Q: 我不小心将我的大肠杆菌slyD-Extract，大肠杆菌反应缓冲液（E. coli Reaction Buffer）（-A.A.）和补料缓冲液（feed buffer）保存在室温下。我还能继续使用它么？

很抱歉，这样的操作会导致产品失活。

Question 1

我不小心将我的大肠杆菌slyD-Extract，大肠杆菌反应缓冲液（E. coli Reaction Buffer）（-A.A.）和补料缓冲液（feed buffer）保存在室温下。我还能继续使用它么？

Accepted Answer

很抱歉，这样的操作会导致产品失活。

Question 2

我的T7 RNA聚合酶已用完。你们建议在后续使用什么产品？

Accepted Answer

我们推荐您使用T7 RNA聚合酶（货号18033019，50 U/µL）。请在50 µL反应系统中使用1–1.5 µL本产品。

Question 3

我的凝胶中的样品出现弥散的情况。这可能是由什么原因所导致的？

Accepted Answer

样品弥散情况可能由以下原因所导致：

•未使用丙酮来沉淀样本——使用丙酮来沉淀蛋白将帮助去除背景弥散情况。
•蛋白上样量过多——减少蛋白样品的用量。
•凝胶自身不够干净——在胶片曝光之前对凝胶进行简单的润洗。
•蛋白合成反应中包含乙醇——请确保在DNA纯化过程中去除了残留的乙醇。
•检查预制胶的保质期——请勿使用过期的凝胶产品。

Question 4

使用Expressway系统反应后，在跑胶时我观察到小分子量产物形成了一条梯带。怎么会发生此种情况？

Accepted Answer

这种现象可能由多种原因所导致。最常见的原因包括：蛋白酶解，DNA和/或RNA模板的降解（截短型的模板将生成截短的蛋白产物），内部启始（如果基因内部含有多个甲硫氨酸和RBS类似序列，核糖体就有可能在错误的甲硫氨酸位点启始翻译过程），提前终止，翻译暂停，过于频繁地应用罕见密码子，复杂的二级RNA结构，等等。蛋白变性时间过长，或1X SDS-PAGE样品缓冲液中加入的SDS不足，也可能会发生这种现象。

Question 5

我获得的蛋白得率不错，但其生物活性很低。我该如何处理？

Accepted Answer

•您的蛋白可能未正确折叠——请尝试在蛋白合成过程中将孵育温度降低至25°C。
•您可能需要对蛋白进行翻译后修饰———Expressway系统不会为重组蛋白进行翻译后修饰。
•您所合成的蛋白可能需要辅助因子才能发挥全部活力——因此请尝试在合成反应中加入所需的辅助因子。

Question 6

我的对照组反应不成功。我该如何处理？

Accepted Answer

如果您的对照组反应未生成蛋白，则可能是试剂丧失活性或受RNA酶的污染。请检查储存条件和试剂的保质期。请小心进行Expressway大肠杆菌slyD-Extract，Expressway 2.5X IVPS大肠杆菌反应缓冲液和Expressway 2X IVPS补料缓冲液（Feed Buffer）的冻融操作。一或两次的冻/融循环是可行的，但应避免多次冻融循环。

Question 7

我表达大分子量蛋白的得率很低。我应如何提升蛋白得率？

Accepted Answer

蛋白得率通常随着蛋白分子量的增加而减小。您可于蛋白合成阶段尝试降低孵育温度至25–30°C。

Question 8

我的蛋白形成了聚集物。我该如何处理？

Accepted Answer

您可在蛋白合成阶段尝试降低孵育温度至25–30°C。此外，也可向反应体系和补料缓冲液中加入温和的去垢剂（如至多0.05%的Triton-X 100,0.025%的十二烷基麦芽苷钠，0.1% CHAPS或0.05% Brij-58）。您也可尝试向反应体系中加入分子伴侣。

Question 9

我的蛋白得率很低或完全不表达，但我的对照组成功生成了蛋白。我该如何处理？

Accepted Answer

请查看下述建议：

•检查您的载体序列（ATG起始密码子，读码框的一致性，等等）。
•如果您的载体包含N端或C端标签，这些序列可能会影响RNA结构和降低翻译水平。可尝试去除融合标签或其他末端序列。
•请确保DNA模板的纯度，且不含酒精、钠盐、乙酸铵或RNA酶等污染物。
•不要使用琼脂糖凝胶中纯化出的DNA，它们会抑制表达反应。
•我们推荐您在2 mL的蛋白合成反应体系中使用10–15 µg的模板DNA。如果您表达的蛋白分子量较大，则可将蛋白合成反应中的DNA模板用量增至20 µg。
•请确保使用恒温混匀仪或带有振荡功能的孵箱来进行反应，请勿使用不带振荡功能的孵箱或水浴。
•多次补料有助于提升蛋白得率。在蛋白合成反应起始后，您可通过更频繁地以小体积多次向样本加入补料缓冲液（feed buffer），来代替在反应初始阶段的30分钟时间点进行一次性补料的操作（即在3小时的时间内，每隔45分钟就向1 mL样本总加入0.25 mL补料缓冲液）。

