Champion™ pET151 Directional TOPO™ Expression Kit with BL21 Star™ (DE3) One Shot™ Chemically Competent E. coli - FAQs

我的目的基因对细菌细胞有毒性作用。可采取哪些预防措施？

有多种措施可防止由毒性基因本底水平表达带来的问题。这些方法均基于T7或Champion表达质粒的设计和构建是正确的：

•在不含T7 RNA聚合酶（即，DH5α）的菌株中繁殖和维持表达质粒。
•如果使用BL21 (DE3)菌株，应在室温而非37°C下生长24-48小时。
•新鲜转化严格调控的大肠杆菌菌株，如BL21-AI菌株。
•转化实验后，将转化反应接种在含100 μg/mL氨苄青霉素和0.1%葡萄糖的LB板上。葡萄糖可抑制T7 RNA聚合酶的本底表达。
•完成BL21-AI菌株的转化后，挑选3或4个转化株直接接种到新制备的含100 μg/mL 氨苄青霉素或50 μg/mL 羧苄青霉素（以及0.1%葡萄糖，如果需要的话）的LB培养基中。当培养物的OD600值达到0.4时，可加入终浓度为0.2%的L-阿拉伯糖来诱导重组蛋白的表达。
•在表达实验中，在生长培养基中补充0.1%葡萄糖和0.2%阿拉伯糖。
•尝试使用调控型细菌表达系统，如我们的pBAD系统。

如何能够知道蛋白质发生降解或者存在密码子使用偏好？

通常，如果您看见1-2条主带，则表示密码子使用偏好导致了翻译过早终止。发生降解时，通常可看到梯度条带。发生降解时，可尝试在裂解缓冲液中加入蛋白酶抑制剂有助于防止降解。如果是这种情况，可通过时间点实验来确定收细胞的最佳时间。

我得到了包涵体，该怎么办？

如果存在溶解性问题，可尝试在诱导时降低温度或减少IPTG用量。也可尝试使用不同的、更严格的菌株进行表达。此外，在表达期间，也可将细菌培养基的葡萄糖含量增加至1%。

我的蛋白得率很低，应该如何进行改善？

使用新鲜的细菌培养物进行接种，因为新鲜的细菌菌落通常可得到较高的蛋白得率。

检查重组蛋白序列中的不常用密码子。利用常用密码子代替稀有密码子，可显著提高表达水平。例如，精氨酸密码子AGG和AGA是大肠杆菌的不常用的密码子，所以这些密码子的tRNA水平低。

进行蛋白纯化时，在缓冲液中加入蛋白酶抑制剂，如PMSF。使用新鲜配制的PMSF，因为PMSF被稀释到水溶液中后30分钟内会失效。

如果您在表达实验中使用氨苄青霉素进行筛选，则可能会因为筛选条件缺失而出现质粒不稳定。这是因为氨苄青霉素被β-内酰胺酶破坏，或者在由细菌代谢物造成的酸性培养基条件下发生水解。在转化和表达实验中，可使用羧苄青霉素替代氨苄青霉素。

重组蛋白可能对细菌细胞具有毒性作用。应选择更严格调控的系统用于表达，如BL21-AI。也可考虑尝试使用不同的表达系统，如pBAD系统。

我的细胞生长非常缓慢，也没有得到任何蛋白表达。我该如何解决这个问题？

当您的目的基因具有毒性时这种情况很常见。尝试使用更严格调控的系统，如BL21 (DE3) (pLysS)或BL21 (DE3) (pLysE)或BL21(AI)。

我没有得到表达，但细胞可正常生长。我该怎么办？

请查看以下可能的原因和解决方案：

•载体中存在移码或提前终止密码子；应检查序列。
•使用了错误的菌株进行表达。
•使用甘油菌可能会改变质粒的完整性，因为大多数用于表达的菌株基因型不是RecA-和EndA-。应使用新鲜转化的细胞。
•蛋白质存在于不溶的组分中；应检查细胞裂解物，而不仅是上清液。
•目的基因使用了稀有密码子——检查所用密码子。
（http://nihserver.mbi.ucla.edu/RACC）
•在培养过程中，细胞剔除了质粒——这在氨苄青霉素抗性质粒中较为常见。尝试在培养基中使用羧苄青霉素代替氨苄青霉素；接种前，使用含新鲜氨苄青霉素/羧苄青霉素的LB培养基清洗和重悬过夜培养物。

