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查看更多产品信息 ViraPower™ HiPerform™ T-REx™ Gateway™ Vector Kit - FAQs (A11144)
55 个常见问题解答
在BP/LR Clonase反应的一步法实验方案中，不建议用BP Clonase酶和LR Clonase酶替代BP Clonase II 酶/LR Clonase II酶，因为这样的重组效率非常低。
有的，我们能提供针对BP/LR Clonase反应的一步式实验方案DNA可以在一步反应后被克隆到目的载体中，从而节省了您的时间和金钱。
建议使用一个供体载体进行一次BP反应以获得一个入门克隆。然后将这一入门克隆和目的载体进行一次LR反应以获得新的表达克隆。
5X LR Clonase缓冲液或5X BP Clonase缓冲液不作为单独产品出售。它们作为酶试剂盒的一部分进行销售。
我们不提供任何用于在植物内表达的Gateway载体。
这里列举了一些可能的原因与解决方案：
- 相对表达目的基因的病毒而言，Lenti3.3/TR病毒转导的MOI值过低：向哺乳动物细胞转导Lenti3.3/TR病毒时需使用比表达质粒病毒（一般MOI=1-5）更高的MOI值（如MOI=10）。
- 转导Lenti3.3/TR病毒之后和转导Lenti6.3/TO/V5-DEST病毒之前，未等待足够长的时间：向哺乳动物中转导Lenti3.3/TR病毒后等待24小时再转导Lenti6.3/TO/V5-DEST病毒。
这可能是由Lenti6.3/TO/V5-DEST载体转导进不表达Tet抑制子的细胞所致。首先建立一株ViraPower T-REx细胞系，之后将其作为Lenti6.3/TO/V5-DEST的宿主。您可能需要确认在细胞生长培养基中未使用含较高四环素水平的FBS。
这里列举了一些可能的原因与解决方案：
- 加入四环素后过早收获和分析细胞：加入四环素后将细胞培养更长时间，再分析重组蛋白的表达情况。请勿在加入四环素24小时之内收获细胞。
-Lenti6.3/TO/V5-DEST慢病毒母液未经滴度测定：使用 手册第21页的步骤测定慢病毒母液。
- Lenti6.3/TO/V5-DEST慢病毒母液未经正确保存：分装母液并将其保存于–80°C条件下；请勿将病毒母液冻融超过3次；如果储存时间超过6个月，请在使用前重新测定病毒滴度。
这里列举了一些可能的原因与解决方案：
-Lenti3.3/TR载体整合到基因组的失活区：筛选其他的Geneticin抗性克隆。选择一株表现出Tet抑制子最高表达水平的细胞克隆，来作为Lenti6.3/TO/V5-DEST载体的宿主。
-将Lenti3.3/TR转进CMV启动子下调的哺乳动物细胞系中：换用另一种哺乳动物细胞系来进行转导实验。
我们提供了将ViraPower HiPerform慢病毒与T-REx技术整合在一起的ViraPower HiPerform T-REx Gateway表达系统（货号A11141），本试剂盒用于在分裂或非分裂型哺乳动物细胞中提升慢病毒介导的目的基因的调控性高表达。ViraPower HiPerform T-REx Gateway载体试剂盒可单独购买（货号A11144）。
这里列举了一些可能的原因与解决方案：
•所用检测法可能不适当或不够灵敏： ◦我们推荐您优化检测方案或寻找更为灵敏的方法。如果使用考马斯亮蓝染色/银染法检测过该蛋白，我们则推荐您使用免疫印迹法来增加检测灵敏度。裂解产物中存在的内源蛋白可能会在考马斯亮蓝染色/银染过程中掩盖目的蛋白。如果可能，我们推荐您在免疫印迹实验中包括一个阳性对照。
•筛选到的克隆数不够：至少筛选出20个克隆。
•在稳转筛选中使用了不适当的抗生素浓度：请确保正确获取了抗生素的杀死曲线。由于某一既定抗生素的效力依赖于细胞类型，血清，培养基和培养技术，因此必须在每次进行稳定筛选的时候确定抗生素的用量。如果采用的培养基或血清条件明显不同，则即使是我们所提供的稳转细胞系对于我们推荐的剂量也可能出现更敏感或更不敏感的情况。
•基因产物（即使低水平）的表达可能与该细胞系的生长不相容（如毒性基因）：使用一个可诱导的表达系统。
•阴性克隆可能由基因表达的关键载体位点处优先发生了线性化所致：在一个不影响表达的位点实施载体线性化，如在细菌抗药性标志物序列中。
这里列举了一些可能的原因与解决方案：
•尝试试剂盒自带的表达对照。
•可能的检测问题：
◦检测瞬转的表达蛋白可能有难度，因为转染效率可能过低，以致用于整个转染群体的评估手段无法成功实现检测。我们推荐您通过稳转筛选或采用能够逐个检测单一细胞的技术手段来优化您的转染操作。您也可尝试通过改变启动子或细胞类型来提高表达水平。
