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查看更多产品信息 pBAD/His Kit - FAQs (V43001)
35 个常见问题解答
L-阿拉伯糖的用量取决于您的表达实验，我们建议在初试表达试验中采用多种不同用量的L-阿拉伯糖，范围为0.00002%至0.2%。
TOP10
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：
-该菌株不是蛋白酶缺陷型的；因此，蛋白质可能被降解。
LMG194
优点：
- 可在最低限培养基中生长良好，M9除外。
- 为了表达质粒必须转化到细胞中。
-含葡萄糖的RM培养基，保证了蛋白质的低基础表达水平。
缺点：
- 不是蛋白酶缺陷型的；因此，蛋白质可能被降解。
-甘油储液可能不稳定，因为这些菌株的基因型不是recA-或endA-。
我们建议使用基因型为araBADC-和araEFGH+的感受态细胞株，这种细胞可转运L-阿拉伯糖，而不会将其代谢。这对于表达研究很重要，因为在细胞内的L-阿拉伯糖水平是恒定不变的，不会随时间降低。我们可提供我们的TOP10感受态细胞或LMG194大肠杆菌菌株。
pBAD载体含有正向和反向pBAD引物，位于目的基因上下游。这些序列如下：
pBAD正向引物：5’-ATGCCATAGCATTTTTATCC-3’
pBAD反向引物：5’-GATTTAATCTGTATCAGG-3’
pBAD/His A 红色
pBAD/His B 橙色
pBAD/His C 黄色
pBAD/His LacZ 绿色
pBad/gIII A 黄色
pBad/gIII B 绿色
pBad/gIII C 蓝色
pBAD/gIII/calmodulin 紫色
araC基因产物可正负调控BL21-AI中用于控制T7 RNA聚合酶表达的araBAD启动子（pBAD）（Ogden et al., 1980; Schleif, 1992）。AraC是一种转录调节因子，可与L-阿拉伯糖形成复合物。当缺乏L-阿拉伯糖时，AraC二聚体与araBAD操纵子的O2和I1半位点接触，形成一个210 bp的DNA环（见下图）。实现最大程度的转录激活，需要2个条件。
•L-阿拉伯糖与AraC结合，使蛋白质释放O2位点并结合I1位点相邻的I2位点。这会释放DNA环，开始转录。
•cAMP激活蛋白（CAP）#31;-cAMP复合物与DNA结合，刺激AraC与I1和I2的结合。
请注意，在生长培养基中加入葡萄糖会抑制基础表达水平。葡萄糖发挥作用是通过降低cAMP水平，进而减少CAP的结合。cAMP水平的降低，会减少转录活性。
使用pBAD表达系统时，我们推荐使用L-阿拉伯糖来调节目的基因的表达。当存在L-阿拉伯糖时，可诱导pBAD的表达，而缺乏L-阿拉伯糖时，会产生非常低的pBAD转录水平(Lee, 1980（http://www.ncbi.nlm.nih.gov/pubmed/?term=Lee%2C+1980+pbad）； Lee et al., 1987（http://www.ncbi.nlm.nih.gov/pubmed/?term=Lee%2C+1987+arbinose）)。
pBAD表达系统可通过调控特定碳源，如葡萄糖、甘油和阿拉伯糖，可实现严格的蛋白表达调控，表达量可调节。pBAD非常适用于在大肠杆菌中表达毒性蛋白质以及优化蛋白质溶解性。该系统有助于使蛋白生成水平恰好低于其不溶解的阈值。pBAD载体利用大肠杆菌阿拉伯糖操纵子的调控元件，可精确调节异源表达水平，从而优化目的蛋白产量。pBAD载体骨架上具有调节蛋白AraC，可实现对pBAD的调控。
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).
While the amount of L-arabinose can vary depending on your expression experiment, we suggest performing a pilot expression experiment with varying amounts of L-arabinose from 0.00002% to 0.2%.
Top10
Advantages:
- Saves time, can go directly from cloning to expression.
- The glycerol stock is more stable because these strains are endA- and recA-.
Disdvantages:
- This strain is not protease-deficient. Therefore, the protein may be degraded.
LMG194
Advantages:
- Grows well in minimal media, except M9.
-Have to transform the plasmid into the cells just for expression.
-RM medium with glucose to ensure low basal level of protein.
Disadvantages:
- Not protease-deficient. Therefore, the protein may be degraded.
- The glycerol stock may not be stable because this cell strain is not recA- or endA-.
We recommend using a competent cell strain that is araBADC- and araEFGH+, allowing transportation of L-arabinose, but not metabolizing it. This is important for expression studies, as the level of L-arabinose will be constant inside the cell and will not decrease over time. We offer our TOP10 competent cells, or our LMG194 E. coli strain.