我的蛋白质溶解性存在问题。你们有何建议？

•降低诱导温度至30°C、25°C或18°C，有助于增加溶解性和减少包涵体形成。温度越低，诱导所需时间越长（即，30°C需3-4小时，25°C需3-5小时，18°C需过夜）。
•在较高温度下生长（30°C或37°C）并达到合适的OD，加入诱导剂，然后降低温度。
•尝试不同剂量的IPTG（1 mM-0.1 mM IPTG）。
•使用低拷贝数的质粒。
•使用营养不丰富的培养基，例如以M9基本培养基代替LB。
•如果蛋白质需要辅因子，如金属，则在培养基中加入辅因子。
•增加葡萄糖至1%。
•尝试使用BL21-AI菌株并使用不同剂量的阿拉伯糖。

我的细菌表达实验没有得到菌落。我该怎么办？

请查看以下关于无菌落生成的可能原因和建议：

•检查所用的抗生素
•转化pUC19对照载体，检验感受态细胞。
•如果目的基因具有毒性，并且启动子为T7启动子，可尝试使用BL21 (DE3) (pLysS)或(pLysE)或BL21 (AI)感受态细胞。您也可尝试在培养基中加入葡萄糖。

如何制备转化菌的甘油储液？

我们建议您在获得正确的构建质粒后，将克隆以甘油储液的形式进行保存。请遵循以下步骤来制备甘油储液：

•在含有50-100 μg/mL氨苄青霉素的LB培养基中，使1-2 mL菌株生长至饱和（12-16小时；OD600 = 1-2）
•取0.85 mL培养物，与0.15 mL无菌甘油混合。
•通过涡旋，混合溶液。
•转移至合适的冻存管中，盖上管帽。
•在乙醇/干冰浴或液氮中冷冻，然后转移至–80°C长期保存。

我在尝试表达一种毒性蛋白，你们建议使用哪种感受态细胞株？

BL21 AI大肠杆菌菌株可对使用T7启动子表达毒性蛋白进行最严格的调控。BL21 AI细胞株与传统BL21 (DE3)细胞株具有完全不同的诱导机制。该细胞株利用克隆到T7 RNA聚合酶上游的araBAD启动子，取代了驱动T7 RNA聚合酶等基因的lacUV5启动子，消除了传统BL21 (DE3)表达系统的渗漏。这消除了对pLysS和pLysE质粒的需求。通常，该细胞株的表达得率与其他BL21细胞株相近。

“渗漏表达”是什么意思？

渗漏表达是指存在一些基础水平表达。例如，在所有BL21（DE3）细胞株中， T7 RNA聚合酶总是存在一些基础水平表达。如果毒性基因被克隆到T7启动子的下游，则这种“渗漏表达”可能导致生长率降低、细胞死亡或质粒不稳定。

用于细菌表达的转化质粒，最大是多少？

我们尝试使用过的最大质粒为16kb。但是，从理论上来说，在效率开始下降之前，您可能可以转化高达30kb的质粒。

Champion pET表达系统与T7表达系统有何区别？

T7和Champion pET表达载体都具有强力的噬菌体T7启动子。使用IPTG诱导后，T7 RNA聚合酶会与T7启动子结合，使目的基因发生转录和翻译。研究表明，即使在无诱导剂的情况下，也一直会存在一些由λDE3溶源菌中lacUV5启动子介导的T7 RNA聚合酶基础表达（Studier and Moffatt, 1986（http://www.ncbi.nlm.nih.gov/pubmed/3537305））。通常，这不会带来问题，但是，如果目的基因对大肠杆菌宿主具有毒性，则目的基因的基础表达可能会导致质粒不稳定和/或细胞死亡。为解决这个问题，经过设计的Champion pET 载体含有T7lac启动子，可驱动目的基因的表达。T7lac启动子包含T7 启动子及其下游的lac操纵子序列。lac操纵子作为lac阻遏物（由lacl基因编码）的结合位点，可进一步抑制BL21 Star（DE3）细胞中由T7 RNA 聚合酶诱导的目的基因的基础转录。

