Expressway™ Tag-On-Demand™ Cell-Free E. coli Expression System *DISCONTINUED* - FAQs

本试剂盒中的甲硫氨酸是单独提供的，这样您就可在重组蛋白中整合非天然氨基酸，也可在蛋白合成反应中调整氨基酸的浓度。基于您的具体应用，您可以选用以下类型的非天然氨基酸：

•放射性标记的甲硫氨酸：在表达与纯化研究中使用35S-甲硫氨酸生成放射性标记蛋白。请参见使用手册（https://tools.thermofisher.com/content/sfs/manuals/expressway_system_man.pdf）第21页中的“开展蛋白合成反应”部分中关于标记型与非标记型甲硫氨酸的推荐用量。
•重金属标记的甲硫氨酸：使用硒代蛋氨酸（Budisa等，1995（http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374119/）； Doublie，1997（http://www.ncbi.nlm.nih.gov/pubmed/9048379）；Hendrickson等，1990（http://www.ncbi.nlm.nih.gov/pubmed/2184035?dopt=Abstract））来生产标签蛋白，并将其应用于X射线晶体学研究。请参见使用手册第21页中的“开展蛋白合成反应”部分中关于标记型甲硫氨酸的推荐用量。注意：选用硒代蛋氨酸时，请勿在蛋白合成反应过程中使用任何一种非标记型甲硫氨酸。

在配制蛋白合成反应体系的过程中：

•如需使用35S-甲硫氨酸生成放射性标记的蛋白，请使用2 μL 35S-甲硫氨酸和1 μL 75 mM非标记型甲硫氨酸。
•如需使用硒代蛋氨酸来生成标记蛋白，请只使用2 μL硒代蛋氨酸；请勿添加非标记型的甲硫氨酸。

您最短可在加入补料2.5小时后获得目的蛋白（一共3小时）。很多反应在3小时的时间内能够合成80-90%的总蛋白。不过，如需获得最高得率，我们推荐您将反应体系一共孵育6小时。

此外，在蛋白合成反应起始后的30分钟时间点添加1/2的补料缓冲液，之后在起始的2小时时间点再加入1/2的补料缓冲液，可能获得更高的蛋白得率。

我们推荐在30–37°C温度区间内孵育蛋白合成反应体系。最佳的反应温度依赖于重组蛋白的溶解性，需根据实际经验来确定。更高的温度（即37°C）一般有助于获得更高的蛋白得率；不过较低的温度（即30°C）通常有助于获得更好的蛋白溶解度。

为了获得最佳的蛋白得率，在孵育时间段内对反应体系进行彻底混匀是至关重要的。我们推荐用户使用1200 rpm的恒温混匀仪或300 rpm的振荡孵箱。请勿使用烘箱或水浴锅一类的静止孵箱，使用这样的设备会使蛋白得率降低30-50%。

对于筛选反应而言，标准的体积为100 µL（50 µL初始反应体积 + 50 µL 补料缓冲液），不过这一反应体系可按比例缩减至25 µL或扩容至2 mL的体积。请注意，蛋白得率可能在很大程度上依赖于其天然性质和所用模板。

标准的反应时间为2小时。不过，将此时间增至4小时能够提升蛋白得率。对于低溶解性的蛋白而言，这一延长的孵育操作可在室温进行。

否，这些抽提物中不含糖基化修饰元件。

某些Invitrogen pET载体在本系统中工作得很好；不过由于其中T7lac启动子的存在，其最终得率可能会比其他载体低一些。即使反应体系中加入了IPTG，lac抑制子也会与lac操纵子位点相结合，并对表达过程形成干扰。载体的最佳选择为pEXP-DEST载体，pEXP5-TOPO载体和pRSET载体。除T7启动子之外，这些载体还包含了一段能够提升翻译效率的基因序列。

不是的，这些大肠杆菌菌株并非源自supF或supE大肠杆菌菌株，这些抽提物的抑制子活性极低。因此，本系统很适合于引入修饰氨基酸的操作。

是的。我们推荐您以高拷贝数的质粒开始实验。这样，研究人员就可直接凭借小抽试剂盒的产物开展Expressway反应了。许多pET载体都是低拷贝载体，在应用于体外翻译系统之前需要进行浓缩操作。

