pQE-TriSystem Vector

用于使用单一构建体在 E. coli、哺乳动物细胞和杆状病毒感染的昆虫细胞中平行表达 His 标签蛋白

S_1126_4_pQE_TriSystem_Vector

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pQE-TriSystem Vector

Cat. No. / ID:  33903

25 µg pQE-TriSystem Vector DNA
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pQE-TriSystem Vector 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • 无需耗时的亚克隆程序
  • 在昆虫或哺乳动物细胞中实现翻译后修饰
  • 一个构建体在三个表达系统中提供高效表达

Product Details

pQE TriSystem Vector 允许利用单一载体在三个不同表达系统中进行 His 标签蛋白的高水平表达。T5 启动子/lac 操作子转录–翻译系统用于在 E. coli 中进行表达,p10 启动子用于在昆虫细胞中进行基于杆状病毒的表达,CAG(CMV/肌动蛋白/珠蛋白)启动子用于在哺乳动物细胞中进行表达。

Performance

pQE-TriSystem 载体包含 CAG、T5 和 p10 启动子,分别支持在哺乳动物、E. coli 和杆状病毒感染的昆虫细胞中进行 6xHis 标签蛋白表达(参见图片  pQE TriSystem)。可以使用强 T5 启动子在细菌表达系统中进行初步研究,该启动子可被 E. coli 聚合酶识别,并允许在任何 E. coli 菌株中高效表达蛋白质。如果需要在哺乳动物或昆虫细胞中表达——例如,为了实现翻译后修饰——可以使用相同的构建体,而不需要耗时的亚克隆程序。
See figures

Principle

QIAexpress pQE 载体将强效的噬菌体 T5 启动子(可被 E. coli RNA 聚合酶识别)与双 lac 操作子抑制模块相结合,以在 E. coli 中提供严格调控的高水平重组蛋白表达。在存在高水平 lac 阻遏物的情况下,蛋白质合成被有效阻断,并且细胞毒性构建体的稳定性得以增强。pQE 载体(参见表格和图片  pQE 载体)能够将 6xHis 标签置入重组蛋白的 N-末端或 C-末端。

QIAexpress pQE 载体中存在的元件
元件 说明
1.优化的启动子/操作子元件

由噬菌体 T5 启动子和两个 lac 操作子序列组成,

这增加了 lac 阻遏物结合的可能性,并确保

强效 T5 启动子的有效抑制

2.合成核糖体结合位点 RBSII 用于高效翻译
3.His 标签编码序列 5' 或 3' 到多位点人工接头克隆区
4.翻译终止密码子 在所有阅读框架中,用于方便地制备表达构建体
5.两个强转录终止子

t0 来自噬菌体 λ,T1 来自 E. coli 的 rrnB 操纵子,以阻止

连读转录并确保表达构建体的稳定性

6.ColE1 复制起点

来自 pBR322
7. β-内酰胺酶基因 (bla) 赋予氨苄青霉素耐药性
See figures

Procedure

将编码目标蛋白质的插入片段克隆到合适的构建体中,并转化至合适的 E. coli 菌株中进行表达。通过添加 IPTG 来诱导表达。载体 pQE TriSystem 构建体可以转化至 E. coli 中,用作昆虫细胞中重组蛋白表达的穿梭载体,或转染至哺乳动物细胞中。

Applications

QIAexpress Expression System 提供适用于许多应用的蛋白质高水平表达,包括:

  • 功能性构象活性蛋白质的纯化
  • 在变性条件下进行纯化用于抗体生产
  • 结晶用于三维结构测定
  • 涉及蛋白质-蛋白质和蛋白质-DNA 相互作用的检测

Supporting data and figures

Specifications

FeaturesSpecifications
In-frame cloning necessary
Expression体内
Tag removal sequence
Expression speciesE.coli,哺乳动物和昆虫细胞
Tag6xHis 标签
N- or C-terminal tagC-末端标签
All three reading frames provided

Resources

安全数据表 (1)
Download Safety Data Sheets for QIAGEN product components.
产品选择指南 (1)
试剂盒操作手册 (1)
A handbook for high-level expression and purification of 6xHis-tagged proteins
Safety Data Sheets (1)
Certificates of Analysis (1)
Kit Handbooks (1)
A handbook for high-level expression and purification of 6xHis-tagged proteins
Selection Guides (1)

FAQ

Is it possible to use QIAGEN's pQE-TriSystem Vectors with the EasyXpress Protein Synthesis Insect Kit?

No, the pQE-TriSystem vectors contain a T5 Promoter. The expression plasmid used with the EasyXpress Protein Synthesis Insect Kit must contain a T7 Promoter.

FAQ ID -876
How can I increase expression of my 6xHis-tagged protein in E. coli?

Low-level expression can occur because the protein is toxic or unstable, or because the expression construct is not maintained in the cells during growth. In some cases, the 5' end of the inserted DNA sequence may encode elements that interfere with transcription or translation (e.g., masking of the Shine-Dalgarno sequence by stem-loop structures resulting from inverted repeats). In these instances, the sequence being expressed should be checked and modified if necessary. Modifications of growth media and different host strains may also have an effect on expression.