Question 10

在使用Expressway表达系统时，我应如何在蛋白合成反应结束后对样本进行分析？

Accepted Answer

蛋白合成反应结束后可通过多种方式分析您的样本，包括：考马斯亮蓝蛋白凝胶染色分析，免疫印迹分析，酶活分析或亲和纯化（如有可用的亲和标签）。如果您计划使用聚丙烯酰胺凝胶电泳来分析样本，您可首先以丙酮沉淀蛋白的操作来去除弥散背景。 使用手册（https://tools.thermofisher.com/content/sfs/manuals/expressway_system_man.pdf）第22页中提供了丙酮沉淀及其他通用性的凝胶电泳方案。

Question 11

我担心蛋白发生降解。我能使用蛋白酶抑制剂么？

Accepted Answer

如果有必要，我们推荐用户在Expressway反应起始阶段向体系中加入PMSF（终浓度为0.5-1.0 mM）。最好使用异丙醇而非乙醇来溶解PMSF，因为乙醇对蛋白合成效率会造成不良影响。您也可在转录/翻译反应过程中使用Pafablock C（AEBSF终浓度为0.1–0.2 mM）。这两种物质均为丝氨酸蛋白酶抑制剂。

Question 12

我希望获得糖基化的蛋白。使用Expressway系统能实现么？

Accepted Answer

否，遗憾的是这些抽提物中不含糖基化修饰元件。

Question 13

富含GC的蛋白包含如此多的精氨酸，Expressway系统表达这些蛋白时有困难么？加入额外的精氨酸会对反应有帮助么？

Accepted Answer

我们没有针对此问题进行过专门测试，然而我们知道tRNA会成为限制因素。基于此种原因，简单加入特定种类的氨基酸不会对表达反应有什么帮助。

Question 14

我能对Expressway系统中生成的蛋白进行纯化操作么？

Accepted Answer

我们实际上未曾使用His标签对抽提物中的蛋白进行过纯化。不过，这一操作是可行的，特别是在变性条件下。

Question 15

Expressway系统中生成的蛋白中会形成二硫键么？

Accepted Answer

否。本系统中并不会形成二硫键。不过，用户可添加碘乙酰胺来促进二硫键的形成（Biotechnol Bioeng 2004, 86(2):188–195）。推荐在室温下使用3mM 碘乙酰胺与抽提物预孵育30分钟。反应体系中已包含了1mM DTT（相当于2mM巯基）；因此，1mM 碘乙酰胺就已过量。