我们观察过两种蛋白：一种在天然的大肠杆菌中完全不能溶解，另一种在大肠杆菌中只能部分溶解。对于这两种蛋白，使用Expressway系统我们都至少观察到某些可溶性蛋白的生成。在这些案例中，Expressway系统的确在某种程度上提升了蛋白产物的溶解度。不过，体外合成蛋白并不会彻底改变蛋白的溶解性质。

否，我们不推荐您这样做，因为我们发现此类操作会抑制蛋白合成反应。相反，您可使用商业化的DNA纯化试剂盒（如我们的PureLink HQ小量质粒纯化试剂盒或CsCl密度梯度离心法来纯化您的DNA模板。

您可使用超螺旋的质粒DNA、线性DNA或PCR产物作为模板。为了获得良好的表达效果，所有的模板都须含有一个T7启动子，一个起始密码子，以及目的基因上游的原核Shine-Dalgarno核糖体结合位点（RBS）。如果您正在设计一款您自己的表达质粒，我们推荐您在DNA模板中加入以下元件：

•目的基因应置于一个T7启动子和一个核糖体结合位点（RBS）下游。目的基因需含有一个ATG起始密码子和一个终止密码子。
•T7启动子上游最少应连接有6-10核苷酸（nt）的序列以辅助启动子实现高效结合（需要线性化的PCR产物）。这一序列应具有广泛的兼容性。
•T7启动子下游应连接不小于15-20 nt的序列，以形成Studier等在1990年所描述的潜在茎环结构。
•RBS与ATG起始密码子之间应有7-9 nt的序列，来帮助获得目的基因最优的翻译效率。这一序列应具有广泛的兼容性。
•目的基因下游4-100 nt处应包含T7终止子，来提高转录终止的效率和信使RNA的稳定性。

•需通过T7启动子（而非T7lac启动子）驱动目的基因的转录。通常情况下使用T7lac启动子的得率会更低，因为由lacl基因编码的lac抑制子会对这一启动子的转录过程形成抑制作用。
•T7终止子对于超螺旋质粒的高效体外转录过程是至关重要的。如果不含终止子，就会形成长链的非特异性RNA产物，后者会夺取dNTP并生成大量的焦磷酸盐。
•gene10序列能够提升体外表达序列的稳定性。该序列会介导一种特异性茎环结构的形成；后者会稳定mRNA并增加翻译效率。
•（Trc载体中的）小型顺反子也能够通过编码一条小型基因产生一条短肽来促进翻译过程。由于这一元件能够将翻译机器引导至目的基因的起始位点附近，因此有助于下游系统的起始。
•我们推荐您以高拷贝数的质粒开始实验。这样，小抽所获得的质粒就可直接应用于Expressway系统。
•RBS与ATG之间的间隔距离对于高效的翻译过程十分重要。
•RBS将增加蛋白得率并提升翻译保真度。

大肠杆菌，麦胚提取物（WGE），或兔网织红细胞裂解物（RRL）都适用于体外翻译。通常情况下，RRL能够有效翻译大于30 kDa的蛋白。WGE能够兼容任何尺寸的蛋白（包括那些15–30 kDa范围内的蛋白），尽管RRL系统可能对于大蛋白的翻译更有效。我们的Expressway产品选用的是大肠杆菌。

slyD是大肠杆菌的内源基因表达产物，富含半胱氨酸，因此能够与Lumio检测试剂发生相互作用。

Expressway Lumio系统将Expressway无细胞系统和Lumio技术的优势整合在一起。在使用Lumio试剂盒的过程中，您的目的基因会与Lumio标签融合在一起，这样就可在聚丙烯酰胺凝胶中灵敏和特异性地检测Lumio-标记的融合蛋白，而无需进行染色或免疫印迹实验了。您也可通过标准的荧光计实时监控Lumio-标记蛋白的合成情况。 

•过表达产物对宿主细胞的毒性
•表达产物不可溶，以及形成包涵体
•表达的蛋白被蛋白酶快速降解
•整合非天然或经修饰的氨基酸
•向蛋白中整合荧光探针
•需要对蛋白产物进行高通量分析

