N-Terminus pQE Vector Set

Para expresión de alto nivel de proteínas con etiqueta His del extremo N

✓ Procesamiento automático sin interrupción de pedidos en línea

✓ Servicio técnico y para productos experto y profesional

✓ Realización y repetición de pedidos rápidas y fiables

N-Terminus pQE Vector Set

Cat. No. / ID: 32915

25 µg de cada uno: pQE-9, pQE-30, pQE-31, pQE-32, pQE-40

El N-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.

✓ Procesamiento automático sin interrupción de pedidos en línea

✓ Servicio técnico y para productos experto y profesional

✓ Realización y repetición de pedidos rápidas y fiables

Características

Los vectores pQE-30 con SCM en los tres marcos de lectura para una clonación rápida
Vector pQE-40 para proteínas de escasa expresión, péptidos cortos
El vector pQE-40 expresa fusiones de DHFR, lo que mejora la estabilidad y inmunogenicidad

Detalles del producto

Este paquete incluye 5 vectores (pQE-9, pQE-30, pQE-31, pQE-32 y pQE-40) para la expresión de proteínas con etiquetas His del extremo N. pQE-30, pQE-31 y pQE-32 proporcionan el sitio de clonación múltiple (SCM) en los tres marcos de lectura, mientras que pQE-9 tiene un sitio de clonación múltiple alternativo más corto. pQE-40 está diseñado para la expresión de proteínas de fusión de DHFR y se recomienda para la expresión de proteínas de expresión escasa o péptidos cortos, que suelen ser propensas a la proteólisis, ya que la DHFR mejora tanto la estabilidad como la inmunogenicidad. Dado que la propia DHFR muestra poca inmunogenicidad en ratón y rata, las proteínas de fusión de DHFR son ideales para el cribado de epítopos.

Principio

Elementos presentes en QIAexpress pQE Vectors
Elemento	Descripción
Elemento promotor/operador optimizado	Se compone del promotor T5 de fago y dos secuencias de operón lac, que aumentan la probabilidad de unión del represor lac y garantizan la represión eficaz del potente promotor T5
Sitio sintético de unión al ribosoma RBSII	Para traducción eficaz
Secuencia de codificación con etiqueta 6xHis	5' o 3' a la región de clonación del polilinker
Codones de parada traslacionales	En todos los marcos de lectura para preparar cómodamente los constructos de expresión
Dos terminadores transcripcionales fuertes,	t₀ del fago lambda y T1 del operón rrnB de E. coli, para evitar la transcripción de lectura y garantizar la estabilidad de los constructos de expresión
Origen de replicación ColE1	De pBR322
Gen beta-lactamasa (bla)	Confiere resistencia a la ampicilina

Procedimiento

Los insertos que codifican proteínas de interés se clonan en constructos apropiados (para más información, véase la sección Manual de QIAexpressionist) y se transforman en una cepa de E. coli para su expresión. La expresión se induce añadiendo IPTG. Los constructos del pQE-TriSystem Vector pueden transformarse en E. coli, utilizarse como vector lanzadera para la expresión de proteínas recombinantes en células de insecto o transfectarse en células de mamífero.

Aplicaciones

El QIAexpress Expression System proporciona un alto nivel de expresión de proteínas idóneo
para muchas aplicaciones, como por ejemplo:

Purificación de proteínas funcionales con configuración activa
Purificación en condiciones desnaturalizantes para la producción de anticuerpos
Cristalización para la determinación de estructuras tridimensionales
Ensayos que implican interacciones proteína-proteína y proteína-ADN

Datos y cifras de respaldo

Vectores pQE.

Specifications

Features	Specifications
Expression	In vivo
Tag	Etiqueta 6xHis
N- or C-terminal tag	Etiqueta N-terminal
Expression species	E. coli
Tag removal sequence	No
In-frame cloning necessary	Sí
All three reading frames provided	Sí

Recursos

A handbook for high-level expression and purification of 6xHis-tagged proteins

For the pQE-40 vector

For the pQE 9 vector

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

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

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

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

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

No, since there is no cleavage site included in pQE40 to remove the DHFR.

FAQ ID -513

All pQE vectors (except pQE-TriSystem) can be sequenced using any of the primers described on page 118 of the QIAexpressionist.

FAQ ID -343

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

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

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

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 lacI^q 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 lacI^q.

LacI^q 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 lacI^q repression module, allows use of any E. coli host strain.

FAQ ID -58

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

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