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查看更多产品信息 BaculoDirect™ N-Term Transfection Kit - FAQs (12562062)
17 个常见问题解答
请查看以下建议：
•所用MOI不正确；应确保对病毒储液用量进行正确计算；所用MOI为5-10。您可能需要根据重组蛋白表达的动力学，测试MOI的范围。
•蛋白质可能在细胞裂解过程中丢失；如果您检测的是细胞内蛋白，则应该对上清液进行分析，从而确定蛋白质是否因细胞裂解而丢失。
•蛋白质降解了或不稳定；在细胞裂解物中加入蛋白酶抑制剂，和/或检验mRNA水平。
•目标蛋白对细胞具有毒性作用；在较早的时间点收获细胞（如，感染后18-24小时）。
利用杆状病毒信号序列，已有效分泌蛋白。请查看以下参考文献：
•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.
研究者已成功使用蜂毒素分泌序列。请查看以下参考文献：
•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.
TK基因是利用更昔洛韦对非重组病毒进行负筛选。
我们建议使用Sf9或Sf21细胞，生成高滴度病毒储液。我们不推荐使用High Five细胞生成病毒储液，因为其转染效率较低。生成高滴度病毒储液后，您可使用Sf9、Sf21、High Five或Mimic Sf9细胞进行蛋白表达。
开始BaculoDirect实验时，将目的基因克隆到Gateway入门载体中，随后利用LR Clonase反应克隆到BaculoDirect载体中。将该载体转染到您的细胞中并生长3天。在第4天，收集P1病毒储液。再次感染细胞，生长3天。在第7天，收集P2病毒储液。感染细胞，在第10天收集蛋白并纯化。
尽管Kozak序列在哺乳细胞翻译起始中的重要性已被证明，但昆虫细胞中是否也严格遵循Kozak规则仍有争议。确定其重要性的唯一方法是，对相同蛋白从不同起始序列的表达进行直接对比。即使这样，一种蛋白实现最佳表达所使用的规则可能并不适合其它蛋白。以下四篇文献证明了Kozak序列对昆虫细胞中的表达效率无任何影响：
•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通常对于高效的翻译启始是足够的，尽管翻译效率要视目的基因而定。最佳的建议应是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有N-端融合型表达载体都已包含了一个RBS或翻译起始位点。
原核生物mRNA含有Shine-Dalgarno序列，也称为核糖体结合位点(RBS)，它是由AUG起始密码子5’端的多嘌呤序列AGGAGG组成。该序列与16S rRNA 3’端的互补，有助于mRNA有效结合到核糖体上。同理，真核生物（特别是哺乳动物）mRNA也含有完成有效翻译所需的重要序列信息。然而，Kozak序列不是真正的核糖体结合位点，而是一种翻译起始增强子。Kozak共有序列是ACCAUGG，其中AUG是起始密码子。-3位的嘌呤(A/G)具有重要作用；若-3位是一个嘧啶(C/T)，翻译过程会对-1、-2和+4位的改变更敏感。当-3位从嘌呤变为嘧啶时，可使表达水平降低多达95%。+4位对表达水平的影响相对较小，可以使表达水平降低约50%。
注：果蝇的最佳Kozak序列稍有不同，酵母完全不遵循这些规则。见下列参考文献：
•Foreign Gene Expression in Yeast: a Review. Yeast, vol. 8, p. 423-488 (1992).
•Caveneer, Nucleic Acids Research, vol. 15, no. 4, p. 1353-1361 (1987).
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.