QIAexpress Type IV Kit

N末端Hisタグ付加タンパク質の高レベル発現およびワンステップ精製のために

S_1126_8_QIAexpress_Type_IV_Kit

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QIAexpress Type IV Kit

Cat. No. / ID:  32149

各5 µg:pQE-30、pQE-31、pQE-32 (N末端6xHis)、10 ml Ni-NTA Agarose
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QIAexpress Type IV Kit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

  • 3つのリーディングフレームすべてを網羅するベクター
  • Hisタグ付加タンパク質の発現および精製のための完全なキット
  • N末端Hisタグ付加タンパク質が高レベルで発現
  • ワンステップ精製および高感度の検出のための多用途で完全なシステム

Product Details

QIAexpress Systemは、Hisタグ付加タンパク質の発現、精製、検出およびアッセイの材料を提供します。

Performance

QIAexpress Type IV Kitにより、全細胞タンパク質の最大50%までの高発現が得られます。

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)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( 図 pQE Vectors および表を参照)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 Hisタグの配置を可能にします。

QIAexpress pQE Vector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確実にします
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 λファージ由来のt0、およびE. coliのrrnBオペロン由来のT1はリードする―転写を防ぎ、発現コンストラクトの安定性を確保する
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株内に形質転換されます。発現は、IPTGの添加によって誘導されます。QIAexpress Type IV Kit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
Applicationsプロテオミクス
Yield総タンパク質の50%未満
特殊機能ワンステップ精製のための多用途で完全なシステム
Tag6xHisタグ
Processing手動
Start material細胞溶解物
N- or C-terminal tagN末端タグ

Resources

セレクションガイド (1)
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)

Publications

YtsJ has the major physiological role of the four paralogous malic enzyme isoforms in Bacillus subtilis.
Lerondel G; Doan T; Zamboni N; Sauer U; Aymerich S;
J Bacteriol; 2006; 188 (13):4727-36 2006 Jul PMID:16788182
Diurnal and seasonal variation of isoprene biosynthesis-related genes in grey poplar leaves.
Mayrhofer S; Teuber M; Zimmer I; Louis S; Fischbach RJ; Schnitzler JP;
Plant Physiol; 2005; 139 (1):474-84 2005 Aug 26 PMID:16126852
MtdC, a novel class of methylene tetrahydromethanopterin dehydrogenases.
Vorholt JA; Kalyuzhnaya MG; Hagemeier CH; Lidstrom ME; Chistoserdova L;
J Bacteriol; 2005; 187 (17):6069-74 2005 Sep PMID:16109948
Differential gene expression associated with euryhalinity in sea bream (Sparus sarba).
Deane EE; Woo NY;
Am J Physiol Regul Integr Comp Physiol; 2004; 287 (5):R1054-63 2004 Jul 8 PMID:15242828

FAQ

Are the buffers in the Ni-NTA Fast Start Kit the same as the ones for use with Ni-NTA purchased separately?

The buffers of the Ni-NTA Fast Start Kit are based on recipes for the respective buffers for purification of 6xHis-tagged proteins under native or denaturing conditions listed in the QIAexpressionist handbook. Specific components have been added for optimized performance. The exact composition of the buffers in the Ni-NTA Fast Start Kit is confidential. However, the buffers listed in the Appendix Section of the QIAexpressionist are compatible with the Ni-NTA Fast Start Kit, and can also be used.

FAQ ID -791
What is the composition of PBS?

The composition of PBS is:

  • 50 mM potassium phosphate
  • 150 mM NaCl; pH 7.2

PBS solution is not a component of any QIAGEN kit, but is used in protocols for recombinant protein purification with the QIAexpress System, and in some protocols for DNA and RNA isolation with various QIAGEN Kits.

 

FAQ ID -361
How can I remove imidazole from a protein sample?
Imidazole does not interfere with most downstream applications and therefore does not need to be removed. If it is necessary to remove the imidazole (e.g., for some sensitive enzyme assays), it can be easily achieved by dialysis, precipitation (e.g., ammonium sulfate), or ultrafiltration.
FAQ ID -91
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
What are the features and benefits of the QIAexpress 6xHis Tag System?

FEATURES BENEFITS
The interaction of the 6xHis tag with Ni-NTA matrices is conformation independent One-step purification can be carried out under native or denaturing conditions
Mild elution conditions can be used Binding, washing, and elution are highly reproducible, and have no effect on protein structure. Pure protein products are ready for direct use in downstream applications
The 6xHis tag is much smaller than other commonly used tags 6xHis tags can be used in any expression system. The Tag does not interfere with the structure and function of the recombinant protein
The 6xHis tag is uncharged at physiological pH The 6xHis tag does not interfere with secretion
The 6xHis tag is poorly immunogenic The recombinant protein can be used without prior removal of the tag as an antigen to generate antibodies against the protein of interest
Using Factor Xa Protease, 6xHis tag can be easily and efficiently removed The detagged protein can be used for crystallographical or NMR studies where removal of the 6xHis tag may be preferred
Some QIAexpress vectors feature a 6xHis-dihydrofolate reductase tag (6xHis-DHFR tag) Small peptides fused to the 6xHis DHFR tag are stabilized while being expressed. The 6xHis-DHFR tag is not highly immunogenic in mouse and rat, so that peptides fused to the tag can be used directly for immunizations or epitope mapping

 

FAQ ID -193
Can I use HEPES buffer instead of phosphate in my Ni-NTA column?
Although we do not strongly recommend it, up to 100 mM has been successfully used in some cases. Buffers that contain secondary or tertiary amines will reduce nickel ions and affect binding of 6xHis tagged proteins to the Ni-NTA resin.
FAQ ID -291
How does imidazole affect my quantitation of protein?

