Invitrogen™ pMIB/V5-His Vector Kit

Catalog No.	Quantity
V803001	1 kit

Catalog No. V803001 Supplier Invitrogen™ Supplier No. V803001

Description

Includes

20μg each of supercoiled, lyophilized pMIB/V5-His A, B and C vectors, pMIB/V5-His/CAT control vector, and 2μg each of the OpIE2 Forward and Reverse Sequencing primers.

3.6kb vector designed for constitutive expression and secretion of recombinant proteins from Sf9, Sf21, and High Five insect cells

• Secretion of proteins into serum-free medium simplifies purification, making it easier to harvest protein from cultured cells
• The OpIE2 promoter for constitutive expression
• The honeybee melittin (HBM) secretion signal to allow secreted expression of the protein
• The Blasticidin resistance gene for rapid selection of stably transfected cell lines in two weeks
• C-terminal V5-epitope and polyhistidine tag for detection with Anti-V5 antibody and purification with nickel-chelating resin

Constitutive Protein Expression in Insect Cells, Insect Expression, Protein Expression, Proteins, Expression, Isolation and Analysis, Secreted Protein Expression in Insect Cells

Specifications

• Promoter: OplE2
• Product Type: Insect Cell Expression Vector
• Content And Storage: The pMIB/V5-His Vector Kit contains 20 μg each of supercoiled, lyophilized pMIB/V5-His A, B, and C vectors, pMIB/V5-His/CAT control vector, and 2 μg each of the OpIE2 Forward and Reverse Sequencing primers. Store at -20°C. All reagents are guaranteed stable for 6 months when properly stored.
• Protein Tag Position (to your gene): C-terminal
• Protein Tag: His Tag (6x), V5 Epitope Tag
• Cloning Method: Restriction Enzyme/MCS
• Quantity: 1 kit
• Vector: pMIB
• Product Line: InsectSelect

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.

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.

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