Question 16

是否需要向Expressway系统抽提物中添加伴侣蛋白以辅助蛋白正确折叠？

Accepted Answer

大肠杆菌细胞中的确含有一些能够辅助蛋白折叠的伴侣蛋白分子。不过，抽提物中并无额外添加伴侣蛋白。

Question 17

在Expressway反应中，我能使用其他类型的非天然氨基酸替代甲硫氨酸么？

Accepted Answer

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。

Question 18

在使用Expressway系统的过程中，我怎样做才能获得最高的蛋白得率？

Accepted Answer

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。

Question 19

在使用Expressway系统的过程中，我应以何种温度进行蛋白合成反应？

Accepted Answer

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。

Question 20

我只有一台静止的孵箱。它适用于开展Expressway系统实验么？

Accepted Answer

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%。

Question 21

在使用Expressway系统的过程中，反应体积能够按比例扩大或缩小么？

Accepted Answer

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。

Question 22

Expressway系统中的蛋白合成反应时间能延长至2小时以上么？

Accepted Answer

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。

Question 23

Expressway系统所生成的蛋白具有糖基化修饰么？

Accepted Answer

否，这些抽提物中不含糖基化修饰元件。

Question 24

Invitrogen pET载体适用于Expressway系统么？如果不兼容，你们推荐使用何种载体？

Accepted Answer

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。

Question 25

Expressway S30抽提物是源自supF或supE大肠杆菌菌株么？Expressway系统是否能够兼容经修饰的氨基酸？

Accepted Answer

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。

Question 26

Expressway系统中可使用高拷贝数的质粒么？

Accepted Answer

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。

Question 27

Expressway系统对于提升蛋白的溶解性有帮助么？

Accepted Answer

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。

Question 28

在使用Expressway系统的过程中，我是否应在蛋白合成反应之前对我的DNA模板进行凝胶纯化？

Accepted Answer

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。

Question 29

您应在Expressway系统中使用何种表达模板？

Accepted Answer

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。

Question 30

在使用Expressway系统设计载体的过程中，有哪些需要考虑在内的注意事项？

Accepted Answer

•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。

Question 31

体外蛋白表达系统可使用何种裂解系统？

Accepted Answer

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。

Question 32

为何Expressway系统所使用的大肠杆菌抽提物标记了“sly D-”的字样？

Accepted Answer

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。

Question 33

Expressway无细胞表达系统与Expressway Lumio无细胞大肠杆菌表达系统之间的区别是什么？

Accepted Answer

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。

Question 34

Expressway小量无细胞表达系统（Mini Cell-Free Expression System，货号K990100）与Expressway大量无细胞表达系统，货号K990096/K990097）之间的区别是什么？

Accepted Answer

Expressway小量无细胞表达系统经设计，能够胜任二十次50 µL反应或单次的1 mL反应。Expressway大量无细胞表达系统足够200 x 25 µL形式的反应（通过2 x 96孔板来完成）。这两款产品均包含IVPS E. coli Extract（大肠杆菌提取物），IVPS反应缓冲液，补料缓冲液（feed buffer），10种氨基酸的混合物，甲硫氨酸，不含DNA酶/RNA酶的水，RNA酶A，T7酶混合物，2 mL反应管和一个阳性表达对照载体。货号为K990096的产品同时包含了pEXP5-NT/TOPO和pEXP5-CT/TOPO表达载体。

Question 35

Expressway无细胞大肠杆菌表达试剂盒（Cell-Free E. coli Expression Kit）能够克服哪些体内表达系统的缺点？

Accepted Answer

•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

Question 36

无细胞表达系统拥有哪些优势？

Accepted Answer

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。

Question 37

你们能提供一个关于Expressway系统的概述么？

Accepted Answer

•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

Question 38

I accidentally stored my E. coli slyD-Extract, E. coli Reaction Buffer (-A.A.), and 2X feed buffer at room temperature. Can I still use them?

Accepted Answer

Unfortunately, this may result in a loss of activity.

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

Question 39

I've run out of the T7 RNA polymerase for my cell-free expression. What do you suggest I use?

Accepted Answer

We would recommend using T7 RNA polymerase (Cat. No. 18033019, 50 U/µL). Use 1-1.5 µL in a 50 µL reaction system.

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

Question 40

I'm getting smearing after running my cell-free expression reaction on a gel.. What could be the cause of this?

Accepted Answer

Smearing may occur if samples for the following reasons:

- Samples were not precipitated with acetone: precipitate proteins with acetone to remove background smearing.
- Too much protein was loaded: reduce the amount used.
- The gel itself was not clean: rinse the gel briefly before exposing to film.
- Ethanol was present in the protein synthesis reaction: make sure that any residual ethanol is removed during DNA purification.
- Check the date of your pre-cast gels: do not use gels after the expiration date.

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

Question 41

I'm seeing a ladder of small-sized products after running my reaction on a gel when using the Expressway system. Why is this?

Accepted Answer

There may be several reasons for why this is occurring. The most common are: proteolysis, degradation of DNA and/or RNA templates (truncated templates will generate truncated protein products), internal initiation (if there are many methionines and internal RBS-like sequences in the gene, the ribosome may initiate translation from the wrong methionine), premature termination, translational pausing, frequent rare codon usage, complicated secondary structure of RNA, and others. This can also happen if proteins are denatured for too long, or not enough SDS was added to the 1X SDS-PAGE sample buffer.

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

Question 42

With a cell-free expression system, I'm getting good protein yield, but it has low biological activity. What can I do?

Accepted Answer

- Your protein may not be folding properly: try to reduce the incubation temperature to as low as 25 degrees C during synthesis.
- You may require post-translational modification of your protein: the Expressway system will not introduce post-translational modifications to the recombinant protein.
- Your synthetic protein may require co-factors for complete activity: try adding required co-factors to the protein synthesis reaction.

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

Question 43

I am using using a cell-free expression system and my control reaction is not working. What should I do?

