S_1125_2_C_Terminus_pQE_Vector_Set

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C-Terminus pQE Vector Set

Cat. No. / ID:  32903

各 25 µg:pQE-16、pQE-60、pQE-70
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C-Terminus pQE Vector Set 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

  • C 端 6xHis 标签可确保只纯化全长蛋白
  • 用于将表达不良的蛋白质表达为 DHFR 融合的 pQE-16 载体
  • 用于将短肽表达为 DHFR 融合的 pQE-16 载体

Product Details

此载体组提供 3 种载体(pQE-16、pQE-60 和 pQE-70),用于表达 C 端 6xHis 标签蛋白,建议用于带有“暂停位点”的开放阅读框,这类位点可能导致提前终止。pQE-60 和 pQE-70 允许编码片段的原始起始密码子取代 pQE 载体中的 ATG,从而保留蛋白的真实 N 端。这两个构建体是通过 PCR 或诱变在插入片段的 ATG 密码子处分别引入 NcoI 和 SphI 限制酶切位点而创建的。pQE-16 允许表达 C 端 6xHis 标记 DHFR 融合蛋白。DHFR(二氢叶酸还原酶)可增强抗原性和稳定性,建议用于表达不良的蛋白质或易被蛋白水解的短肽。由于 DHFR 本身在小鼠和大鼠中几乎没有显示出免疫原性,因此 DHFR 融合蛋白是表位筛选的理想选择。

Principle

QIAexpress pQE 载体将强大的噬菌体 T5 启动子(由大肠杆菌 RNA 聚合酶识别)与 doublelac 操作子抑制模块相结合,在大肠杆菌中提供严格调控的高水平重组蛋白表达(见图“ QIAexpress pQE 载体”)。存在高浓度的 lac 抑制剂时,蛋白合成被有效阻断,细胞毒性构建体的稳定性得到增强。 pQE 载体可将 6xHis 标签置于重组蛋白的 N 端或 C 端。

 

QIAexpress pQE 载体中存在的元素
元素 说明
经过优化的启动子/操作子元素 由噬菌体 T5 启动子和两个 lac 操作子序列组成,可提高 lac 抑制剂结合的概率,并确保有效抑制强大的 T5 启动子
合成核糖体结合位点 RBSII 用于高效翻译
6xHis-tag 编码序列 多接头克隆区的 5' 或 3'
翻译终止密码子 在所有阅读框中,以方便制备表达构建体
两个强转录终止子 来自噬菌体 λ 的 t0 和来自大肠杆菌rrnB 操纵子的 T1,用于防止通读转录并确保表达构建体的稳定性
ColE1 复制起始点 来自 pBR322
β-内酰胺酶基因 (bla) 赋予氨苄西林抗药性
See figures

Procedure

编码目标蛋白的插入片段被克隆到适当的构建体中(有关详细信息,请参阅 QIAexpressionist 手册),并转化为合适的大肠杆菌菌株进行表达。通过添加 IPTG 诱导表达。载体 pQE-TriSystem 构建体可以转化为大肠杆菌,作为穿梭载体用于在昆虫细胞中表达重组蛋白,或转染到哺乳动物细胞中。

Applications

QIAexpress 表达系统可提供适合许多应用的高水平蛋白表达,
其中包括:

  • 纯化具有构象活性的功能性蛋白
  • 在变性条件下进行纯化以生产抗体
  • 结晶以确定三维结构
  • 涉及蛋白间和蛋白与 DNA 之间相互作用的检测

Supporting data and figures

Specifications

FeaturesSpecifications
Expression species大肠杆菌
In-frame cloning necessary
N- or C-terminal tagC 端标签
Expression体内
Special features基于 T5 启动子转录-翻译系统
Tag6xHis 标签
Tag removal sequence
All three reading frames provided

Resources

产品选择指南 (1)
载体序列 (3)
For the pQE-16 vector
For the pQE-70 vector
For the pQE-60 vector
安全数据表 (1)
Download Safety Data Sheets for QIAGEN product components.
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)
Vector Sequences & Maps (3)
For the pQE-16 vector
For the pQE-60 vector
For the pQE-70 vector

FAQ

Why is the pQE DNA provided in QIAexpress Kits blue in color?
The blue color results from bromophenol blue added to the plasmid DNA prior to lyophilization to make the DNA more easily visible. After the DNA pellet is resuspended in water or Tris-HCl, the dye will not inhibit downstream applications such as transformation of bacterial cells or restriction enzyme digestion.
FAQ ID -487
What is the size, charge and isoelectric point of the DHFR protein in the pQE vectors?

The native Dihydrofolate Reductase (DHFR) protein has the following technical features:

  • Molecular Weight: 21.5 kDa
  • Isoelectric Point: 8.5
  • Charge at pH 7.0: 2.2

DHFR expressed in QIAexpress vectors pQE-16 and pOE-40 has the following technical features:

pQE-16

  • Molecular Weight: 23.1 kDa
  • Isoelectric Point: 9.1
  • Charge at pH 7.0: 5.2

pQE-40

  • Molecular Weight: 24.5 kDa
  • Isoelectric Point: 9.2
  • Charge at pH 7.0: 7.2
FAQ ID -470
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
Which primers can I use for sequencing pQE-expression vector constructs?
All pQE vectors (except pQE-TriSystem) can be sequenced using any of the primers described on page 118 of the QIAexpressionist.
FAQ ID -343
Is dihydrofolate reductase (DHFR) immunogenic?

Murine DHFR, used as a fusion protein to enable expression of very small proteins and peptides with the QIAexpress System, is poorly immunogenic in mouse and rats. It may however lead to an immune response in rabbits and goat. DHFR protein is encoded in the QIAexpress vectors pQE-16 or pQE-40.

FAQ ID -471
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