C-Terminus pQE Vector Set

C末端6xHisタグ付きタンパク質の高レベル発現用

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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¥122,000
C-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

  • C末端6xHisタグにより、全長タンパク質のみが確実に精製されます
  • 低発現タンパク質をDHFR融合体として発現させるためのpQE-16ベクター
  • 短いペプチドをDHFR融合体として発現させるためのpQE-16ベクター

Product Details

このセットには、C末端6xHisタグ付きタンパク質発現用の3つのベクター(pQE-16、pQE-60、pQE-70)が含まれており、早期終了を引き起こす可能性がある「一時停止サイト」を持つオープンリーディングフレームに推奨されます。pQE-60とpQE-70は、コード化断片の元の開始コドンをpQEベクターのATGに置き換えることができ、タンパク質の本来のN末端を保持します。これら2つのコンストラクトは、PCRまたは突然変異誘発により、インサートのATGコドンにそれぞれNcoIおよびSphI制限部位を導入することで作成されます。pQE-16は、C末端6xHisタグ付きDHFR融合タンパク質の発現を可能にします。DHFR (ジヒドロ葉酸還元酵素)は抗原性と安定性を強化するので、発現量の少ないタンパク質やタンパク質分解を受けやすい短いペプチドに推奨されます。DHFR自体はマウスやラットではほとんど免疫原性を示さないため、DHFR融合タンパク質はエピトープスクリーニングに最適です。

Principle

QIAexpress pQEベクターは、強力なファージT5プロモーター(E. coli RNAポリメラーゼによって認識される)とdoublelac オペレーター抑制モジュールを組み合わせ、E. coliで、組み換えタンパク質の厳密に制御された高レベル発現を実現します(図  QIAexpress pQEベクターを参照)。タンパク質合成は、高レベルlacリプレッサー存在下で効果的に阻害され、細胞毒性コンストラクトの安定性が強化されます。 pQEベクターは、組み換えタンパク質のN末端またはC末端のいずれかに6xHisタグを配置できます。

 

QIAexpress pQEベクターに存在する要素
要素 説明
最適化したプロモーター/オペレーター要素 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、lacリプレッサー結合確率を高め、強力なT5プロモーターの効率的な抑制を保証します
合成リボゾーム結合部位RBSII 効率的な翻訳用
6xHisタグコード化配列 ポリリンカークローニング領域の5'または3'のいずれか
翻訳停止コドン 発現コンストラクトの調製に便利な全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 λファージ由来のt0E. colirrnBオペロン由来のT1は、リードスルー転写を防ぎ、発現コンストラクトの安定性を確保します
ColE1複製起点 pBR322から
β-ラクタマーゼ遺伝子(bla アンピシリン耐性を付与
See figures

Procedure

目的のタンパク質をコードするインサートを適切なコンストラクトにクローニングし(詳細情報については、QIAexpressionistハンドブックを参照)、発現に適したE. coli株に形質転換します。発現はIPTGを添加すると誘導されます。ベクターpQE-TriSystemコンストラクトは、E. coliへ形質転換されるか、昆虫細胞で組み換えタンパク質発現用のシャトルベクターとして使用されるか、哺乳動物細胞にトランスフェクションされます。

Applications

QIAexpress Expressionシステムは、以下のような
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。

  • 機能的で立体構造的に活性なタンパク質の精製
  • 抗体生産のための変性条件下での精製
  • 三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
Expression speciesE. coli
In-frame cloning necessaryはい
N- or C-terminal tagC末端タグ
ExpressionIn vivo
Special featuresT5プロモーター転写翻訳システムに基づく
Tag6xHisタグ
Tag removal sequenceいいえ
All three reading frames providedはい

Resources

セレクションガイド (1)
Vector Sequences & Maps (3)
For the pQE-16 vector
For the pQE-60 vector
For the pQE-70 vector
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
キットハンドブック (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

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