在转化/T7表达实验中，能否用羧苄青霉素代替氨苄青霉素？

可以。实际上，羧苄青霉素通常比氨苄青霉素更稳定，可防止pET质粒的丢失从而可能会有助于增加蛋白表达水平。

T7启动子表达系统的得率预期会是多少？

T7启动子表达系统通常可产生相当高的得率，可轻松进行放大。得率取决于所表达的蛋白，范围通常为每升培养物可得到100 µg至10 mg蛋白。

我能否使用你们的带有T7启动子的哺乳动物表达载体，在在大肠杆菌中进行蛋白表达？

尽管可生成转录本，但是没有可起始翻译的核糖体结合位点或Shine Dalgarno序列；因此，不会生成蛋白质。

哪些因素会影响诱导型T7系统的表达？

有多种因素可影响表达，包括：

•诱导剂（IPTG）的加入量
•诱导的开始时间点（进行诱导的最佳OD600值为0.4-0.6）
•诱导持续时长
•诱导温度
•载体本身

T7表达系统的优势和原理是什么？

T7表达系统使用强力的噬菌体T7启动子可进行高水平表达。该系统依赖于T7 RNA聚合酶。尽管该聚合酶不是细菌内源产生的，但一些大肠杆菌菌株（如BL21（DE3）和BL21（DE3）pLysS）经过基因工程改造已携带可编码该RNA聚合酶的基因，称之为DE3噬菌体λ溶原菌。该λ溶原菌含有lacl基因、受lacUV5启动子控制的T7 RNA聚合酶基因以及一小部分lacZ基因。这个lac基因被插入到int基因中，从而使其失活。破坏int基因可防止在缺乏辅助噬菌体的情况下发生噬菌体切割（即，溶菌）。lac阻遏物可抑制T7 RNA聚合酶的表达。因此，当这些细胞处于正常情况时，lac阻遏物（lacl产物）结合到 lacUV5启动子与编码T7 RNA聚合酶的基因之间的lac操纵子区域。这有效阻止了T7 RNA聚合酶基因的转录。当然，总会存在少量的基础表达水平的T7 RNA聚合酶。这是因为lac阻遏物与lac操纵子区域处于平衡状态，使操纵子位点大部分被占据，但并不是一直被占据。

加入可阻止lac阻遏物与lac操纵子结合的化合物后，可诱导蛋白质表达。该化合物为异丙基硫代半乳糖苷（IPTG）。IPTG与lac阻遏物结合，改变其构象，从而使其不能与lac操纵子结合。这会导致细胞大量生成T7 RNA聚合酶。由于T7 RNA聚合酶对T7启动子（仅存在于转化质粒中）具有特异性，质粒编码的蛋白将发生过表达。

我在使用T7启动子原核表达系统时应在什么温度进行诱导？

诱导的温度范围很大，可以从37°C到30°C乃至室温。较低温度通常需要较长的生长时间。

我我在使用T7启动子原核表达系统，应在OD值达到多少时开始诱导蛋白表达？

诱导时间差异很大。在使用T7系统的实验中，当OD600为0.1–1.2时进行诱导，获得了成功的诱导结果。一般来说，在高OD值进行诱导，会产生较低的表达得率，因为细胞密度过高会使细胞立即停止生长。进行诱导的最佳OD600为0.4-0.6。

