Search
Search
View additional product information for BaculoDirect™ N-Term Transfection Kit - FAQs (12562062)
9 product FAQs found
Please see the following suggestions:
- The incorrect MOI was used; ensure that the amount of viral stock was calculated correctly, and that an MOI of 5-10 was used. You may need to test a range of MOIs depending on the kinetics of expression of your recombinant protein.
- The protein may be lost during cell lysis; if you are trying to detect an intracellular protein, analyze the supernatant to determine if the protein is being lost due to cell lysis.
- The protein is being degraded or unstable; add protease inhibitors to your cell lysates and/or check mRNA levels.
- The protein of interest is toxic to the cells; harvest the cells at earlier time points (e.g., 18-24 hr post-infection).
Proteins have been efficiently secreted utilizing baculovirus signal sequences. Please see the following references:
- Kuhn S, Zipfel PF (1995) The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins. Gene 162:225-229.
- Krol BJ, Murad S, Walker LC, Marshall MK, Clark WL, Pinnell SR, Yeowell HN (1996) The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system. J Invest Dermatol 106:11-16.
Investigators have been successful with the honeybee melittin secretion sequence. Please see the following references:
- Tessier DC, Thomas DY, Khouri HE, Laliberté F, Vernet T (1991) Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide. Gene 98:177-183.
- Garnier L, Cahoreau C, Devauchelle G, Cérutti M (1995) The intracellular domain of the rabbit prolactin receptor is able to promote the secretion of a passenger protein via an unusual secretory pathway in lepidopteran cells. BioTechnology 13:1101-1104.
- Vihko P, Kurkela R, Porvari K, Herrala A, Lindfors A, Lindqvist Y, Schneider G (1993) Rat acid phosphatase: Overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization. Proc Natl Acad Sci U S A 90:799-803.
- Mroczkowski BS, Huvar A, Lernhardt W, Misono K, Nielson K, Scott B (1994) Secretion of thermostable DNA polymerase using a novel baculovirus vector. J Biol Chem 269:13522-13528.
The TK gene is for negative selection of non-recombinant virus using ganciclovir.
We recommend using Sf9 or Sf21 cells to generate high-titer viral stocks. We do not recommend using High Five cells to generate viral stocks due to lower transfection efficiency. Once you have generated your high-titer viral stocks, you can use Sf9, Sf21, High Five, or Mimic Sf9 cells for protein expression.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Begin your BaculoDirect experiments by cloning in your gene of interest into your Gateway Entry Vector, followed by the LR Clonase reaction into the BaculoDirect vector. Transfect this vector into your cells and grow for 3 days. On the 4th day, collect P1 viral stock. Re-infect cells, and grow for 3 days. On the 7th day, collect P2 viral stock. Infect cells, followed by harvesting of protein and purification on the 10th day.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
A precipitate may sometimes appear after thawing the ganciclovir solution. If this occurs, heat the solution to 37C in a water bath for 5-10 minutes and vortex a few times. The precipitate should go back into solution. For future use, it is best to aliquot the remainder and store at -20C.
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.