N-Terminus pQE Vector Set

N末端Hisタグ付加タンパク質を高レベルで発現させるために

S_1125_8_N_Terminus_pQE_Vector_Set

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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は分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • 高速クローニングのための、3つのリーディングフレームすべてにMCSを持つpQE-30ベクター
  • 発現レベルの低いタンパク質、短いペプチド向けのpQE-40ベクター
  • pQE-40ベクターは、DHFR融合型を発現し、安定性と抗原性を高めます

Product Details

このセットは、N末端にHisタグのあるタンパク質の発現用に、5つのベクター(pQE-9、pQE-30、pQE-31、pQE-32、pQE-40)を提供します。pQE-30、pQE-31、pQE-32は、3つのリーディングフレームすべてにマルチプルクローニングサイト(MCS)を提供しますが、pQE-9は、代わりにより短いマルチプルクローニングサイトを持っています。pQE-40は、DHFR融合タンパク質の発現向けに設計されており、発現レベルの低いタンパク質または短いペプチドの発現用にお勧めします。それらは、タンパク質分解されやすいのですが、それはDHFRが安定性と抗原性を高めることによります。DHFR自体は、マウスやラットではほとんど免疫性がないため、DHFR融合タンパク質はエピトープのスクリーニングに最適です。

Principle

QIAexpress pQEベクターに存在するエレメント
要素 説明
最適化されたプロモーター/オペレーターエレメント ファージT5プロモーターと2つのlacオペレーター配列で構成され、それによりlacリプレッサー結合の可能性が上がり、強力なT5プロモーターの効率的な抑制を保証します
合成リボソーム結合部位RBSII 効率的な翻訳のために
6xHisタグコード配列 ポリリンカークローニング領域への5’または3’のいずれか
翻訳終止コドン 発現コンストラクトの便利な調製のため、すべてのリーディングフレーム内
2つの強力な転写ターミネーター 読み過ごし転写を防ぎ、発現コンストラクトの安定性を保証するため、ファージλ由来のt0、およびE. colirrnBオペロン由来のT1
ColE1複製開始点 pBR322由来
βラクタマーゼ遺伝子(bla アンピシリン耐性を与えます

 

 

 

Procedure

目的のタンパク質をコードするインサートは、適切なコンストラクト内にクローニングされ(詳しくは、QIAexpressionist ハンドブックを参照)、発現のために適切なE. coli株内に転換されます。発現は、IPTGの添加によって誘導されます。Vector pQE-TriSystemのコンストラクトは、E.coli内に転換されたり、昆虫細胞内での遺伝子組み換えタンパク質の発現のためにシャトルベクターとして使用されたり、哺乳類細胞内に導入されたりします。

Applications

QIAexpress Expressionシステムは、以下をはじめとする
数多くのアプリケーションに適したタンパク質の高レベル発現を実現します。

  • 機能的にも構造的にも活性のあるタンパク質の精製
  • 抗体産生のための変性条件下での精製
  • 3次元構造決定のための結晶化
  • タンパク質–タンパク質およびタンパク質–DNA相互作用を含むアッセイ

Supporting data and figures

Specifications

FeaturesSpecifications
ExpressionIn vivo
Tag6xHisタグ
N- or C-terminal tagN末端タグ
Expression species大腸菌
Tag removal sequenceいいえ
In-frame cloning necessaryはい
All three reading frames providedはい

Resources

MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
セレクションガイド (1)
キットハンドブック (1)
A handbook for high-level expression and purification of 6xHis-tagged proteins
Vector Sequences & Maps (2)
For the pQE-40 vector
For the pQE 9 vector
テクニカルインフォメーション (1)
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)

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

How can I express toxic protein in E. coli?

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
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
If pQE40 is used to express a fusion protein with DHFR, can the DHFR be cleaved afterwards?
No, since there is no cleavage site included in pQE40 to remove the DHFR.
FAQ ID -513
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