我在使用T7启动子原核表达系统，应使用多少IPTG诱导表达？

IPTG的用量差异很大，我们通常建议的起始范围为0.1-5 mM IPTG。

当克隆到细菌表达载体中时，核糖体结合位点（RBS）与ATG之间的最佳间距是多少？

RBS与ATG之间的序列应该为8-12 bp，并且不含有任何回文序列。

能否在大肠杆菌（E. coli）中共表达2种蛋白？

若要在大肠杆菌中同时表达2种蛋白，我们建议使用双启动子载体或同时使用2种不同的可兼容的载体。例如，您可尝试同时使用1个pET载体和1个pRSET载体，它们含有不同的复制起点（ORI），分别为pBR322和pUC复制起点。唯一的问题是，pRSET载体是高拷贝数的，而pET不是；因此，如果您将它们转化到同一个宿主细胞中，从pRSET中得到的蛋白表达可能明显多于pET。

我需要在克隆目的基因时在其中包含一个核糖体结合位点（RBS）或Kozak序列吗？

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。

对TOPO TA克隆来说最佳的插入片段：载体比例是多少？是否有公式来计算用量？

我们建议起始摩尔比为1:1(插入片段：载体)，范围为0.5：1到2：1(插入片段：载体)。对TOPO克隆来说，2kb大小的 PCR产物的ng值应在5- 10ng之间。

计算公式：< br / > 插入片段长度（bp）/ 载体长度（bp）x 载体用量（ng）= 插入片段：载体比为1：1时所需的插入片段的用量（ng）。

插入片段：载体的最佳比例是多少？是否有公式可以进行计算？

您可能需要尝试不同的插入片段:载体比例，范围从1:1至15:1。

公式：
length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio (插入片段长度 (bp) X 载体重量(ng) ) / 载体长度 (bp) = 插入片段:载体比例为1:1时所需的插入片段重量(ng)

我可以使用Taq聚合酶生成我的目的基因用于定向TOPO克隆吗？

不，你的基因必须用一种校对活性聚合酶例如Platinum SuperFi DNA聚合酶或AccuPrime Pfx DNA聚合酶进行扩增以获得平末端才可用于定向TOPO克隆。

定向TOPO 克隆引物设计的要求是什么？

在引物设计时请考虑以下问题：

• 3’PCR引物不能含有与5’辅助序列GTGG同源的序列。
•使用的酶必须产生平末端的PCR产物。
•引物不能含有5’磷酸基团，它将抑制5’ OH亲核反应基团。
•设计引物时必须考虑读码框。

你们推荐在TA/Blunt/D-TOPO克隆中使用哪种PCR聚合酶，为什么？

TA克隆

这种克隆方法最初是为配合纯Taq聚合酶（天然的、重组的、热启动）使用而设计的；然而，某些高保真Taq酶和Taq酶混合物通常也适合TA克隆。即使Taq与具有校正能力的聚合酶以10:1或15:1的比例，仍可以产生足够的3’ A突出端去做TA克隆。

推荐使用的我公司的聚合酶包括Platinum Taq、Accuprime Taq、Platinum或Accuprime Taq High Fidelity、AmpliTaq、AmpliTaq Gold或AmpliTaq Gold 360等。

平末端克隆

使用Platinum SuperFi DNA聚合酶等具有校对能力的酶。

定向TOPO克隆

Platinum SuperFi DNA聚合酶效果良好。

Shine-Dalgarno和Kozak序列有何区别？

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).

My gene of interest is toxic to bacterial cells. Are there any precautions you can suggest?

Several precautions may be taken to prevent problems resulting from basal level expression of a toxic gene of interest. These methods all assume that the T7-based or Champion-based expression plasmid has been correctly designed and created.