The pBAD vectors contain a forward and reverse pBAD primer flanking the gene of interest. The sequences are as follows:
pBAD forward primer:
5'-ATGCCATAGCATTTTTATCC-3'
pBAD reverse primer:
5'-GATTTAATCTGTATCAGG-3'
Here are the cap colors:
- pBAD/His A: Red
- pBAD/His B: Orange
- pBAD/His C: Yellow
- pBAD/His LacZ: Green
- pBad/gIII A: Yellow
- pBad/gIII B: Green
- pBad/gIII C: Blue
- pBAD/gIII/calmodulin: Purple
The araBAD promoter (pBAD) used to control expression of T7 RNA polymerase in BL21-AI is both positively and negatively regulated by the product of the araC gene (Ogden et al., 1980; Schleif, 1992). AraC is a transcriptional regulator that forms a complex with L-arabinose. In the absence of L-arabinose, the AraC dimer contacts the O2 and I1 half sites of the araBAD operon, forming a 210 bp DNA loop. For maximum transcriptional activation, two events are required.
- L-arabinose binds to AraC and causes the protein to release the O2 site and bind the I2 site that is adjacent to the I1 site. This releases the DNA loop and allows transcription to begin.
- The cAMP activator protein (CAP)-cAMP complex binds to the DNA and stimulates binding of AraC to I1 and I2.
Please note, basal expression levels can be repressed by introducing glucose to the growth medium. Glucose acts by lowering cAMP levels, which in turn decreases the binding of CAP. As cAMP levels are lowered, transcriptional activation is decreased.
We recommend using L-arabinose to regulate expression of your gene when using the pBAD expression system. In the presence of L-arabinose, expression from pBAD is turned on while the absence of L-arabinose produces very low levels of transcription from pBAD (Lee, 1980 [http://www.ncbi.nlm.nih.gov/pubmed/?term=Lee%2C+1980+pbad]; Lee et al., 1987 [http://www.ncbi.nlm.nih.gov/pubmed/?term=Lee%2C+1987+arbinose]).
The pBAD expression system allows tightly controlled, titratable expression of your protein through the regulation of specific carbon sources such as glucose, glycerol, and arabinose. pBAD is ideal for expressing toxic proteins and optimizing protein solubility in E. coli. The system helps to produce proteins at a level just below the threshold at which they become insoluble. The regulatory elements of the E. coli arabinose operon are used in the pBAD vectors to precisely modulate heterologous expression levels, allowing optimization of the yields of the protein of interest. The regulatory protein, AraC, is provided on the pBAD vector backbone, allowing for regulation of pBAD.
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.
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.
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 RBS, which stands for Ribosomal Binding Site, is required for translational initiation in E. coli, and consists primarily of purines. The consensus core region, or Shine-Dalgarno sequence (AGGAGG) is frequently located 8-12 base pairs upstream of the initiating codon AUG. This sequence forms base pairs with a complementary sequence located at the 3' end of the 16S rRNA molecule of the ribosome and assists in the recognition of the proper AUG of translation initiation.
In prokaryotes, such as E. coli, the promoter consists of two short regulatory sequences located 10 and 35 nucleotides upstream of the gene, respectively. The sequence located at -10 is called the Pribnow box (consensus sequence: TATAAT) and is absolutely essential for the transcription initiation. The sequence at -35 (consensus sequence: TTGACA) facilitates high transcription rate. Both regions are recognized by the sigma subunit of RNA polymerase, and instruct the holoenzyme where to start transcription. Once polymerization begins, the sigma subunit dissociates, and the core enzyme continues to transcribe the DNA template.
No, the exact start site of the pBAD promoter has not been determined experimentally.
If the construct is growing under maximal repression (i.e. in LMG194 with D-glucose in RM media), the cells should be harvested and resuspended in RM medium containing 0.2% glycerol and the appropriate concentration of arabinose (determined empirically). If cells are growing in LB medium, then arabinose may be added directly to the medium. Note that TOP10 cells will not grow in RM medium.
Any E. coli strain that is araBADC- and araEFGH+ is suitable for use with the pBAD promoter. Suitable strains that can be used include TOP10, TOP10F', and DH10B. Cells that are not araBADC- and therefore cannot be used with pBAD constructs, include DH5alpha, OmniMAX, Mach1, BL21(DE3) and INValphaF'.
LMG194 will grow in LB and RM medium that contains M9 salts. LMG194 will not grow in M9 salts alone. RM medium is used to ensure low basal expression levels from the pBAD promoter.
Please note that we do not support the use of LMG194 cells for any applications other than with the pBAD expression system.
The LMG194 strain is provided to allow for growth of your plasmid construct under conditions of maximal repression. This strain is capable of growth on minimal media that includes glucose as the sole carbon source. Glucose provides maximal repression of the pBAD promoter. Use of the LMG194 strain is not absolutely necessary since the TOP10 strain may also be used for expression.
Note: all suitable strains must have mutations in the ara gene locus to prevent the breakdown of arabinose when it is added to the medium.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The LMG194 strain can be selected on LB plates containing 25 ug/mL tetracycline.
The molecular weight of arabinose is 150.1 g/mol.
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.