亚基样品的制备

JH Jing Huang

CB Christopher J. Bley

DR Dustin P. Rand

JC Julian J.-L. Chen

Ming Lei

发布: 2015年08月20日第5卷第16期 DOI: 10.21769/BioProtoc.1565 浏览次数: 8231

评审: Arsalan DaudiAnonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Nature Structural & Molecular Biology, featuring study using the protocol.

Jun 2014

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

结合His标签辅助的弹性蛋白样多肽（ELP）新型蛋白纯化方法

Young Kee Chae and Han Bin Shin

2025年06月20日 2003 阅读

嗜热栖热菌CRISPR Cas6的异源表达与纯化方法

Junwei Wei [...] Yingjun Li

2025年07月20日 1106 阅读

hSox2-HMG结构域的原核表达与纯化

Lijie Yang [...] Jingjun Hong

2025年08月20日 1157 阅读

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 Brunswick^TM Innova 44 )
Centrifuge (Eppendorf, model: 5810R )
Ultracentrifuge (Beckman Coulter, model: Optima ^TM)
45Ti rotor (Beckman Coulter)
Sonicator (Branson Sonifier)
AKTA purifier (GE)

Procedure

English

中文翻译

文章信息

版权信息

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.