Since imidazole absorbs UV radiation at 280 nm, an elution profile measured at 280 nm while purifying a 6xHis tagged protein by FPLC will show an increase in absorbance above the background signal allowing quantitation of your protein. The absorbance of imidazole can vary depending on its source and purity, but elution buffer containing 250 mM imidazole usually has an A280 of 0.2–0.4.

To quantitate proteins in eluates containing imidazole, we recommend the Bradford protein assay, which is based on the ability of Coomassie Brilliant Blue R250 to form strong complexes with proteins. This assay is more tolerant of higher imidazole concentrations than the Lowry and the biuret assay, which are more sensitive to imidazole because they involve the reduction of copper. For accurate measurements, always use elution buffer without protein as the reagent blank.

FAQ ID -132
What are the most commonly used protease inhibitors?

The most commonly used protease inhibitors and their working concentrations are included in the table below:

Protease inhibitor Inhibits Suggested working concentration Stock solution (200x)
PMSF* Serine proteases and cysteine proteases such as papain 85 µg/ml (0.5 mM) 17 mg/ml (200 mM) in ethanol or isopropanol
Leupeptin Serine and thiol proteases 0.5 µg/ml (1 µM) 0.1 mg/ml (200 µM) in water
Pepstatin Aspartic proteases 0.7 µg/ml (1 µM) 0.14 mg/ml (200 µM) in ethanol
Aprotinin Serine proteases 1 µg/ml (0.15 µM) 0.2 mg/ml (30 µM) in water
Pefabloc® Serine proteases 0.5 mg/ml (2 mM) 100 mg/ml (400 mM) in water
Na2-EDTA Metalloproteases 0.35 mg/ml (0.75 mM) 70 mg/ml (150 mM) in water, pH 8.0

* PMSF is inactivated in aqueous solutions and should be added to buffers immediately before use. The half-life of an aqueous solution of PMSF is around 30 minutes at pH 8.

All these protease inhibitors are normally prepared as stock solutions and stored in aliquots at –20°C. Stock solutions are usually stable for up to six months. In addition, ready-to-use mixtures of protease inhibitors are available. Complete Protease Inhibitor Cocktail Tablets (Roche Molecular Biochemicals) are available in an EDTA-free formulation and are recommended for use during the purification of 6xHis-tagged proteins under native conditions.

FAQ ID -53
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
What is the difference between Ni-NTA Agarose and Ni-NTA Superflow?

The binding capacity of both resins is the same: up to 50mg/ ml mg 6xHis-tagged protein per ml of resin (2500 nmol @ ~20 kDa). The difference between them is the bead support, which determines pressure resistance and flow rate:

Ni-NTA Agarose:

  • Sepharose CL-6B (bead size 45–165 µm)
  • max. volumetric: 0.5–1.0 ml/min
  • max. pressure: 2.8 psi/(0.2bar)
  • for use with gravity flow only

Ni-NTA Superflow:

  • Superflow (bead size 60–160 µm)
  • max. volumetric: 20 ml/min
  • max. pressure: 140 psi/(10bar)
  • for use with gravity flow or FPLC

You can find a detailed comparison table in the Appendix at the back of the QIAexpressionist Handbook under the title 'Ni-NTA Matrices'.

FAQ ID -764
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 do I prepare an insert for pQE vectors?

In general, it is only necessary to prepare the insert by restriction digestion and gel purification. The fragment can be ligated into the vector directly. If there is no appropriate restriction site, if it is desirable to minimize the number of extra codons, or if the construct must be optimized in some other way, more complicated manipulations may be necessary. The ends of coding fragments can be modified by PCR, by in vitro mutagenesis, or by the addition of linkers. Please refer to standard laboratory manuals for details on such procedures.

If the 6xHis tag needs to be integrated into other vectors, please follow the recommendations in the 'Cloning' chapter of the QIAexpressionist handbook, section "Integration of 6xHis tags in other vectors".

FAQ ID -185
How can I improve the expression of proteins containing hydrophobic regions?
The addition of either 1% Octylglycoside or 1% Dodecylmaltoside to the lysate might help to solubilize proteins containing large hydrophobic regions. The detergents should be added directly to the reaction mix.
FAQ ID -339
How can I eliminate contaminating protein in my Ni-NTA 6xHis-tag protein purification?
  • Use 10-20 mM imidazole in the lysis and wash buffers (both for native and denaturing conditions). Optimal imidazole concentrations have to be determined empirically.
  • Increase the NaCl concentration (up to 2 M) in the purification buffers to reduce the binding of contaminants as a result of nonspecific ionic interactions.
  • Add ß-mercaptoethanol (up to 20 mM) to the lysis buffer to prevent copurification of host proteins that may have formed disulfide bonds with the protein of interest during cell lysis.
  • Add detergents such as Triton X-100 and Tween 20 (up to 2%), or additives such as glycerol (up to 50%) or ethanol (up to 20%) to reduce nonspecific binding to the matrix due to nonspecific hydrophobic interactions.
  • Reduce the amount of Ni-NTA matrix. Low-affinity binding of background proteins will be reduced by matching the total binding capacity of Ni-NTA matrix with the expected amount of 6xHis-tagged protein.
FAQ ID -102
Why do you recommend using Triton X for the purification of 6xHis-tagged protein?

Nonionic detergents such as Triton X-100 (0.1 - 1%) and Tween 20 (up to 2%) can be used to reduce non-specific binding of contaminating proteins due to non-specific hydrophobic or ionic interactions. They will have no effect on the binding of 6xHis-tagged protein to the Ni-NTA resin when used within the recommended concentration range.

Optimal concentrations for these additives to binding and wash buffers should be determined empirically for each purification protocol and protein.

-100
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