Expression and Purification of the Thermus thermophilus Argonaute Protein
嗜热细菌Argonaute蛋白的表达和纯化
发布: 2014年10月05日第4卷第19期 DOI: 10.21769/BioProtoc.1253 浏览次数: 10606
评审: Arsalan DaudiAnonymous reviewer(s)
下载 PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Mar 2014
Abstract
The Argonaute protein of Thermus thermophilus (TtAgo) has recently been studied in detail. For its in vitro characterization, TtAgo was purified after heterologous expression in Escherichia coli (E. coli). As TtAgo expression is toxic, a tightly controlled system was used for protein expression. The expression strain E. coli KRX carries a chromosomal T7 RNA polymerase gene under control of a rhamnose promoter. The ago gene is expressed via an IPTG-inducible T7 promoter. This allows for tightly (double) controlled expression of (toxic) TtAgo. Here, we describe the steps required for controlled expression and purification of this toxic protein.
Materials and Reagents
- Expression of TtAgo in E. coli KRX
- Glycerol stock of Escherichia coli KRX (Promega corporation, catalog number: L3002 ) [encodes a T7 RNA polymerase gene under control of a rhamnose promoter], transformed with plasmids pRARE (EMD Millipore, plasmid of RosettaTM (DE3) Competent Cells, catalog number: 70954-3 ) [encodes tRNAs for rare codons to enhance protein translation efficiency] and pWUR702 [pCDF-1b derivative, with T. thermophilus HB27 TT_P0026 ago gene insert fused to an N-terminal strep(II)-tag, expression under control of an IPTG-inducible T7 promoter] (Addgene, catalog number: 53079 ).
Note: The plasmid pWUR703 (Addgene, catalog number: 53082 ) can be used for expression of TtAgoDM (Double Mutant, D478A, D546A). Expression and purification of this protein is identical to TtAgo expression and purification.
- 1,000x chloramphenicol solution (34 mg/ml) dissolved in 100% ethanol
- 1,000x streptomycin solution (50 mg/ml) dissolved in MilliQ H2O
- 20% D-glucose solution dissolved in MilliQ H2O (sterile filtered)
- 20% L-rhamnose solution dissolved in MilliQ H2O (sterile filtered)
- 1 M IPTG dissolved in MilliQ H2O (sterile filtered)
- LB medium (see Recipes)
- Glycerol stock of Escherichia coli KRX (Promega corporation, catalog number: L3002 ) [encodes a T7 RNA polymerase gene under control of a rhamnose promoter], transformed with plasmids pRARE (EMD Millipore, plasmid of RosettaTM (DE3) Competent Cells, catalog number: 70954-3 ) [encodes tRNAs for rare codons to enhance protein translation efficiency] and pWUR702 [pCDF-1b derivative, with T. thermophilus HB27 TT_P0026 ago gene insert fused to an N-terminal strep(II)-tag, expression under control of an IPTG-inducible T7 promoter] (Addgene, catalog number: 53079 ).
- Purification of TtAgo
- Cell pellet from o/n TtAgo expression (see above)
- Strep-Tactin Sepharose® 50% suspension (IBA, catalog number: 2-1201-XXX )
- 1.2 ml Bio-Spin® Chromatography Columns (Bio-Rad Laboratories, catalog number: 732-6008 )
- Buffer I (see Recipes)
- Buffer II (see Recipes)
- Buffer III (see Recipes)
- Cell pellet from o/n TtAgo expression (see above)
Equipment
- Expression of TtAgo in E. coli KRX
- 50 ml Greiner tube
- 2.5 L Erlenmeyer flask
- Centrifuge
- 37 °C shaker incubator
- 20 °C shaker incubator
- Ice-water bath (water and ice mixed)
- 50 ml Greiner tube
- Purification of TtAgo
- French-Press, sonicator, or equivalent for cell disruption
- Centrifuge
- French-Press, sonicator, or equivalent for cell disruption
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Swarts, D. C., Jore, M. M. and Oost, J. V. D. (2014). Expression and Purification of the Thermus thermophilus Argonaute Protein. Bio-protocol 4(19): e1253. DOI: 10.21769/BioProtoc.1253.
Download Citation in RIS Format
分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 分离和纯化
生物化学 > 蛋白质 > 表达
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