◦细胞中的蛋白表达水平对于所选择的检测方法来说可能过低。我们推荐您优化检测方案或寻找更为灵敏的方法。如果使用考马斯亮蓝染色/银染法检测过该蛋白，我们则推荐您使用免疫印迹法来增加检测灵敏度。裂解产物中存在的内源蛋白可能会在考马斯亮蓝染色/银染过程中掩盖目的蛋白。如果可能，我们推荐您在免疫印迹实验中包括一个阳性对照。
◾蛋白可能降解或截短了：使用Northern杂交进行检测。
◾可能的时程问题：由于蛋白表达随时间延长而发生的变化依赖该蛋白的天然属性，我们一般推荐您先获取一份表达的时程曲线。尝试进行一次时程分析将帮助您确定最优的表达时间窗。
◾可能的克隆问题：通过限制性酶切和/或测序来验证克隆。
不可以；新霉素对哺乳动物细胞有毒性。我们推荐您使用Geneticin（又称 G418硫酸盐)，这一产品的毒性较低，是在哺乳动物细胞中进行有效筛选的新霉素的替代品。
即使缺乏Kozak序列，翻译也还是会在核糖体遇到的第一个ATG处启始，不过启始效率可能相对较低。只要处于最初ATG的阅读框内，任何下游的插入序列都可能表达为融合蛋白，不过如果这里没有Kozak保守序列，则蛋白的表达水平预期会比较低。如果载体中包含一个非Kozak型的保守ATG，我们则推荐您将基因克隆至该ATG上游，再包含一个Kozak序列来优化表达效果。
当执行共转染时，用户无法在稳转细胞系中同时完成功能性TetR或GeneSwitch蛋白的双重测试。另一方面，如果执行连续转染，用户就可对所生成的T-REx或GeneSwitch细胞系进行功能性测试，他们可将LacZ对照表达质粒瞬转进入细胞，并挑取那些在诱导剂缺乏条件下表达LacZ的本底水平最低，而在含诱导剂条件下LacZ表达水平最高的克隆。之后可对这一克隆进行扩增，并按需用于T-REx或GeneSwitch表达载体的转染实验。
使用GeneSwitch系统能够确保目的基因处于极低的基础表达水平，而T-REx系统可能会有少量渗漏表达，因为FBS中不可避免的存在着一些四环素。GeneSwitch系统的诱导表达水平可能甚至高于CMV启动子。GeneSwitch系统的劣势在于，尽管该系统能够在转基因条件下以优异的性能工作，但在培养系统中关闭表达的操作不是很容易实现。而另一方面，T-REx系统可通过加入和去除诱导剂来切换开关状态。
Flp-In T-REx系统将Flp-In系统的靶向整合与T-REx系统的强大诱导表达能力整合在一起。该系统能够生成同基因，可诱导的稳定表达细胞系，并能够针对这些细胞系实行多克隆筛选。一旦建立了包含整合FRT位点的Flp-In T-REx宿主细胞系，后续建立表达目的基因的Flp-In T-REx细胞系就变得迅速高效了。
强力霉素可作为T-REx系统中诱导剂的代替品。它的作用机理与四环素相近，并在T-REx系统中表现出与四环素相似的剂量效应和诱导性质。强力霉素显示出比四环素更长的半衰期（分别为48小时和24小时）。我们未提供强力霉素，但用户可从Sigma（货号D9891）进行购买。
我们提供三类独特的哺乳动物表达系统来帮助用户实现目的基因可诱导/调控性的表达。
•T-REx系统
•Flp-In T-REx系统
•GeneSwitch系统
请参见下表中的比较结果：
系统 --基础表达水平--诱导表达水平--表达最大化的响应时间--转基因应用
T-REx系统--低--最高--高--合适
Flp-In T-REx系统--较低--高--24-48小时--合适
GeneSwitch系统--最低--高--24-48小时--合适
我们提供pJTI R4 Exp CMV EmGFP pA载体，货号A14146，您可使用这一产品来监控转染和表达情况。
我们推荐使用One Shot ccdB Survival 2 T1^R 感受态细胞，货号A10460。该菌株能够耐受ccdB基因的毒性效应。
注意： 请勿使用常规的大肠杆菌克隆株 - 包括TOP10或DH5α - 来进行扩增和培养，因为这些菌株均对ccdB的效应很敏感。
在小鼠细胞系中，人们已知CMV启动子的效率会随时间延长而逐渐下降。因此，我们推荐您使用一款非CMV型的载体，如EF1α或UbC启动子，以在小鼠细胞系中长时间表达蛋白。
保守的Kozak序列为A/G NNATGG，其中的ATG表示起始密码子。ATG周围的核苷酸点突变会影响翻译效率。尽管我们通常情况下都推荐加入一段Kozak保守序列，不过这一操作的必要性还是基于具体的目的基因，一般只需ATG就足以高效地启始翻译过程。最佳的建议是保持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，所有具有N-融合表达的表达载体都已经包含了一个翻译起始位点。
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).