无细胞表达系统非常适合于毒性靶标蛋白的表达，因为不需要依赖细胞就能获得蛋白。体外蛋白表达系统在单一反应管中利用必要的细胞学组份来驱动基因表达。

•通过PCR或以质粒载体制备一个DNA模板
•纯化模板
•进行合成反应
•通过考马斯染色，免疫印迹等手段分析样品

Methionine is supplied separately in the kit to allow you to incorporate unnatural amino acids into your recombinant protein and adjust the amino acid concentration in the protein synthesis reaction. Depending on your application, you may use the following unnatural amino acids:

- Radiolabeled methionine: Use ³⁵S-methionine to produce radiolabeled protein for use in expression and purification studies. See “Performing the Protein Synthesis Reaction” on page 21 of the manual (http://tools.thermofisher.com/content/sfs/manuals/expressway_system_man.pdf) for recommended amounts of labeled and unlabeled methionine.

- Heavy metal-labeled methionine: Use selenomethionine (Budisa et al., 1995 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374119/]; Doublie, 1997 [http://www.ncbi.nlm.nih.gov/pubmed/9048379]; Hendrickson et al., 1990 [http://www.ncbi.nlm.nih.gov/pubmed/2184035?dopt=Abstract]) to produce labeled protein for use in X-ray crystallographic studies. See “Performing the Protein Synthesis Reaction” on page 21 of the manual (http://tools.thermofisher.com/content/sfs/manuals/expressway_system_man.pdf) for recommended amounts of labeled methionine. Note: When using selenomethionine, do not use any unlabeled methionine in the protein synthesis reaction.

When setting up the protein synthesis reaction:

- To generate radiolabeled protein using ³⁵S-methionine, use 2 µL of ³⁵S-methionine and 1 µL of unlabeled 75 mM methionine.

- To generate labeled protein using selenomethionine, use 2 µL of selenomethionine only; do not add unlabeled methionine.

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

You may obtain your protein of interest in as little as 2.5 hours of incubation after feeding (3 hours total). Many reactions yield 80-90% of total protein within 3 hours. However, for maximum yield, we recommend incubating the reaction for a full 6 hours.

Additionally, higher protein yields may be obtained by adding one half-volume of feed buffer at 30 minutes and one half-volume of feed buffer again at 2 hours after initiating the protein synthesis reaction.

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

We recommend incubating the protein synthesis reaction at a temperature range from 30-37 degrees C. The optimal temperature to use depends on the solubility of your recombinant protein, and should be determined empirically. Higher protein yields are generally obtained with incubation at higher temperatures (i.e., 37 degrees C); however, protein solubility generally improves with incubation at lower temperatures (i.e., 30 degrees C).

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

To obtain optimal protein yield, it is critical to mix the reaction thoroughly throughout the incubation period. We recommend using a thermomixer incubator set to 1,200 rpm or a shaking incubator set to 300 rpm. Do not use stationary incubators such as incubator ovens or water baths, as protein yields may be reduced by up to 30-50%.

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

For screening reactions, the standard volume is 100 µL (50 µL initial reaction + 50 µL feed buffer), but this can be decreased to 25 µL reaction volume and increased up to 2 mL reaction volume. Note that protein yields may vary depending on the nature of the protein expressed and the template used.

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

The standard reaction time is 2 hours. However, increasing the time to 4 hours may increase the yield of protein. For less soluble proteins, this longer incubation should be carried out at ambient temperature.

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

No, the proper machinery for glycosylation is not present in these extracts.

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Some Invitrogen pET vectors work well in this system; however, the yields might be lower than that found with other vectors due to the presence of the T7lac promoter. The lac repressor can bind to the lac operator site and interfere with expression even when IPTG has been added to the reaction. The best vectors to choose are the pEXP-DEST vectors, pEXP5-TOPO vectors, and pRSET vectors. In addition to the T7 promoter, these also have a gene sequence that enhances translatability.

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

No, the E. coli strain is not a supF or supE strain, and it has very little suppressor activity. Therefore, it should be useful for introducing modified amino acids.

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Yes. We recommend starting out with a high copy number plasmid. This way, a researcher can go right from a miniprep kit directly into the Expressway reaction. Many of the pET vectors are low copy, and need to be concentrated before being used in an in vitro expression system.

