C-Terminus pQE Vector Set

Para la expresión de alto nivel de proteínas marcadas con 6xHis en el extremo C-terminal

S_1125_2_C_Terminus_pQE_Vector_Set

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C-Terminus pQE Vector Set

Cat. No. / ID:  32903

25 µg cada uno: pQE-16, pQE-60, pQE-70
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El C-Terminus pQE Vector Set está concebido para su uso en aplicaciones de biología molecular. Este producto no está concebido para el diagnóstico, la prevención ni el tratamiento de enfermedades.

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✓ Knowledgeable and professional Product & Technical Support

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Features

  • La etiqueta 6xHis del extremo C-terminal garantiza que solo se purifica la proteína de duración completa
  • Vector pQE-16 para la expresión de proteínas poco expresadas como fusiones de DHFR
  • Vector pQE-16 para la expresión de péptidos cortos como fusiones de DHFR

Product Details

Este conjunto proporciona 3 vectores (pQE-16, pQE-60 y pQE-70) para la expresión de proteínas de extremo C-terminal marcadas con 6xHis y está recomendado para los marcos de lectura abiertos con «sitios de pausa», que pueden causar una finalización prematura. pQE-60 y pQE-70 permiten que el codón original de inicio del fragmento de codificación sustituya al ATG en el vector pQE, conservando el N-terminal auténtico de la proteína. Estas dos construcciones se crean introduciendo un sitio de restricción NcoI y SphI, respectivamente, en el codón ATG del inserto mediante PCR o mutagénesis. pQE-16 permite la expresión de proteínas de fusión de DHFR de extremo C-terminal marcadas con 6xHis. La DHFR (dihidrofolato reductasa) mejora la antigenicidad y la estabilidad y se recomienda para las proteínas poco expresadas o péptidos cortos propensos a la proteólisis. Como la propia DHFR muestra poco inmunogenicidad en ratones y ratas, las proteínas de fusión de DHFR son ideales para el cribado de epítopos.

Principle

Los vectores pQE de QIAexpress pQE combinan un potente promotor del fago T5 (reconocido por la ARN polimerasa de E. coli) con un módulo de represión de doble operador lac para proporcionar una expresión estrictamente regulada y de alto nivel de las proteínas recombinantes en E. coli (consulta la figura « Vector pQE de QIAexpress»). La síntesis de proteínas se bloquea de forma efectiva en presencia de altos niveles del represor lac y mejora la estabilidad de preparados citotóxicos. Los  vectores pQE permiten la colocación del marcado 6xHis ya sea en el extremo N-terminal o en el C-terminal de la proteína recombinante.

 

Elementos presentes en los vectores pQE de QIAexpress
Elemento Descripción
Elemento optimizado del promotor/operador Consta del promotor del fago T5 y dos secuencias del operador lac, que aumentan la probabilidad de la unión del represor lac y garantizan una represión eficiente del potente promotor T5
Sitio de unión ribosómica sintética RBSII Para una traslación eficiente
Secuencia de codificación marcada con 6xHis 5' o 3’ a la región de clonación del policonector
Codones de parada traslacionales En todos los marcos de lectura, para la preparación práctica de preparados de expresión
Dos potentes terminadores transcripcionales t0 del fago lambda y T1 del operón rrnB de E. coli, para evitar la transcripción de lectura y garantizar la estabilidad de la construcción de expresión
Origen de replicación de ColE1 A partir de pBR322
Gen de la betalactamasa (bla) Confiere resistencia a la ampicilina
See figures

Procedure

Los insertos que codifican proteínas de interés se clonan en construcciones adecuadas (para obtener información detallada, consulte El Manual QIAexpressionist) y se transformaron en una cepa de E.coli para la expresión. La expresión se induce mediante la adición de IPTG. Los preparados del vector pQE TriSystem se pueden transformar en E.coli, utilizarse como vector lanzadera para la expresión de proteína recombinante en células de insecto o transmitirse a células de mamíferos.

Applications

El sistema Expression QIAexpress proporciona una expresión de proteínas de alto nivel adecuado para
muchas aplicaciones, entre las que se incluyen:

  • Purificación de proteínas funcionales y conformacionalmente activas
  • Purificación en condiciones desnaturalizantes para la producción de anticuerpos
  • Cristalización para la determinación de la estructura tridimensional
  • Ensayos relacionados con interacciones proteína-proteína y proteína-ADN

Supporting data and figures

Specifications

FeaturesSpecifications
Expression speciesE. coli
In-frame cloning necessary
N- or C-terminal tagMarcador de extremo C-terminal
ExpressionIn vivo
Special featuresBasado en el sistema de transcripción-traducción del promotor T5
TagEtiqueta 6xHis
Tag removal sequenceNo
All three reading frames provided

Resources

Selection Guides (1)
Vector Sequences & Maps (3)
For the pQE-16 vector
For the pQE-60 vector
For the pQE-70 vector
Safety Data Sheets (1)
Kit Handbooks (1)
A handbook for high-level expression and purification of 6xHis-tagged proteins
Certificates of Analysis (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