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查看更多产品信息 pTrcHis A, B, & C Bacterial Expression Vectors - FAQs (V36020)
22 个常见问题解答
TOP10、DH5α、其他克隆菌株
优点：
- 省时，可直接从克隆到表达。
-甘油储液更稳定，因为这些菌株的基因型是endA-和recA-。
缺点：
-如果目的基因具有毒性，则克隆步骤会变得困难。
-这些克隆菌株不是蛋白酶缺陷型的；因此，蛋白质可能被降解。
BL21 Sta（DE3）或BL21（DE3）
优点：
-这些表达菌株是蛋白酶缺陷型的。
缺点：
-您需要将质粒转化到表达菌株中。
-甘油储液可能不稳定，因为这些表达菌株的基因型不是endA-和recA-。
-（DE3）部分是多余的，因为启动子不需要T7 RNA聚合酶。
pTrc启动子可被大肠杆菌RNA聚合酶识别，而不是T7聚合酶，因此，pTrc启动子可在任何大肠杆菌菌株中表达，而不仅仅是BL21菌株。所以，您可使用Top10、DH5α等进行表达。但是，如果您的目的基因具有毒性，则会因表达渗漏而出现克隆困难。
一种被称为CAP（代谢产物活化蛋白）的转录激活蛋白，通常可结合到trc启动子的上游并激活转录。该蛋白需要cAMP才能与DNA结合。在培养基中加入葡萄糖可降低细胞内cAMP水平。在LB培养基和琼脂板中补充葡萄糖，会抑制trc启动子的基础水平转录。为确保插入片段的稳定性，我们建议您在选择培养基中加入25 mM或0.5%葡萄糖。
pTrc系统中的pTrc启动子是一种强杂合启动子，由trp启动子的-35区域和lacUV5启动子/操纵子的-10区域构成。pTrc表达受Lacl蛋白抑制，受IPTG诱导。
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).
Top10, DH5α, other cloning strains
Advantages:
- Saves time, can go directly from cloning to expression.
- The glycerol stock is more stable because these strains are endA- and recA-.
Disdvantages:
- If GOI is toxic, the cloning step can be difficult.
- These cloning strains are not protease-deficient; therefore, the protein may be degraded.
BL21 Star(DE3) or BL21 (DE3)
Advantages:
- These expression strains are protease-deficient.
- You have to transform the plasmid into an expression strain.
- The glycerol stock may be unstable because these expression strains are not endA- and recA-.
- The (DE3) part is wasted because the promoter does not need T7 RNA polymerase.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The pTrc promoter is recognized by E. coli RNA polymerase, not T7 polymerase, and therefore can be expressed in any E. coli strain, not just BL21 strains. Therefore, you can use Top10, DH5α, etc. for expression. However, if your gene is toxic, the cloning step can be difficult as there is leakiness in expression.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
A transcriptional activator protein called CAP (catabolite activator protein) normally binds upstream of the trc promoter and activates transcription. This protein requires cAMP to bind the DNA. Adding glucose to the medium can reduce intracellular cAMP levels. Supplementing LB medium and agar plates with glucose will repress basal level transcription from the trc promoter. We recommend that you include 25 mM, 0.5% glucose in the selection medium to ensure stability of your insert.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The pTrc promoter in the pTrc system is a strong hybrid promoter composed of the -35 region of the trp promoter and the -10 region of the lacUV5 promoter/operator. Expression of pTrc is repressed by the LacI protein and induced by IPTG.
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.
BL21 strains are deficient in ompT and lon proteases, making them desirable as protein expression strains. However, many BL21(DE3)-based strains express T7 RNA polymerase under the control of the lacUV5 promoter, which is IPTG inducible, as is the Trc promoter in the vector. Therefore, we do not recommend the use of any BL21(DE3)-based strain [BL21(DE3), BL21(DE3)pLysE, BL21 Star(DE3), BL21(DE3)pLysS], due to the possibility of competing RNA polymerase activity after induction with IPTG.
BL21-AI is a strain which utilizes an arabinose-inducible promoter to drive T7 RNA polymerase. The BL21-AI strain is not induced by IPTG so it is a better choice for pTrc vectors.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
The EM7 promoter is a synthetic promoter derived from the T7 promoter. The promoter is typically used to drive expression of antibiotic resistance genes for selection in E. coli.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
IPTG is added to induce expression of the T7 RNA polymerase gene under control of the lac promoter in BL21 cells. Increasing the amount of IPTG further usually does not affect expression levels because of the high efficiency of T7 RNA polymerase. In the pTrc system, 1 mM is sufficient to inactivate the lacIq protein expressed in the cell.
The gene 10 leader sequence is from the T7 gene 10 capsid protein and is used to stabilize the expression of foreign genes in E. coli.
We've expressed CAT and Beta-Gal in volumes from 2 ml to 50 ml and get about 20-50 µg protein/ml of culture. Protein yield is dependent on the type of protein being expressed and the culturing conditions.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The chirality of arabinose used for induction is very important. L-arabinose works great, but D-arabinose doesn't induce at all. L-arabinose is available from Sigma (catalog# A3256).
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
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. The Shine-Dalgarno sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.