- Propagate and maintain your expression plasmid in a strain that does not contain T7 RNA polymerase (i.e., DH5α).
- If using BL21 (DE3) cells, try growing cells at room temperature rather than 37 degrees C for 24-48 hr.
- Perform a fresh transformation using a tightly regulated E. coli strain, such as BL21-AI cells.
- After following the transformation protocol, plate the transformation reaction on LB plates containing 100 µg/mL ampicillin and 0.1% glucose. The presence of glucose represses basal expression of T7 RNA polymerase.
- Following transformation of BL21-AI cells, pick 3 or 4 transformants and inoculate directly into fresh LB medium containing 100 µg/mL ampicillin or 50 µg/mL carbenicillin (and 0.1% glucose, if desired). When the culture reaches an OD600 of 0.4, induce expression of the recombinant protein by adding L-arabinose to a final concentration of 0.2%.
- When performing expression experiments, supplement the growth medium with 0.1% glucose in addition to 0.2% arabinose.
- Try a regulated bacterial expression system such as our pBAD system.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm trying to express my protein using a bacterial expression system. How do I know if I'm seeing degradation of my protein or if what I’m seeing is codon usage bias?

Typically, if you see 1-2 dominant bands, translation stopped prematurely due to codon usage bias. With degradation, you usually see a ladder of bands. With degradation, you can try using a protease inhibitor and add it to the lysis buffer to help prevent degradation. If degradation is the issue, a time point experiment can be done to determine the best time to harvest the cells.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm trying to express my protein using a bacterial expression system and am getting inclusion bodies. What should I do?

If you are having a solubility issue, try to decrease the temperature or decrease the amount of IPTG used for induction. You can also try a different, more stringent cell strain for expression. Adding 1% glucose to the bacterial culture medium during expression can also help.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting low protein yield from my bacterial expression system. What can I do to improve this?

- Inoculate from fresh bacterial cultures, since higher protein yields are generally obtained from a fresh bacterial colony.

- Check the codon usage in the recombinant protein sequence for infrequently used codons. Replacing the rare codons with more commonly used codons can significantly increase expression levels. For example, the arginine codons AGG and AGA are used infrequently by E. coli, so the level of tRNAs for these codons is low.

- Add protease inhibitors, such as PMSF, to buffers during protein purification. Use freshly made PMSF, since PMSF loses effectiveness within 30 min of dilution into an aqueous solution.

- If you are using ampicillin for selection in your expression experiments, you may be experiencing plasmid instability due to the absence of selective conditions. This occurs as the ampicillin is destroyed by β-lactamase or hydrolyzed under the acidic media conditions generated by bacterial metabolism. You may want to substitute carbenicillin for ampicillin in your transformation and expression experiments.

- The recombinant protein may be toxic to bacterial cells. Try a tighter regulation system for competent cell expression such as BL21-AI. You may also consider trying a different expression system such as the pBAD system.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

My cells are growing very slowly, and I'm not getting any protein expression from my baterial expression system. What can I do to fix this?

This typically occurs when your gene of interest is toxic. Try using a tighter regulation system, such as BL21 (DE3) (pLysS) or BL21 (DE3) (pLysE), or BL21(AI).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting no expression from my bacterial expression vector, but my cells are growing normally. What should I do?

Please view the possible causes and solutions to try:

- Frame shifts or a premature stop codon is present in the construct; check the sequence.
- The wrong cell strain was used for expression.
- If using a glycerol stock, the integrity of the plasmid can change because most cell strains for expression are not RecA and EndA-. Use freshly transformed cells.
- The protein is in the insoluble fraction; check cell lysates and not just the supernatant.
- Check the codon usage in the recombinant protein sequence for infrequently used codons. Replacing the rare codons with more commonly used codons can significantly increase expression levels. For example, the arginine codons AGG and AGA are used infrequently by E. coli, so the level of tRNAs for these codons is low.

- Rare codons were used in the gene of interest: check the codon usage. (http://nihserver.mbi.ucla.edu/RACC)
- The cells may be kicking out the plasmid during culture: this is more common in plasmids that are ampicillin resistant. This occurs as the ampicillin is destroyed by β-lactamase or hydrolyzed under the acidic media conditions generated by bacterial metabolism. Try using carbenicillin instead of ampicillin in the medium; wash and resuspend the overnight culture with LB containing fresh amp/carb before inoculation.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm having problems with protein solubility using a T7 promoter-based vector. What would you recommend to try?

