Citations et références (54)

Invitrogen™

pTrcHis A, B, & C Bacterial Expression Vectors

Nos vecteurs pTrcHis A, B et C sont conçus pour offrir une initiation à la translation améliorée et une expressionAfficher plus

Référence	Quantité
V36020	20 μg

Référence V36020

Prix (EUR)

1 258,00

20 µg

Quantité:

20 μg

Prix (EUR)

1 258,00

20 µg

Nos vecteurs pTrcHis A, B et C sont conçus pour offrir une initiation à la translation améliorée et une expression de haut niveau dans E. coli. Ces vecteurs comprennent :

• Transcription régulée de haut niveau à partir du promoteur trc
• Amélioration de l’efficacité de la translation des gènes eucaryotes dans E. coli
• L’opérateur lacO et le gène répresseur lacIq pour la régulation transcriptionnelle dans toute souche d’E. coli

Ce vecteur particulier offre :

• Marqueur de polyhistidine d’extrémité N-terminale (6xHis) pour une rapide purification à l’aide de résine de chélation de nickel et une détection à l’aide d’un anticorps anti-HisG
• Épitope Xpress™ d’extrémité N-terminale pour une détection facile avec un anticorps anti-Xpress™
• Site de clivage de l’entérokinase pour l’élimination du marqueur de fusion

Pour le marqueur de polyhistidine d’extrémité C-terminale et l’épitope c-myc, veuillez consulter notre vecteur pTrcHis2 A, B et C.

Usage exclusivement réservé à la recherche. Ne pas utiliser pour des procédures de diagnostic.

Spécifications

Résistance aux antibiotiques des bactériesAmpicilline (AmpR)

ClivageSite de reconnaissance d’EK (entérokinase)

Système constitutif ou inductibleInductible

Agent d’inductionIPTG

Type de produitVecteur d’expression bactérienne

Quantité20 μg

Agent de sélection (eucaryotique)Néant

VecteurpTrc

Méthode de clonageEnzyme de restriction / MCS

AccélérateurTrc, lacO

Marqueur de protéineThiorédoxine

Unit Size20 µg

Contenu et stockage

Tous les vecteurs sont fournis en suspension. Un stab d’E. coli TOP10 et un contrôle d’expression positive sont également fournis. Conserver les vecteurs à -20°C. Stocker le stab à température ambiante. La stabilité du vecteur est garantie pendant 6 mois lorsqu’il est correctement conservé.

Foire aux questions (FAQ)

What are the advantages and/or disadvantages of using TOP10, DH5α, or other cloning strains versus BL21 Star (DE3) or BL21 (DE3) cells for expression with the pTrc system?

Top10, DH5α, other cloning strains

Advantages:
- Saves time, can go directly from cloning to expression.
- The glycerol stock is more stable because these strains are endA- and recA-.

Disdvantages:
- If GOI is toxic, the cloning step can be difficult.
- These cloning strains are not protease-deficient; therefore, the protein may be degraded.

BL21 Star(DE3) or BL21 (DE3)

Advantages:
- These expression strains are protease-deficient.
- You have to transform the plasmid into an expression strain.
- The glycerol stock may be unstable because these expression strains are not endA- and recA-.
- The (DE3) part is wasted because the promoter does not need T7 RNA polymerase.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What competent cell strain can I use for expression with my pTrc Expression System?

The pTrc promoter is recognized by E. coli RNA polymerase, not T7 polymerase, and therefore can be expressed in any E. coli strain, not just BL21 strains. Therefore, you can use Top10, DH5α, etc. for expression. However, if your gene is toxic, the cloning step can be difficult as there is leakiness in expression.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How does glucose repress the pTrc promoter?

A transcriptional activator protein called CAP (catabolite activator protein) normally binds upstream of the trc promoter and activates transcription. This protein requires cAMP to bind the DNA. Adding glucose to the medium can reduce intracellular cAMP levels. Supplementing LB medium and agar plates with glucose will repress basal level transcription from the trc promoter. We recommend that you include 25 mM, 0.5% glucose in the selection medium to ensure stability of your insert.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What are the benefits of the pTrc expression system?

