N-Terminus pQE Vector Set

用于 N-末端 His 标签蛋白的高水平表达

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N-Terminus pQE Vector Set

Cat. No. / ID: 32915

各 25 µg：pQE-9、pQE-30、pQE-31、pQE-32、pQE-40

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N-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。

✓ 全天候自动处理在线订单

✓ 博学专业的产品和技术支持

✓ 快速可靠的（再）订购

特点

在所有三个阅读框中具有 MCS 的 pQE-30 载体可用于快速克隆
pQE-40 载体可用于低表达蛋白质和短肽
pQE-40 载体可表达 DHFR 融合蛋白，从而增强稳定性和抗原性

产品详情

该套组提供 5 种载体（pQE-9、pQE-30、pQE-31、pQE-32 和 pQE-40），用于 N-末端 His 标签蛋白的表达。pQE-30、pQE-31 和 pQE-32 在所有三个阅读框中提供多克隆位点 (multiple cloning site, MCS)，而 pQE-9 仅具有一个较短的替代多克隆位点。pQE-40 设计用于 DHFR 融合蛋白的表达，并且被推荐用于低表达蛋白质或短肽的表达，这些蛋白质或短肽通常易于蛋白水解，因为 DHFR 增强了稳定性和抗原性。由于 DHFR 本身在小鼠和大鼠中几乎没有免疫原性，因此 DHFR 融合蛋白非常适合用于表位筛选。

原理

QIAexpress pQE 载体中存在的元件
元件	说明
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
合成核糖体结合位点 RBSII	用于高效翻译
6xHis 标签编码序列	5' 或 3' 到多位点人工接头克隆区
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
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
ColE1 复制起点	来自 pBR322
β-内酰胺酶基因 (bla)	赋予氨苄青霉素耐药性

程序

将编码目标蛋白质的插入片段克隆到合适的构建体中（详细信息请参阅《QIAexpressionist 手册》），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。

应用

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

辅助数据和图表

pQE 载体。

Specifications

Features	Specifications
Expression	体内
Tag	6xHis 标签
N- or C-terminal tag	N-末端标签
Expression species	E. coli
Tag removal sequence	否
In-frame cloning necessary	是
All three reading frames provided	是

资源

Download Safety Data Sheets for QIAGEN product components.

A handbook for high-level expression and purification of 6xHis-tagged proteins

For the pQE-40 vector

For the pQE 9 vector

A handbook for high-level expression and purification of 6xHis-tagged proteins

For the pQE-40 vector

For the pQE 9 vector

Publications

Modulating RssB activity: IraP, a novel regulator of sigma(S) stability in Escherichia coli.

Bougdour A; Wickner S; Gottesman S;

Genes Dev; 2006; 20 (7):884-97 2006 Apr 1 PMID:16600914

Characterization of the putative alpha subunit of a heterotrimeric G protein in rice.

Iwasaki Y; Kato T; Kaidoh T; Ishikawa A; Asahi T;

Plant Mol Biol; 1997; 34 (4):563-72 1997 Jul PMID:9247538

FAQ

To reduce the effects of protein toxicity on bacterial cell growth prior to induction, the level of basal transcription that occurs in the absence of induction (“leakiness”) should be repressed as much as possible, and the number of generations before induction should be kept to a minimum.

For very toxic proteins, we recommend using the pQE-80L series of expression vectors in the M15[pREP4] E. coli host strain. The pQE-80L vectors have a cis-lacIq gene that overexpresses the lac repressor, in addition to a lacI repressor gene present in trans on a separate pREP4 plasmid. This combination of two repressor modules results in highly efficient suppression of recombinant protein expression prior to induction and gives the best chance of successful expression of toxic proteins. Furthermore, induction of the toxic protein should be done at a time point as late as possible, and the IPTG concentration used for induction should be reduced. Conditions have to be optimized for each application.

Another alternative is to use cell-free protein biosynthesis systems , like the EasyXpress Protein Synthesis Kit. The advantages of cell-free expression systems include time savings, the possibility to produce proteins that are toxic and the ability to adapt reaction conditions to the requirements of the synthesized protein.

FAQ ID -373

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

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

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

No, since there is no cleavage site included in pQE40 to remove the DHFR.

FAQ ID -513

All pQE vectors (except pQE-TriSystem) can be sequenced using any of the primers described on page 118 of the QIAexpressionist.

FAQ ID -343

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

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

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

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 lacI^q 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 lacI^q.

LacI^q 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 lacI^q repression module, allows use of any E. coli host strain.

FAQ ID -58

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

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