pIZ/V5-His Vector Kit
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Invitrogen™

pIZ/V5-His Vector Kit

The InsectSelect™ Kit features the pIZ/V5-His vector for high-level expression in a variety of insect cells. This exceptionally small vectorRead more
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Catalog NumberQuantity
V800001
also known as V8000-01
1 kit
Catalog number V800001
also known as V8000-01
Price (USD)
1,218.00
1 kit
Add to cart
Quantity:
1 kit
Price (USD)
1,218.00
1 kit
Add to cart
The InsectSelect™ Kit features the pIZ/V5-His vector for high-level expression in a variety of insect cells. This exceptionally small vector has several features that facilitate expression, analysis, and detection of recombinant protein in insect cells:

• The OpIE2 promoter for constitutive expression
• The Zeocin™ resistance gene for rapid selection of stably transfected cell lines
• C-terminal V5 epitope and polyhistidine (6xHis) sequence for detection with Invitrogen's Anti-V5 Antibody and rapid purification using nickel-chelating resin
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Product TypeInsect Cell Expression Vector
Protein Tag Position (to your gene)C-terminal
Quantity1 kit
VectorpIZ
Cloning MethodRestriction Enzyme/MCS
Product LineInsectSelect™
PromoterOplE2
Protein TagHis Tag (6x), V5 Epitope Tag
Unit Size1 kit
Contents & Storage
The InsectSelect™ Kit includes 20 μg each of supercoiled, lyophilized pIZ/V5-His and pIZ/V5-His/CAT, 2 μg of lyophilized OpIE2 Reverse Sequencing primer, 1 g Zeocin™, Cellfectin™ Reagent, and 1 L of medium for the insect cell line chosen. Sf9 cells are supplied with GIBCO™ Grace's Insect Media, High Five™ Cells are supplied with GIBCO™ Express Five™ Serum-Free Medium. The pIZ/V5-His Vector Kit contains only the expression vectors and primer as described above. Store frozen insect cells in liquid nitrogen. Store medium and Cellfectin™ Reagent at +4°C. Store all other reagents at -20°C. All reagents are guaranteed stable for 6 months when properly stored.

Frequently asked questions (FAQs)

Do I need to include a Kozak sequence for expression of recombinant proteins in insect cells?

While the importance of a Kozak consensus sequence in translation initiation has been demonstrated in mammalian cells, there seems to be some debate as to whether the Kozak rules are as stringent in insect cells. The only way to determine its importance would be a direct comparison of expression of the same protein from different initiation sequences. Even then, the rules for optimal expression of one protein may not hold for another. Here are two references which indicate that a Kozak consensus sequence does not have any effect on efficiency of expression in insect cells:

- Hills D, Crane-Robinson C (1995) Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression.
- Biochim Biophys Acta 1260(1):14-20.
- Ranjan A, Hasnain SE (1995) Influence of codon usage and translational initiation codon context in the AcNPV-based expression system: computer analysis using homologous and heterologous genes. Virus Genes 9(2):149-153.

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.

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.

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