- Lower the induction temperature to 30 degrees C, 25 degrees C, or 18 degrees C to help increase solubility and reduce the formation of inclusion bodies. The lower the temperature, the more time needed to do the induction (i.e., 30 degrees C for 3-4 hours, 25 degrees C for 3-5 hours, or 18 degrees C for overnight).
- Grow at a higher temperature (30 degrees C or 37 degrees C) to reach the proper OD, add inducer, then shift to the lower temperature.
- Try different amounts of IPTG (1 mM-0.1 mM IPTG).
- Use a low copy number plasmid.
- Use a less rich medium, such as M9 minimal medium instead of LB.
- If the protein requires a cofactor, such as a metal, add the cofactor to the medium.
- Add glucose to 1%.
- Try the BL21-AI strain and use different amounts of arabinose.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting no colonies with my T7 promoter-based bacterial expression system. What can I do?

Please check the following possibilities and suggestions for getting no colonies:

- Check the antibiotic used.
- Check the competent cells with pUC19 control reaction.
- If your gene of interest is toxic, try using BL21 (DE3) (pLysS) or (pLysE) or BL21 (AI) cells if the promoter is the T7 promoter. You can also try adding glucose to the medium.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How can I make a glycerol stock of my desired construct?

Once you have obtained your desired construct, we recommend that you store your clone as a glycerol stock. Please follow these steps to create a glycerol stock:

- Grow 1 to 2 mL of the strain to saturation (12-16 hours; OD600 = 1-2) in LB containing 50-100 µg/mL ampicillin
- Combine 0.85 mL of the culture with 0.15 mL of sterile glycerol
- Mix the solution by vortexing
- Transfer to an appropriate vial for freezing and cap
- Freeze in an ethanol/dry ice bath or liquid nitrogen and then transfer to –80 degrees C for long-term storage.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm trying to express a toxic protein using a T7 promoter-based vector. What competent cell strain would you suggest?

The BL21 AI E. coli strain offers the tightest regulation of expression for production of toxic proteins using the T7 promoter. The BL21 AI line uses a completely different mechanism of induction from that of the traditional BL21 (DE3) lines. This cell line utilizes an araBAD promoter cloned upstream of T7 RNA polymerase. This replaces the lacUV5 promoter driving the T7 RNA polymerase gene and all but eliminates the leakiness of the traditional BL21 (DE3) expression systems. This eliminates the need for pLysS and pLysE plasmids. In general, the expression yields from this strain are similar to that of other BL21 strains.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I am trying to express my protein using a T7 promoter-based vector. What does "leaky expression" mean?

Leaky expression means there is some basal level expression seen. For example, in all BL21 (DE3) cell lines, there is always some basal level expression of T7 RNA polymerase. This “leaky expression” could lead to reduced growth rates, cell death, or plasmid instability if a toxic gene is cloned downstream of the T7 promoter.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What is the maximum size plasmid transformed for bacterial expression?

The largest size plasmid we've tried is 16 kb, but you should theoretically be able to transform a plasmid as large as 30 kb before efficiency begins to drop.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What is the difference between the Champion pET expression systems and the T7 expression systems?

Both the T7 and Champion pET expression vectors contain a strong bacteriophage T7 promoter. After induction with IPTG, T7 RNA polymerase will bind the T7 promoter, leading to transcription and translation of your gene of interest. Studies have shown that there is always some basal expression of T7 RNA polymerase from the lacUV5 promoter in lambda DE3 lysogens, even in the absence of inducer (Studier and Moffatt, 1986 [http://www.ncbi.nlm.nih.gov/pubmed/3537305]). In general, this is not a problem, but if the gene of interest is toxic to the E. coli host, basal expression of the gene of interest may lead to plasmid instability and/or cell death. To address this problem, the Champion pET vectors have been designed to contain a T7lac promoter to drive expression of the gene of interest. The T7lac promoter consists of a lac operator sequence placed downstream of the T7 promoter. The lac operator serves as a binding site for the lac repressor (encoded by the lacI gene) and functions to further repress T7 RNA polymerase-induced basal transcription of the gene of interest in BL21 Star (DE3) cells.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Can I use carbenicillin in place of ampicillin in my transformation/T7 expression experiments?