Please review the section "Specific considerations" in the Chapter 'Expression in E. coli' of the QIAexpressionist Handbook and refer to standard literature in protein science (e.g., Current Protocols in Protein Science, eds. John Wiley and Sons, New York) for additional information.

FAQ ID -63
How can I increase the amount of soluble recombinant protein in E. coli expression?

The QIAexpress Protein Purification System allows easy solubilization of 6xHis-tagged proteins sequestered into insoluble inclusion bodies by using denaturants such as 6 M Guanidine-HCl or 8 M Urea, or a variety of detergents. Proteins purified under denaturing conditions can then be refolded if necessary before use (please see: Wingfield, P. T., Palmer, I., and Liang, S.-M. (1995). Folding and purification of insoluble (inclusion-body) proteins from Escherichia coli. In: Current Protocols in Protein Science, vol. 1, Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P.T. eds. Wiley and Sons, Inc. New York, pp. 6.5.1–6.5.27.). The QIAexpressionist also contains recommendations for refolding proteins prior to the Appendix section of the handbook.

To increase levels of soluble protein, here are a few recommendations:

  • a reduction in growth temperature following induction may be helpful. Growth temperature often directly affects both expression levels and protein solubility, and lower temperatures will reduce expression levels leading to a higher amount of soluble protein.
  • the culture can be grown to a higher cell density before induction and the expression period can be kept to a minimum.
  • The IPTG concentration can be reduced from 1 mM to 0.005 mM, which would reduce the expression level by 90–95%.
  • it may be sufficient to change the host strain used, since certain strains tolerate some proteins better than others and allow higher levels of expression before forming inclusion bodies.
  • many proteins require metal cofactors in order to remain soluble, and the addition of metal salts to the culture media may be helpful. If the metal requirements of the protein are not known, a number of different supplements should be tested. Note that some divalent cations may interfere with protein binding to Ni-NTA.

 

 

FAQ ID -64
What is the origin of replication and the plasmid copy number of the pQE vectors?
The QIAexpress pQE vectors contain a pBR322 derived ColE1 origin of replication and are classified as low-copy plasmids (by our estimate, approximately 20-30 copies per cell; exact numbers have not been determined). The pQE-TriSystem Vector has a pUC origin of replication and is classified as a high-copy vector. Please see also FAQ 350 for general information on replication origins and copy numbers of various commonly used plasmids.
FAQ ID -338
How should I propagate pQE expression plasmids?

QIAexpress pQE vectors and constructs can be maintained in any E. coli strain that is ampicillin-sensitive and carries the pREP4 repressor plasmid, or harbors the lacIq gene on the F-factor episome.

M15 and SG13009 E. coli host strains carry lacI on the plasmid pREP4, while XL1-Blue or the JM series contain an episomal copy of lacIq.

LacIq is a mutation of lacI that produces very high levels of the lac repressor. Initial cloning and propagation using XL1-Blue is recommended because plasmid preparations derived from QIAexpress host strains will also contain pREP4 DNA, which could make clone analysis more difficult.

Alternatively, the pQE-80L series of expression vectors which encodes a lacIq repression module, allows use of any E. coli host strain.

FAQ ID -58
Do pQE vectors contain the CAT gene?
The chloramphenicol acetyl transferase gene (CAT) present between t0 and T1 has no promoter and is not normally expressed. Depending on the bacterial strain and insert,low CAT activities may be detectable
FAQ ID -362
How can I be sure that I am harvesting my induced bacterial culture at the best time point for protein expression?

To optimize the expression of a given recombinant protein, a time-course analysis of the level of protein expression in the induced culture is recommended. Intracellular protein content is often a balance between the amount of soluble protein in the cells, the formation of inclusion bodies, and protein degradation. By checking the 6xHis-tagged protein present at various times after induction in the soluble and insoluble fractions, the optimal induction period can be established, and the bacterial culture can be harvested at this time. It may be useful to perform plasmid Mini preparations on culture samples during the time-course to enable monitoring of plasmid (expression construct) maintenance.

Below, you can see an example of a time course of recombinant protein expression using the QIAexpress System. You can find this information also in the Section 'Expression in E. coli' in the QIAexpressionist Handbook. The handbook is an important resource for useful background information and protocols. For instructions on how to isolate protein from the soluble and insoluble fractions of induced cultures please see Protocol 14. "Protein minipreps of 6x His-tagged proteins from E. coli under native conditions" and Protocol 19. "6xHis-tagged protein minipreps under denaturing conditions."

 

 

 

Time course of expression using the QIAexpress System. Expression of 6xHis-tagged DHFR was induced with 1 mM IPTG. Aliquots were removed at the times indicated and purified on Ni-NTA Agarose under denaturing conditions. Proteins were visualized by Coomassie staining. Yields per liter culture were 2.8, 5.5,12.3, 33.8, and 53.9 mg, respectively. ■A Crude cell lysate; ■B purification with Ni-NTA. 1: flow-through, 2 & 3: first and second eluates; M: markers; C: noninduced control.

 

 

FAQ ID -788