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Invitrogen™ pTrcHis2 A, B, & C Bacterial Expression Vectors

Catalog No. V36520
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20 μg
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V36520 20 μg
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Catalog No. V36520 Supplier Invitrogen™ Supplier No. V36520
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Description

Includes

All vectors are supercoiled and are provided lyophilized. A TOP10 E. coli stab and positive expression control are provided.

Offer enhanced translation initiation and high-level expression in E. coli

  • These vectors feature high-level regulated transcription from the trc promoter
  • Enhanced translation efficiency of eukaryotic genes in E. coli
  • The lacO operator and lacIq repressor gene for transcriptional regulation in any E. coli strain
  • This particular vector offers C-terminal polyhistidine (6xHis) tag for rapid purification with nickel-chelating resin and detection with an anti-HisG antibody
  • C-terminal Xpress epitope for easy detection with an anti-Xpress antibody
  • Enterokinase cleavage site for removal of fusion tag

Bacterial Expression, Protein Expression, Proteins, Expression, Isolation and Analysis

Specifications

Constitutive or Inducible System Inducible
Inducing Agent IPTG
Promoter Trc, lacO
Product Type Bacterial Expression Vector
Selection Agent (Eukaryotic) None
Content And Storage All vectors are supercoiled and are provided lyophilized. A TOP10 E. coli stab and positive expression control are provided. Store vectors at -20°C. Store stab at room temperature. The vector is guaranteed stable for 6 months when properly stored.
Antibiotic Resistance Bacterial Ampicillin (AmpR)
Cleavage EK (Enterokinase) Recognition Site
Protein Tag Thioredoxin
Cloning Method Restriction Enzyme/MCS
Quantity 20 μg
Vector pTrc
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Frequently Asked Questions (FAQs)

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.
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.

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.

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.

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.
Can BL21 cells be used to express protein(s) from pTrc vectors (pTrcHis, pTrcHis2, pTrcHis-TOPO, pTrcHis2-TOPO)?

BL21 strains are deficient in ompT and lon proteases, making them desirable as protein expression strains. However, many BL21(DE3)-based strains express T7 RNA polymerase under the control of the lacUV5 promoter, which is IPTG inducible, as is the Trc promoter in the vector. Therefore, we do not recommend the use of any BL21(DE3)-based strain [BL21(DE3), BL21(DE3)pLysE, BL21 Star(DE3), BL21(DE3)pLysS], due to the possibility of competing RNA polymerase activity after induction with IPTG.

BL21-AI is a strain which utilizes an arabinose-inducible promoter to drive T7 RNA polymerase.  The BL21-AI strain is not induced by IPTG so it is a better choice for pTrc vectors.
Can you tell me the difference between a Shine-Dalgarno sequence and a Kozak sequence?

Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:

- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.

Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:

- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
What is the source of the EM7 promoter?

The EM7 promoter is a synthetic promoter derived from the T7 promoter. The promoter is typically used to drive expression of antibiotic resistance genes for selection in E. coli.
Will adding more than the recommended 1 mM of IPTG yield better protein expression in the pTrc and T7-based expression systems?

IPTG is added to induce expression of the T7 RNA polymerase gene under control of the lac promoter in BL21 cells. Increasing the amount of IPTG further usually does not affect expression levels because of the high efficiency of T7 RNA polymerase. In the pTrc system, 1 mM is sufficient to inactivate the lacIq protein expressed in the cell.
What protein yields should I expect to get with the pRSET expression vectors?

We've expressed CAT and Beta-Gal in volumes from 2 ml to 50 ml and get about 20-50 µg protein/ml of culture. Protein yield is dependent on the type of protein being expressed and the culturing conditions.
Which form of arabinose should be used for induction in the pBAD Expression System, L- or D-arabinose?

The chirality of arabinose used for induction is very important. L-arabinose works great, but D-arabinose doesn't induce at all. L-arabinose is available from Sigma (catalog# A3256).
I sequenced one of your vectors after PCR amplification and observed a difference from what is provided online (or in the manual). Should I be concerned?

Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.

Are your vectors routinely sequenced?

No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.

How was the reference sequence for your vectors created?

Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.

What is a Shine-Dalgarno sequence?

Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. The Shine-Dalgarno sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA.

For Research Use Only. Not for use in diagnostic procedures.