Yes; in fact, carbenicillin is generally more stable than ampicillin and may help to increase expression levels by preventing loss of the pET plasmids.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What is the yield I should expect from my T7 promoter-based expression system?

The T7 promoter-based expression systems usually give fairly high yield and can be scaled up easily. Yields will vary depending on the protein being expressed, but in general yields range from 100 µg to 10 mg per liter of culture.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Can I use one of your mammalian expression vectors with a T7 promoter for expression in E. coli?

Transcripts can be made, but there is no ribosome binding site or Shine Dalgarno sequence to initiate translation; therefore, little protein will be produced.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What factors can affect expression in an inducible T7 system?

There are several factors that can affect expression including:

- Amount of inducer (IPTG) added
- Time of induction (optimal OD600 for induction is 0.4 to 0.6)
- Duration of induction
- Induction temperature
- The construct itself


Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What are the advantages and mechanism of the T7 expression system?

The T7 expression system allows for high-level expression from the strong bacteriophage T7 promoter. The system relies upon the T7 RNA polymerase. While it is not endogenous to bacteria, some strains of E. coli (such as BL21 (DE3) and BL21 (DE3)pLysS) have been engineered to carry the gene encoding for this RNA polymerase in a piece of DNA called the DE3 bacteriophage lambda lysogen. This lambda lysogen contains the lacI gene, the T7 RNA polymerase gene under control of the lacUV5 promoter, and a small portion of the lacZ gene. This lac construct is inserted into the int gene, thus inactivating it. Disruption of the int gene prevents excision of the phage (i.e., lysis) in the absence of helper phage. The lac repressor represses expression of T7 RNA polymerase. Therefore, under normal circumstances in these cells, the lac repressor (the lacI product) binds to the lac operator region between the lacUV5 promoter and the gene encoding for T7 RNA polymerase. This effectively prevents transcription of the T7 RNA polymerase gene. Of course, there is always a small basal level of T7 RNA polymerase present. This is due to the fact that the lac repressor is in equilibrium with the lac operator region, causing the operator site to be occupied most, but not all of the time.

Adding a substance that prevents the lac repressor from binding to the lac operator then induces protein expression. This compound is isopropyl b-D-thiogalactoside (IPTG). IPTG binds to the lac repressor, changing its conformation in such a way that it is no longer able to bind the lac operator. This enables the cells to make T7 RNA polymerase in much more substantial amounts. As the T7 RNA polymerase is specific for the T7 promoter (which is only found in the transformed plasmid), the protein encoded by the plasmid will be overexpressed.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I am using a bacterial expression vector containing the T7 promoter. At what temperature should I perform my induction?

Induction can be performed at a variety of temperatures, ranging from 37 degrees C to 30 degrees C to room temperature. A lower temperature typically requires a longer growth time.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I am using a bacterial expression vector containing the T7 promoter. At what OD should I induce my cells?

The time of induction can vary widely. Successful experiments using the T7 systems have induced at an OD600 of 0.1-1.2. Generally speaking, induction at high ODs will lead to lower expression yields, as the cells will stop growing rapidly after the density is too high. The optimal OD600 for induction is 0.4 to 0.6.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How much IPTG can I use to induce expression from a T7 promoter containing bacterial expression vector?

This can vary somewhat, but we typically suggest a starting range of 0.5-1 mM IPTG.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What is the optimal spacing between the ribosome-binding site (RBS) and the ATG when cloning into a bacterial expression vector?

The sequence between the RBS and ATG should be between 8-12 bp and should not contain any palindromic sequence.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Can I co-express two proteins in E. coli?

To express two proteins at the same time in E. coli, we suggest using a dual promoter vector or using two different but compatible vectors at the same time. For example, you could try a pET vector with a pRSET vector, which contain different ORI (pBR322 origin and pUC origin, respectively). The only issue is that the pRSET vector is high copy number but pET is not; therefore, you may get significantly more protein expression from pRSET than from pET if you add them into one host cell.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

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.