In the single-step protocol for the BP/LR Clonase reaction, we would not recommend substituting the BP Clonase II/LR Clonase II enzymes with BP Clonase /LR Clonase enzymes as this would result in very low recombination efficiency.
Yes, we have come up with a single-step protocol for BP/LR Clonase reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.
We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.
We do not offer the 5X LR Clonase buffer and 5X BP Clonase buffer as standalone products. They are available as part of the enzyme kits.
We do not offer any Gateway vectors for expression in plants.
Here are some possible causes and solutions:
- Transduced Lenti3.3/TR viral construct at too low of an MOI when compared to the epxression construct; Transduce the Lenti3.3TR viral construct into mammalian cells at a higher MOI (e.g., MOI of 10) than the expression construct (e.g., MOI 1-5)
- Did not wait for a sufficient amount of time after tranducing the Lenti3.3/TR viral construct before transducing the Lenti6.3/TO/V5-DEST viral construct; Transduce mammalian cells with the Lenti6/TR construct, and then wait for 24 hrs before tranducing cells with the Lenti6.3/TO/V5-DEST construct.
This can happen if the Lenti6.3/TO/V5-DEST construct is transduced into cells that do not express the Tet repressor. Generate a ViraPower T-REx cell line first, and then use this cell line as the host for the Lenti6.3/TO/V5-DEST virus. You may also want to confirm that your cells are not being grown in medium with FBS that has high tetracycline levels in it.
Here are some possible causes and solutions:
- Cells harvested and assayed too soon after addition of tetracycline; culture cells for a longer period of time after addition of tetracycline before assaying for recombinant protein epxression (at least 24 hrs)
- Lenti6.3/TO/V5-DEST lentiviral stock not titered; Titer the stock (see page 21 of the manual)
- Lenti6.3/TO/V5-DEST lentiviral stock stored incorrectly; Aliquote and store stocks at -80 degrees C, do not freeze/thaw more than 3 times; re-titer stock before use if stored longer than 6 months
Here are some possible causes and solutions:
- Lenti3.3/TR construct integrated into an inactive region of the genome; Screen other Geneticin-resistant colonies and choose the clone that exhibits the highest level of Tet repressor expression for use as the hose for your Lenti6.3/TO/V5-DEST construct.
- Tranduced Lenti3.3/TR into a mammalian cell line in which the CMV promoter is downregulated; use another mammalian cell line for transduction.
We offer the ViraPower HiPerform T-REx Gateway Expression System (Cat. No. A11141) that combines ViraPower HiPerform Lentiviral and T-REx technologies to facilitate lentiviral-based, regulated, high-level expression of a target gene in dividing or non-dividing mammalian cells. The ViraPower HiPerform T-REx Gateway Vector Kit is available separately (Cat. No. A11144).
Here are possible causes and solutions:
Detection method may not be appropriate or sensitive enough:
- We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
- Insufficient number of clones screened: Screen at least 20 clones.
- Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
- Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.
Here are possible causes and solutions:
- Try the control expression that is included in the kit
Possible detection problem:
- Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
- Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
Protein might be degraded or truncated: Check on a Northern.
Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression.
Possible cloning issues: Verify clones by restriction digestion and/or sequencing.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
No; neomycin is toxic to mammalian cells. We recommend using Geneticin (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells.
Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression.
When a co-transfection is performed, there is no way of testing the double stable cell line for functional TetR or GeneSwitch protein, respectively. On the other hand, when sequential transfection is performed, one can functionally test the generated T-REx or GeneSwitch cell line by transiently transfecting the lacZ expression control plasmid and then picking a clone that shows the lowest basal level of expression of lacZ in the absence of the inducer, and the highest level of lacZ in the presence of the inducer. This clone can then be expanded and used to transfect the T-REx or GeneSwitch expression construct, as the case may be.
With the GeneSwitch system, it is possible to have the absolute lowest basal levels of expression of the gene of interest, whereas the T-REx system may be a little leaky due to the inevitable presence of tetracycline in FBS. The induced level of expression in the GeneSwitch system can be even higher than that seen with the CMV promoter. The disadvantage of the GeneSwitch system is that the expression does not appear to switch off very easily in culture, although it has been demonstrated to function beautifully in transgenics. The T-REx system, on the other hand, can be switched on and off by the addition and removal of the inducer.
The Flp-In T-REx system combines the targeted integration offered by the Flp-In system with the powerful inducible expression offered by the T-REx system. It allows generation of isogenic, inducible, stable cell lines and permits polyclonal selection of these cell lines. Once the Flp-In T-REx host cell line containing an integrated FRT site has been created, subsequent generation of Flp-In T-REx cell lines expressing the gene(s) of interest is rapid and efficient.
Doxycycline may be used as an alternative inducing agent in the T-REx system. It is similar to tetracycline in its mechanism of action, and exhibits similar dose-response and induction characteristics as tetracycline in the T-REx system. Doxycycline has been shown to have a longer half-life than tetracycline (48 hours vs. 24 hours, respectively). We do not offer doxycycline, but it may be obtained from Sigma (Cat. No. D9891).
We offer three unique mammalian expression systems for inducible/regulated expression of the gene of interest:
- T-REx system
- Flp-In T-REx system
- GeneSwitch system
Please see below to see how they compare with one another:
System -- Basal Expression Level -- Induced Expression Level -- Response time to Maximal Expression -- Transgenic Appliation
T-Rex system -- Low -- Highest -- High -- Suitable
Flp-In T-REx system -- Lower -- High -- 24-48 hrs -- Suitable
GeneSwitch system -- Lowest -- High -- 24-48 hrs -- Suitable
We offer pJTI R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.
We recommend using One Shot ccdB Survival 2 T1R Competent Cells, Cat. No. A10460. This strain is resistant to the toxic effects of the ccdB gene. Note: Do not use general E. coli cloning strains, including TOP10 or DH5alpha, for propagation and maintenance, as these strains are sensitive to ccdB effects.
The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1alpha or UbC promoter, for long-term expression in mouse cell lines.
The consensus Kozak sequence is A/G NNATGG, where the ATG indicates the initiation codon. Point mutations in the nucleotides surrounding the ATG have been shown to modulate translation efficiency. Although we make a general recommendation to include a Kozak consensus sequence, the necessity depends on the gene of interest and often, the ATG alone may be sufficient for efficient translation initiation. 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 consensus Kozak. In general, all expression vectors that have an N-terminal fusion will already have an initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
No. The two systems are not compatible since they utilize different strategies for promoter regulation. The T-REx system is designed such that native E. coli tet-repressor protein molecules bind to specific tet-operator sequences (2X TO) just downstream of the TATA box in the full length CMV promoter in the expression vector. This binding keeps the promoter silent simply by preventing the normal transcription machinery from productive assembly at the TATA box. Incidentally, it is this full length CMV promoter region that permits higher induced expression levels relative to other systems.
The recombinant 'repressor' proteins utilized in Clontech's system are actually recombinant fusion proteins which also contain a potent transcriptional transactivator. The Clontech system places operator sequences 5' to the TATA box and relies upon the VP16 transactivator to promote transcription. These repressor-transactivator fusion constructs would have unpredictable and unreliable effects at the CMV promoter in our expression constructs. Additionally, the tet-repressor protein produced from the pCDNA6/TR construct in the T-REx system has no transactivation domain and so would exert little regulatory effect at the minimal promoter region (non-full length CMV) found in the Clontech response plasmids.
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.
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Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.
No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.
Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.
Eukaryotic (and specifically mammalian) mRNA contains sequence information that is 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.