Invitrogen™ pDisplay™ Mammalian Expression Vector

Catalog No.	Quantity
V66020	20 μg

Catalog No. V66020 Supplier Invitrogen™ Supplier No. V66020

Description

Includes

20μg pDisplay vector is supplied supercoiled and lyophilized.

Designed to target recombinant proteins to the surface of mammalian cells

• Displayed proteins can be analyzed for their ability to interact with known or putative ligands
• Proteins of interest are targeted and anchored to cell surface by cloning the gene of interest in frame with vectors unique N-terminal secretion signal and C-terminal transmembrane anchoring domain of platelet-derived growth factor receptor (PDGFR)
• In contrast to phage display vectors which operate exclusively in prokaryotic cells, pDisplay vector offers advantage of having displayed protein of interest processed in mammalian cells
• Therefore, recombinant proteins of eukaryotic origin that are expressed from pDisplay more closely resemble their native form: this may favor more accurate ligand binding interactions
• In addition to N-terminal cell surface targeting signal and C-terminal transmembrane anchoring domain, pDisplay vector contains T7 promoter/priming site for in vitro transcription of sense RNA and for sequencing of inserts
• Neomycin resistance marker for stable selection in mammalian cells using Geneticin™
• SV40 origin for replication and simple vector rescue in cell lines expressing large T antigen (e.g.,COS-1 and COS-7)
• Ampicillin resistance gene for selection in E. coli

Mammalian Two-Hybrid, Protein-Protein Interactions, Proteins, Expression, Isolation and Analysis

Specifications

• Delivery Type: Transfection
• Promoter: CMV
• Product Type: Mammalian Expression Vector
• Selection Agent (Eukaryotic): Geneticin™ (G-418)
• Content And Storage: 20 μg pDisplay vector is supercoiled and supplied as a 0.5 μg/mL solution in TE buffer, pH 8.0. Store at -20°C. Guaranteed stable for 6 months when properly stored.
• Protein Tag: IgK Leader Sequence, PDGFR Transmembrane Domain, c-Myc Epitope Tag, HA Tag
• Cloning Method: Restriction Enzyme/MCS
• Quantity: 20 μg
• Vector: pDisplay
• Product Line: pDISPLAY

Frequently Asked Questions (FAQs)

I used the pDisplay vector to express my protein on the cell surface. I'm able to detect the protein on a western blot using anti-HA antibody, but not with immunofluorescence using anti-myc antibody. Can you please help troubleshoot?

We would recommend checking to make sure that the gene of interest is cloned in-frame with the Platelet-derived Growth Factor Receptor (PDGFR) transmembrane domain.

Can I use FACS cell sorting or magnetic bead-based methods to isolate my cells transfected with the pDisplay vector?

When we tried FACS cell sorting of pDisplay-transfected cells using our anti-myc antibody, the sorting was not very efficient. However, pDisplay-transfected cells can be isolated using magnetic beads by first incubating the cells with anti-myc antibody and then incubating the cell-antibody complex with magnetic beads that have anti-mouse IgG1 conjugated to them. The cell can then be isolated using a magnet.

I used pDisplay vector to express a cell surface receptor protein. Should I be concerned about digestion of the protein during trypsinization of the cells?

Trypsin digests proteins at arginine or lysine residues, and it is widely accepted that the extracellular domains of membrane proteins are digested upon trypsinization of cells. But typically these proteins are rapidly replaced once the trypsin is removed. As an alternative to trypsin, you can remove most adherent cells using Versene (Cat. No. 15040066), which is a sterile 0.5 mM EDTA solution in PBS, and/or scraping. The EDTA chelates any free Mg2+ or Ca2+ ions, which are necessary for maintaining many cell attachments. Cell Dissociation Buffer, Enzyme-free, Hank's-based (Cat. No. 13150016) or PBS-based (Cat. No. 13151014), which are cocktails of chelating agents, are also fine for this application and may be more effective than EDTA alone, and would be unlikely to adversely affect the receptor protein.

If I transfect empty pDisplay vector into mammalian cells, will it result in a cell membrane-targeted polypeptide?

Transfection of pDisplay vector alone into mammalian cells does not result in a displayed polypeptide because the Ig kappa signal peptide and the PDGFR transmembrane domain are not in the same reading frame, and the ORF containing the kappa signal peptide ends 5 bp before the start of what is defined as the PDGFR transmembrane domain.

I heard that you carry a vector to help secrete my protein so it is targeted to the cell membrane. Which one is it?

We offer the pDisplay vector, Cat. No. V66020, designed to target recombinant proteins to the surface of mammalian cells. It contains an N-terminal murine Ig kappa-chain secretion signal and a C-terminal transmembrane anchoring domain from Platelet-derived Growth Factor Receptor (PDGFR) to target and anchor the protein of interest to the cell surface.

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.

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.