What is the best molar ratio of PCR product:vector to use for TOPO TA cloning? Is there an equation to calculate the quantity to use?

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO cloning reaction is typically 5-10 ng of a 2 kb PCR product.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)

What is the best ratio of insert:vector to use for cloning? Is there an equation to calculate this?

The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio

Can I use Taq polymerase to generate my gene of interest for directional TOPO cloning?

No, your gene of interest must be amplified with a proofreading polymerase such as Platinum SuperFi DNA Polymerase or AccuPrime Pfx DNA Polymerase that leaves blunt ends for directional TOPO cloning.

What are the requirements for primer design when using directional TOPO cloning?

Please consider the following when designing your primers:

- The 3' pcr primer cannot contain homology to the 5' flap sequence GTGG.
- The enzyme you use must create a blunt-ended PCR product for cloning.
- Primers cannot contain 5' phosphates, which will block the 5' OH nucleophile reactive group.
- The reading frame must be considered when you are designing your primers.

Which PCR polymerases do you recommend for TA/Blunt/D-TOPO cloning and why?

TA Cloning:
- This cloning method was designed for use with pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 or 15:1 ratio of Taq to proofreader polymerase will still generate enough 3' A overhangs for TA cloning.
- Recommended polymerases include Platinum Taq, Accuprime Taq, Platinum or Accuprime Taq High Fidelity, AmpliTaq, AmpliTaq Gold, or AmpliTaq Gold 360.

Blunt cloning:
- Use a proofreading enzyme such as Platinum SuperFi DNA Polymerase.

Directional TOPO cloning:
- Platinum SuperFi DNA Polymerase works well.

Where is the transcriptional start site of the T7 promoter featured in many of Invitrogen's vectors?

Although Invitrogen has not formally mapped the transcriptional start site of the T7 promoter, the following reference indicates that the start site occurs at the G following the CACTATA sequence found in the promoter: Nucleic Acids Research, Vol.20, No. 20, pp 4626-4634.

What PCR enzyme would you recommend for use with the Directional TOPO Cloning Kits?

For the Directional TOPO Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50 or Accuprime Pfx and Accuprime Pfx Supermix from Thermo Fisher Scientific are recommended for use.

When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.

Does Thermo Fisher Scientific have a vector for co-expression of two proteins in E. coli?

A dual promoter vector is the best option for expressing two proteins at the same time. Unfortunately, we do not offer any prokaryotic expression vectors containing dual promoters. Another option is to use two different but compatible vectors at the same time. For example, you can try using a pET vector such as pET100/D-TOPO with a pRSET vector. Our pET vectors have a pBR322 origin and our pRSET vectors have a pUC origin, so they are able to replicate in E. coli at the same time. The only issue here is that pRSET is a high copy number vector and our pET vectors are not. Therefore, you may get significantly more protein expression from pRSET in comparison to pET if they are expressed in the same host.

Will carbenicillin in place of ampicillin help to increase expression levels from the pET TOPO vectors?

For most purposes, ampicillin works well for selection of transformants and expression experiments. However, degradation of ampicillin is enhanced during the late stages of bacterial growth, when the pH of the culture tends to decrease. This could result in non-selective conditions, resulting in the appearance of satellite colonies on plates, and if the transferred plasmid is unstable it may result in the loss of the plasmid and low expression levels. Carbenicillin is generally more stable under acidic conditions than ampicillin and studies have shown that using carbenicillin in place of ampicillin may help increase expression levels by preventing the loss of the pET TOPO plasmid. Use at a concentration of 50 ug/ml.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Can you tell me the difference between a Shine-Dalgarno sequence and a Kozak sequence?

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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What is the source of the EM7 promoter?

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.

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What protein yields should I expect to get with the pRSET expression vectors?

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.

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I sequenced one of your vectors after PCR amplification and observed a difference from what is provided online (or in the manual). Should I be concerned?

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.

Are your vectors routinely sequenced?

No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.

How was the reference sequence for your vectors created?

Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.

What is a Shine-Dalgarno sequence?

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.