N-Terminus pQE Vector Set

Pour l’expression de haut niveau des protéines marquées à l’histidine sur l’extrémité N-terminale

S_1125_8_N_Terminus_pQE_Vector_Set

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N-Terminus pQE Vector Set

Cat. No. / ID:   32915

25 µg chacun : pQE-9, pQE-30, pQE-31, pQE-32, pQE-40
843,00 €
Connexion Pour ouvrir votre compte.
Le N-Terminus pQE Vector Set est destiné aux applications de biologie moléculaire. Ce produit n’est pas conçu pour le diagnostic, la prévention ou le traitement des maladies.

✓ Traitement automatique des commandes en ligne 24 h/24 7 j/7

✓ Assistance technique et produits pertinente et professionnelle

✓ Commande (ou réapprovisionnement) rapide et fiable

Caractéristiques

  • Vecteurs pQE-30 avec MCS dans les trois cadres de lecture pour un clonage rapide
  • Vecteur pQE-40 pour les protéines faiblement exprimées, peptides courts
  • Vecteur pQE-40 exprimant les fusions DHFR, ce qui améliore la stabilité et l’antigénicité

Détails produit

Ce kit contient 5 vecteurs (pQE-9, pQE-30, pQE-31, pQE-32 et pQE-40) pour l’expression des protéines marquées à l’histidine sur l’extrémité N-terminale. pQE-30, pQE-31 et pQE-32 présentent le site de clonage multiple (MCS) dans les trois cadres de lecture tandis que pQE-9 présente un autre site de clonage multiple, plus court. pQE-40 est conçu pour l’expression des protéines de fusion DHFR, il est recommandé pour l’expression des protéines faiblement exprimées ou des peptides courts, qui sont souvent sujets à la protéolyse, car la DHFR améliore à la fois la stabilité et l’antigénicité. Dans la mesure où la DHFR ne présente en soi que peu d’immunogénicité chez la souris et le rat, les protéines de fusion DHFR sont idéales pour le criblage des épitopes.

Principe

Éléments présents dans les QIAexpress pQE Vectors
Élément Description
Élément promoteur/opérateur optimisé Comprend le promoteur du phage T5 et deux séquences d’opérateurs lac, qui accroissent la probabilité d’une liaison du répresseur lac et assurent une répression performante du promoteur T5 fort
Site de liaison du ribosome synthétique RBSII Pour une traduction performante
Séquence codante du marqueur 6xHis-tag 5' ou 3' vers le site de clonage multiple
Codons stop de traduction Dans tous les cadres de lecture pour une préparation pratique des constructions d’expression
Deux terminateurs de transcription forts t0 à partir du phage lambda, et T1 à partir de l’opéron rrnB d’E. coli, pour empêcher la transcription ininterrompue et garantir la stabilité de la construction de l’expression
Origine de la réplication ColE1 À partir de pBR322
Gène de la bêta-lactamase (bla) Confère une résistance à l’ampicilline

 

 

 

Procédure

Les inserts codant les protéines d’intérêt sont clonés dans des constructions appropriées (pour des informations détaillées, consultez le Manuel QIAexpressionist) puis transformés en une souche d’E. coli adaptée à l’expression. L’expression est induite par ajout d’IPTG. Les constructions Vector pQE-TriSystem peuvent être transformées en E. coli, utilisées comme vecteur navette pour l’expression de protéines recombinantes dans des cellules d’insecte ou transfectées dans des cellules de mammifère.

Applications

Le QIAexpress Expression System permet une expression de haut niveau des protéines adaptée à de
nombreuses applications, notamment :

  • Purification de protéines fonctionnelles, de conformation active
  • Purification en conditions dénaturantes pour la production d’anticorps
  • Cristallisation pour la détermination d’une structure tridimensionnelle
  • Dosages impliquant des interactions protéine–protéine et protéine–ADN

Données et illustrations utiles

Specifications

FeaturesSpecifications
ExpressionIn vivo
TagMarqueur 6xHis-tag
N- or C-terminal tagMarqueur N-terminal
Expression speciesE. coli
Tag removal sequenceNon
In-frame cloning necessaryOui
All three reading frames providedOui

Ressources

Safety Data Sheets (1)
Selection Guides (1)
Kit Handbooks (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
Certificates of Analysis (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