Invitrogen™ pSecTag2 A, B, & C Mammalian Expression Vectors

Catalog No.	Quantity
V90020	20 μg

Catalog No. V90020 Supplier Invitrogen™ Supplier No. V90020

Description

Includes

20μg of each vector and a PSA expression control are supplied supercoiled and lyophilized.

Designed for secretion, purification, and detection of fusion proteins

• Each vector has large multiple cloning site in three reading frames to simplify cloning in frame with N-terminal secretion signal
• Secretion signal from the V-J2-C region of mouse Ig kappa-chain for efficient secretion of recombinant proteins
• Cytomegalovirus (CMV) promoter for high-level constitutive expression
• C-terminal polyhistidine (6xHis) tag for rapid purification with nickel-chelating resin and detection with Anti-His(C-term) Antibody
• C-terminal c-myc epitope for detection with Anti-myc Antibody
• Bovine Growth Hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability
• SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen (e.g., COS-1, COS-7)T
• pSecTag2 vectors carry Zeocin resistance gene for cost-effective selection in mammalian cells.
• Zeocin selection can also be used in E. coli

Constitutive Expression, Mammalian Expression, Protein Expression, Proteins, Expression, Isolation and Analysis, Secreted Expression

Specifications

• Delivery Type: Transfection
• Promoter: CMV
• Product Type: Mammalian Expression Vector
• Selection Agent (Eukaryotic): Zeocin™
• Content And Storage: 20 μg of each vector and a PSA expression control are supplied supercoiled and lyophilized. Store at -20°C. Vectors are guaranteed stable for 6 months when properly stored.
• Protein Tag: His Tag (6x), c-Myc Epitope Tag, IgK Leader Sequence
• Cloning Method: Restriction Enzyme/MCS
• Quantity: 20 μg
• Vector: pSec
• For Use With (Application): Secreted Expression

Frequently Asked Questions (FAQs)

I used the pSecTag2 vector and am seeing cellular but not secreted expression of the protein of interest. Can you offer some tips?

If the gene of interest has a start codon in the context of a perfect Kozak sequence, it may be preferentially translated over the vector’'s initiation codon, resulting in no leader sequence and no glycosylation, and hence no targeting to the endoplasmic reticulum and no secretion. This is rare, but it has been observed. If it is a problem, we recommend using PCR to delete the start codon.

Can I propagate my pSecTag2 vector in E. coli containing the Tn5 gene?

pSectag2 vectors have the Zeocin antibiotic-resistance marker for selection in E. coli, and any E. coli strain that contains the complete Tn5 transposable element (i.e., DH5alphaF'IQ, SURE, SURE2) encodes the ble (bleomycin) resistance gene that confers resistance to the Zeocin antibiotic. Hence, for the most efficient selection, we highly recommend choosing an E. coli strain that does not contain the Tn5 gene.

I see that the pSectag2 vectors carry the Zeocin antibiotic-resistance gene driven by the EM7 promoter. Can I use Zeocin antibiotic for selection when I propagate these vectors in E. coli?

Yes, you can use Zeocin antibiotic for selection in E. coli. However, keep in mind that for Zeocin antibiotic to be active, the salt concentration of the medium must remain low (<90 mM) and the pH must be 7.5. Prepare LB broth and LB agar plates using low-salt (5 g NaCl/liter) LB.

What is the difference between pSecTag2 and pSecTag2/Hygro vectors?

The only difference between these vectors is that the pSecTag2 vectors have the Zeocin antibiotic-resistance gene for stable selection, whereas the pSecTag2/Hygro vectors have the Hygromycin B resistance gene for stable selection.

I would like to get secreted expression of my protein. What are your recommendations?

You can insert an N-terminal secretion signal or leader sequence upstream of your gene and in-frame with the gene sequence to facilitate secreted expression of the protein. We actually offer the pSecTag2 (Cat. No. V90020) and pSecTag2/Hygro (Cat. No. V91020) vectors designed for this purpose. These vectors contain the N-terminal murine Ig kappa-chain secretion signal for secreted expression of the protein of interest.

I performed stable selection but my antibiotic-resistant clones do not express my gene of interest. What could have gone wrong?

Here are possible causes and solutions:

Detection method may not be appropriate or sensitive enough:
- We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
- Insufficient number of clones screened: Screen at least 20 clones.
- Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
- Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.

I used a mammalian expression vector but do not get any expression of my protein. Can you help me troubleshoot?

Here are possible causes and solutions:

- Try the control expression that is included in the kit
Possible detection problem:

- Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
- Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot. Protein might be degraded or truncated: Check on a Northern. Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression. Possible cloning issues: Verify clones by restriction digestion and/or sequencing.

I am using a mammalian expression vector that has the neomycin resistance gene. Can I use neomycin for stable selection in mammalian cells?

No; neomycin is toxic to mammalian cells. We recommend using Geneticin (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells.

Is it okay if my construct has an ATG that is upstream of the ATG in my gene of interest? Will it interfere with translation of my gene?

Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression.

Do you offer a GFP-expressing mammalian expression vector that I can use as a control to monitor my transfection and expression?

We offer pJTI R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.

I am working with a mouse cell line and would like to express my gene at high levels using one of your vectors with the CMV promoter. Do you foresee any problems with this approach?

The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1alpha or UbC promoter, for long-term expression in mouse cell lines.

Do I need to include a consensus Kozak sequence when I clone my gene of interest into one of your mammalian expression vectors?

The consensus Kozak sequence is A/G NNATGG, where the ATG indicates the initiation codon. Point mutations in the nucleotides surrounding the ATG have been shown to modulate translation efficiency. Although we make a general recommendation to include a Kozak consensus sequence, the necessity depends on the gene of interest and often, the ATG alone may be sufficient for efficient translation initiation. 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 consensus Kozak. In general, all expression vectors that have an N-terminal fusion will already have an initiation site for translation.

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.

Do you offer any mammalian expression vectors for secreted expression of the gene of interest?

Yes, we offer two mammalian expression vectors, pSecTag2 (Cat. No. V90020) and pSecTag2/Hygro (Cat. No. V91020), designed for secreted expression, detection, and purification of the protein of interest. Each of these vectors has a large multiple cloning site in three reading frames to simplify cloning in frame with the N-terminal secretion signal. These vectors offer the following features:
-Secretion signal from the V-J2-C region of the mouse Ig kappa chain for efficient secretion of recombinant proteins
-Cytomegalovirus (CMV) promoter for high-level expression
-C-terminal polyhistidine (6xHis) tag for rapid purification with nickel-chelating resin and detection with an Anti-His(C-term) Antibody
-C-terminal c-myc epitope for detection with an Anti-myc Antibody
-Bovine Growth Hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability
-SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen (e.g., COS-1, COS-7)

The pSecTag2 vector has the Zeocin antibiotic resistance gene for selection in E. coli as well as stable selection in mammalian cells whereas the pSecTag2/Hygro has the Hygromycin B resistance gene for stable selection in mammalian cells.

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.

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 the consensus Kozak sequence and what is the function of the Kozak sequence?

Eukaryotic (and specifically mammalian) mRNA contains sequence information that is 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.

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