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

We have looked at two proteins: one that was totally insoluble in intact E. coli and another that was partly insoluble in E. coli. In both cases, we saw at least some soluble protein when synthesized in vitro with the Expressway system. In these instances, it did seem that there is at least some increased solubility with the Expressway system. However, synthesizing protein in vitro does not completely change protein solubility.

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

No, we do not recommend doing so, as we have seen this inhibit the protein synthesis reaction. Instead, you can use commercial DNA purification kits (such as our PureLink HQ Mini Plasmid Purification Kit) or a CsCl gradient centrifugation to purify your DNA template.

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

You can use supercoiled plasmid DNA, linear DNA, or a PCR product as your template. For proper expression, all templates must contain a T7 promoter, an initiation codon, and a prokaryotic Shine-Dalgarno ribosome-binding site (RBS) upstream of the gene of interest. If you are designing your own expression construct, we recommend generating a DNA template with the following elements:

- Gene of interest placed downstream of a T7 promoter and a ribosome-binding site (RBS). The gene of interest must contain an ATG initiation codon and a stop codon.

- Sequence upstream of the T7 promoter containing a minimum of 6-10 nucleotides (nt) for efficient promoter binding (required for linear PCR products). This sequence need not be specific.

- Sequence following the T7 promoter containing a minimum of 15-20 nt, which forms a potential stem-and-loop structure as described by Studier et al., 1990.

- Sequence of 7-9 nt between the RBS and the ATG initiation codon for optimal translation efficiency of the protein of interest. This sequence need not be specific.

- A T7 terminator located 4-100 nt downstream of the gene of interest for efficient transcription termination and message stability.

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

- Transcription of the gene of interest must be driven by a T7 promoter (not the T7lac promoter). Using a T7lac promoter typically renders poor yield, as the lac repressor encoded by the lacI gene binds and represses transcription from this promoter.

- The T7 terminator is important for efficient in vitro transcription from a supercoiled plasmid. If the terminator is absent, long nonspecific RNA products will be produced, which can deprive the reaction of dNTPs and generate a copious amount of pyrophosphate.

- A gene10 sequence enhances the stability of the in vitro expressed sequence. This sequence causes a specific stem-loop structure to form, which helps to stabilize the mRNA and leads to increased translation.

- The mini cistron (in the Trc vectors) also acts to enhance translation by coding for a short gene sequence that encodes a small peptide. Since this brings the translation machinery to the proximity of the start of the gene of interest, it helps to initiate the system downstream.

- We recommend starting with a high copy number plasmid. This way, minipreps can be used directly with the Expressway system.

- Spacing between the RBS and ATG is very important for efficient translation.

- The RBS will increase the yield of protein and increase translation fidelity.

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

E. coli, rabbit reticulocyte lysate (RRL), and HeLa cell lysate can all be used for in vitro translation. Our Expressway system utilizes E. coli. In general, RRL efficiently translates proteins greater than 30 kDa. The 1-Step Human In Vitro Protein Expression Kits are ideal for expressing human proteins.

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slyD is an endogenous gene product from E. coli. slyD is very Cys-rich, which makes it interact with the Lumio detection agent.

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The Expressway Lumio system incorporates the benefits of the Expressway cell-free system and Lumio technology. Using the Lumio kit, your gene of interest is fused to a Lumio tag, enabling sensitive and specific in-gel detection of the Lumio -tagged fusion protein in polyacrylamide gels without the need for staining or western blotting. You can also monitor real-time synthesis of the Lumio -tagged protein using a standard fluorometer.

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- Toxicity to the host cell from over-expressed product
- Product insolubility and formation of inclusion bodies
- Rapid proteolytic degradation of the expressed protein
- Incorporation of unnatural or modified amino acids
- Incorporation of fluorescent probes into the protein
- Requirement of high-throughput analysis of protein products

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

A cell-free expression system is best used when working with a toxic target, as no cells are needed for protein expression. In vitro protein expression utilizes the necessary cellular components to drive expression in a single tube.

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

- Begin by generating a DNA template, either by PCR or in a plasmid vector
- Purify the template
- Perform the synthesis reaction
- Analyze the sample via Coomassie staining, western blot, etc.

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