The pTrc promoter in the pTrc system is a strong hybrid promoter composed of the -35 region of the trp promoter and the -10 region of the lacUV5 promoter/operator. Expression of pTrc is repressed by the LacI protein and induced by IPTG.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

View all pTrcHis A, B, & C Bacterial Expression Vectors FAQs

Citations et références (54)

Citations et références

Abstract

Metaxin is a component of a preprotein import complex in the outer membrane of the mammalian mitochondrion.

Authors:Armstrong LC, Komiya T, Bergman BE, Mihara K, Bornstein P

Journal:J Biol Chem

PubMed ID:9045676

'Metaxin, a novel gene located between the glucocerebrosidase and thrombospondin 3 genes in the mouse, is essential for survival of the postimplantation mouse embryo. In this study, the subcellular location, domain structure, and biochemical function of metaxin were investigated. Anti-recombinant metaxin antibodies recognized 35- and 70- kDa proteins in mitochondria ... More

Dual DNA binding specificity of ADD1/SREBP1 controlled by a single amino acid in the basic helix-loop-helix domain.

Authors:Kim JB, Spotts GD, Halvorsen YD, Shih HM, Ellenberger T, Towle HC, Spiegelman BM

Journal:Mol Cell Biol

PubMed ID:7739539

'Adipocyte determination- and differentiation-dependent factor 1 (ADD1), a member of the basic helix-loop-helix (bHLH) family of transcription factors, has been associated with both adipocyte differentiation and cholesterol homeostasis (in which case it has been termed SREBP1). Using PCR-amplified binding analysis, we demonstrate that ADD1/SREBP1 has dual DNA sequence specificity, binding ... More

Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies.

Authors:Tingley WG, Ehlers MD, Kameyama K, Doherty C, Ptak JB, Riley CT, Huganir RL

Journal:J Biol Chem

PubMed ID:9030583

'Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N- methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine ... More

Ca2+/calmodulin binds to and modulates P/Q-type calcium channels [see comments]

Authors:Lee A, Wong ST, Gallagher D, Li B, Storm DR, Scheuer T, Catterall WA

Journal:Nature

PubMed ID:10335845

'Neurotransmitter release at many central synapses is initiated by an influx of calcium ions through P/Q-type calcium channels, which are densely localized in nerve terminals. Because neurotransmitter release is proportional to the fourth power of calcium concentration, regulation of its entry can profoundly influence neurotransmission. N- and P/Q-type calcium channels ... More

Oligomerization-dependent association of the SAM domains from Schizosaccharomyces pombe Byr2 and Ste4.

Authors: Ramachander Ranjini; Kim Chongwoo A; Phillips Martin L; Mackereth Cameron D; Thanos Christopher D; McIntosh Lawrence P; Bowie James U;

Journal:J Biol Chem

PubMed ID:12171939

'SAM (sterile alpha motif) domains are protein-protein interaction modules found in a large number of regulatory proteins. Byr2 and Ste4 are two SAM domain-containing proteins in the mating pheromone response pathway of the fission yeast, Schizosaccharomyces pombe. Byr2 is a mitogen-activated protein kinase kinase kinase that is regulated by Ste4. ... More

View all pTrcHis A, B, & C Bacterial Expression Vectors citations

pTrcHis A, B, & C Bacterial Expression Vectors

Foire aux questions (FAQ)

What are the advantages and/or disadvantages of using TOP10, DH5&alpha;, or other cloning strains versus BL21 Star (DE3) or BL21 (DE3) cells for expression with the pTrc system?

What competent cell strain can I use for expression with my pTrc Expression System?

How does glucose repress the pTrc promoter?

What are the benefits of the pTrc expression system?

Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

Citations et références (54)

What are the advantages and/or disadvantages of using TOP10, DH5α, or other cloning strains versus BL21 Star (DE3) or BL21 (DE3) cells for expression with the pTrc system?