Learn More
Invitrogen™ DES™-Inducible/Secreted Kit with pCoHygro
Description
Includes
20μg pCoHygro, pMT/BiP/V5-His A, B, C and pMT/BiP/V5-His/GFP, Calcium Phosphate Transfection Kit, forward and reverse primers (2μg each), GIBCO Schneider's Drosophila Medium, frozen S2 cells, and the appropriate selection agent.
- Offers inducible metallothionein promoter that is induced upon addition of copper sulfate or cadmium chloride
- Small size (3.6 kb) to improve DNA yields and increase subcloning efficiency
- C-terminal V5 epitope tag for rapid detection with Anti-V5 Antibody
- C-terminal 6xHis tag for simple purification of recombinant fusion proteins using nickel-ch
- N-terminal signal sequence from insect BiP gene is provided to direct recombinant fusion protein through secretory pathway of S2 cells into culture medium
- Small size (3.6 kb) to improve DNA yields and increase subcloning efficiency
- C-terminal V5 epitope tag for rapid detection with Anti-V5 Antibody
- C-terminal 6xHis tag for simple purification of recombinant fusion proteins using nickel-chelating resin
- To facilitate cloning, set of three vectors, A, B and C is provided; Each vector has multiple cloning site in different reading frame relative to the BiP signal sequence
Inducible Protein Expression in Insect Cells, Insect Expression, Protein Expression, Proteins, Expression, Isolation and Analysis, Secreted Protein Expression in Insect Cells
Specifications
Specifications
| Inducing Agent | Copper Sulfate |
| Product Type | Inducible/Secreted Kit |
| Content And Storage | The DES™ Inducible/Secreted Kits contains 20 μg of each of a selection vector (pCoHygro or pCoBlast), pMT/BiP/V5-His A, B, C and pMT/BiP/V5-His/GFP, Calcium Phosphate Transfection Kit, forward and reverse primers (2 μg each), GIBCO™ Schneider's Drosophila Medium, frozen S2 cells, and the appropriate selection agent. Store S2 cells in liquid nitrogen. Store medium and selection agent at +4°C. Store all other reagents at -20°C. All components are guaranteed stable for 6 months when properly stored. |
| Protein Tag | His Tag (6x), V5 Epitope Tag |
| Cloning Method | Restriction Enzyme/MCS |
| Quantity | 1 kit |
| Vector | pMT |
| Product Line | DES |
Frequently Asked Questions (FAQs)
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.
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.
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.
When grown at the optimal temperature of 27-28ºC, Drosophila S2 cells grow very quickly, with an approximate 14-16 hr doubling time. The cells can also be grown at 22-25ºC with doubling time of 24hrs. Humidity is an issue, so a shallow pan of water can be left in the incubator, but the incubator does not need to be turned on.
Growth medium is also a factor in doubling time - the doubling times listed here are for Sf900 II medium.
The cells can reach maximum densities of 1 to 2.5 x 10E7 cells/ml in shake flask cultures.
For Research Use Only. Not for use in diagnostic procedures.