pQE-TriSystem Vector

Zur parallelen Expression von Proteinen mit His-tag in E. coli- und Säugerzellen sowie in mit Baculoviren infizierten Insektenzellen mit einem einzigen Konstrukt

S_1126_4_pQE_TriSystem_Vector

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pQE-TriSystem Vector

Cat. No. / ID:  33903

25 µg pQE-TriSystem Vektor DNA
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507,00 €
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pQE-TriSystem Vector ist für molekularbiologische Anwendungen vorgesehen. Dieses Produkt ist nicht für die Diagnose, Prävention oder Behandlung einer Krankheit bestimmt.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • Keine zeitaufwändigen Subklonierungsverfahren erforderlich
  • Erhalten Sie posttranslationale Modifikationen in Insekten- und Säugerzellen
  • Ein Konstrukt für effiziente Expression in drei Expressionssystemen

Product Details

Der pQE-TriSystem Vector ermöglicht hohe Expressionslevel von Proteinen mit His-tag mit einem einzigen Vektor in drei verschiedenen Expressionssystemen. Er enthält das T5-Promotor/lac-Operator-Transkriptions-Translationssystem für die Expression in E. coli, den p10-Promotor für die Baculovirus-basierte Expression in Insektenzellen und den CAG-Promotor (CMV/Actin/Globin) für die Expression in Säugerzellen.

Performance

Der pQE-TriSystem Vector enthält CAG-, T5- und p10-Promotoren, die die Expression von Proteinen mit 6xHis-tag in Säuger-, E. coli- und mit Baculoviren infizierten Insektenzellen ermöglichen (siehe Abbildung  pQE TriSystem). Vorstudien können in bakteriellen Expressionssystemen durchgeführt werden, wobei der starke T5-Promotor verwendet wird, der von der E. coli-Polymerase erkannt wird und eine effiziente Expression von Proteinen in jedem E. coli-Stamm ermöglicht. Wenn die Expression in Säuger- oder Insektenzellen erforderlich ist, um z. B. posttranslationale Modifikationen zu erhalten, kann dasselbe Konstrukt verwendet werden - ohne zeitaufwändige Subklonierungsverfahren.
See figures

Principle

QIAexpress pQE-Vektoren kombinieren einen leistungsstarken T5-Phagen-Promotor (der von der E. coli-RNA-Polymerase erkannt wird) mit einem doppelten lac-Operator-Repressionsmodul, um streng regulierte, hohe Expressionslevel rekombinanter Proteine in E. coli zu ermöglichen. Die Proteinsynthese wird in Gegenwart großer Mengen des lac-Repressors wirksam blockiert und die Stabilität der zytotoxischen Konstrukte erhöht. Die pQE-Vektoren (siehe Tabelle und Abbildung  pQE-Vektoren) ermöglichen die Positionierung des 6xHis-tags entweder am N- oder C-Terminus des rekombinanten Proteins.

In QIAexpress pQE-Vektoren enthaltene Elemente
Element Beschreibung
1. Optimiertes Promotor-/Operatorelement

Besteht aus dem T5-Phagen-Promotor und zwei lac-Operatorsequenzen,

die die Bindungswahrscheinlichkeit des lac-Repressors erhöhen und

eine effiziente Unterdrückung des starken T5-Promotors gewährleisten

2. Synthetische ribosomale Bindungsstelle RBSII Für effiziente Translation
3. His-tag-kodierende Sequenz Entweder 5' oder 3' von der Polylinker-Klonierregion
4. Translations-Stoppcodons In allen Leserahmen zur einfachen Herstellung von Expressionskonstrukten
5. Zwei starke Transkriptionsterminatoren

t0 vom Lambda-Phagen und T1 vom rrnB-Operon von E. coli, um

eine Read-Through-Transkription zu verhindern und die Stabilität des Expressionskonstrukts zu gewährleisten

6. ColE1-Replikationsursprung

Aus pBR322
7. Beta-Lactamase-Gen (bla) Verleiht Ampicillin-Resistenz
See figures

Procedure

Inserts, die für die untersuchten Proteine kodieren, werden in geeignete Konstrukte kloniert und zur Expression in einen geeigneten E. coli-Stamm transformiert. Die Expression wird durch Zugabe von IPTG induziert. pQE-TriSystem Vector-Konstrukte können in E. coli transformiert, als Shuttle-Vektor für die rekombinante Proteinexpression in Insektenzellen verwendet oder in Säugerzellen transfiziert werden.

Applications

Das QIAexpress Expressionssystem ermöglicht hohe Expressionslevel von Proteinen, die sich
für viele Anwendungen eignen wie:

  • Aufreinigung funktioneller Proteine mit aktiver Konformation
  • Aufreinigung unter denaturierenden Bedingungen für die Antikörperproduktion
  • Kristallisierung zur Bestimmung der dreidimensionalen Struktur
  • Protein-Protein- und Protein-DNA-Interaktionsassays

Supporting data and figures

Specifications

FeaturesSpecifications
In-frame cloning necessaryJa
ExpressionIn vivo
Tag removal sequenceNein
Expression speciesE. coli, Säugerzellen, Insektenzellen
Tag6xHis-tag
N- or C-terminal tagC-terminaler Tag
All three reading frames providedNein

Resources

Safety Data Sheets (1)
Selection Guides (1)
Kit Handbooks (1)
A handbook for high-level expression and purification of 6xHis-tagged proteins
Certificates of Analysis (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