pQE-TriSystem Vector

単一のコンストラクトを使用した、E. coli、哺乳類細胞、バキュロウイルス感染昆虫細胞でのHisタグタンパク質の並行発現のために

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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$668.00
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pQE-TriSystem Vector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

  • 時間のかかるサブクローニングの手順は不要
  • 昆虫または哺乳類細胞で翻訳後修飾を獲得
  • 1つのコンストラクトが、3つの発現システムで効率的な発現を提供

Product Details

pQE TriSystem Vectorにより、3つの異なる発現システムを含む単一ベクターから、Hisタグf付加タンパク質の高レベルの発現が可能です。E. coliでの発現のためのT5プロモーター/lacオペレーターの転写–翻訳システム、昆虫細胞でのバキュロウイルスベースの発現のためのp10プロモーター、そして哺乳類細胞での発現のためのCAG(CMV/アクチン/グロビン)プロモーターがあります。

Performance

pQE-TriSystemベクターは、哺乳類、E. coli、バキュロウイルス感染昆虫細胞で、6xHisタグ付加タンパク質の発現を可能にするCAG、T5、p10プロモーターをそれぞれ含みます(図  pQE TriSystemを参照)。予備研究を、E. coliポリメラーゼによって認識される強力なT5プロモーターを使用して、細菌発現システムで実行でき、任意のE. coli株におけるタンパク質の効率的な発現を可能にします。たとえば翻訳後修飾を得るために、哺乳類または昆虫細胞での発現が必要な場合、時間のかかるサブクローニング手順を必要とせずに同じコンストラクトを使用できます。
See figures

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)を二重のlacオペレーター抑制モジュールと組み合わせて、E. coliで遺伝子組み換えタンパク質の厳しく制御される、高レベルな発現を実現します。タンパク質合成は、高レベルのlacリプレッサーの存在で効果的にブロックされ、細胞毒性コンストラクトの安定性が向上します。pQEベクター(表と図、 pQE Vectorsを参照)は、遺伝子組み換えタンパク質のN末端またはC末端のいずれかの6xHisタグの配置を可能にします。

QIAexpress pQE Vectorsに存在するエレメント
要素 説明
1. 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プロモーターおよび2つのlacオペレーター配列で構成されます。

これは、lacリプレッサー結合の確率を向上させ、確実にする

強力なT5プロモーターの効率的な抑制を保証します

2. 合成リボソーム結合部位RBSII 効率的な翻訳のために
3. Hisタグをコードする配列 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
4. 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
5. 2つの強力な転写ターミネーター

ファージλ由来のt0、およびE. coliのrrBオペロン由来のT1、防ぐために

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

6. ColE1複製開始点

pBR322由来
7. βラクタマーゼ遺伝子 (bla) アンピシリン耐性を与えます
See figures

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ンされ、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 System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
In-frame cloning necessaryはい
ExpressionIn vivo
Tag removal sequenceいいえ
Expression species大腸菌、哺乳類 & 昆虫細胞
Tag6xHisタグ
N- or C-terminal tagC末端タグ
All three reading frames providedいいえ

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
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