Accepted Answer

If you are getting no protein from your control reaction, the reagents may have lost activity or may be contaminated with RNases. Check the storage conditions and expiration of the reagents. Use care when freezing and thawing the Expressway E. coli slyD-Extract, Expressway 2.5X IVPS E. coli Reaction Buffer, and Expressway 2X IVPS Feed Buffer. One or two freeze/thaw cycles are acceptable, but avoid multiple cycles.

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

Question 44

I am using a cell-free expression system and I'm getting low protein yield with my large protein. Is there anything I can do to increase my yield?

Accepted Answer

Protein yield may decrease as the size of the protein increases. You can try to reduce the incubation temperature to 25-30 degrees C during protein synthesis.

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

Question 45

I am using a cell-free expression system and my protein is forming aggregates. What should I do?

Accepted Answer

You can try to reduce the incubation temperature to 25-30 degrees C during protein synthesis. Additionally, a mild detergent can be added (e.g., up to 0.05% Triton-X-100, 0.025% sodium dodecyl maltoside, 0.1% CHAPS, or 0.05% Brij-58) to the reaction and feed buffer. You can also try to add molecular chaperones to the reaction.

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

Question 46

I'm trying to express my protein in vitro and am getting low to no protein yield, whereas my control reaction is producing protein. What should I do?

Accepted Answer

Please review the following suggestions:

- Check the sequence of your vector (ATG initiation codon, in frame, etc.).
- If working with a N- or C-terminal tag, the tag may be affecting the RNA structure and lowering translation levels. Try moving the fusion tag or the other terminus.
- Ensure that your DNA template is pure, and not contaminated with ethanol, sodium salt, ammonium acetate, or RNases.
- Do not purify your DNA from an agarose gel, as this can inhibit the reaction.
- We recommend using 10-15 µg of template DNA in a 2 mL protein synthesis reaction. If you are expressing a large protein, increase the amount of DNA template used in the protein synthesis reaction to 20 µg.
- Ensure that you are using a thermomixer or incubator with shaking, as opposed to a non-shaking incubator or water bath for the reaction.
- Multiple feeding steps can further improve the protein yield. Instead of doing one feeding at 30 min of the initial reaction, you can feed the reaction multiple times with smaller volumes of feed buffer to the sample more frequently (i.e., 0.25 mL feed buffer to 1 mL sample every 45 min over 3 hours) after initiating protein synthesis.

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

Question 47

How can I analyze my samples after the protein synthesis reaction when using the Expressway expression system?

Accepted Answer

There are several ways to analyze your samples after the protein synthesis reaction, including: Coomassie-stained protein gel analysis, western blot analysis, enzymatic activity assay, or by affinity purification (if an affinity tag is present). If you plan to analyze your sample using polyacrylamide gel electrophoresis, you should first precipitate the proteins with acetone to remove background smearing. A protocol to perform acetone precipitation and other general guidelines for gel electrophoresis are provided in the manual (http://tools.thermofisher.com/content/sfs/manuals/expressway_system_man.pdf) on page 22.

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

Question 48

I am using the Expressway system and I'm worried about protein degradation. Can I use a protease inhibitor?

Accepted Answer

If needed, we recommend the addition of PMSF (final concentration 0.5-1.0 mM) at the beginning of the Expressway reaction. It is better to dissolve the PMSF in isopropanol instead of ethanol, as ethanol has a negative effect on protein synthesis. You can also use Pefabloc SC (final concentration 0.1-0.2 mM AEBSF) in your transcription/translation reaction. Both are serine protease inhibitors.

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

Question 49

I want my proteins to be glycosylated. Can I use the Expressway system?

Accepted Answer

No, unfortunately, the machinery for glycosylation is absent in these extracts.

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

Question 50

Since so many amino acids will code for Arg, will this make it difficult to express GC-rich proteins using the Expressway system? Will the addition of extra Arg to the reaction be helpful?

Accepted Answer

We have not specifically tested for this, although we do know that the limiting factor will be the tRNAs. Because of this, simply adding more of a particular amino acid will not make a difference.

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

Question 51

Can I purify proteins produced from the Expressway system?

Accepted Answer

We have not actually done any purifications with the extracts using the His tag. However, it should work, especially if you do it under denaturing conditions.

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

Question 52

Will proteins made in the Expressway system form disulfide bonds?

Accepted Answer

No. Disulfide bridges will not form in this system. However, the formation of disulfide bridges may be achieved through the addition of iodoacetamide (Biotechnol Bioeng 2004, 86(2):188-195). Pre-incubation of the extract with 3 mM iodoacetamide for 30 min at room temperature is recommended. The reaction already contains 1 mM DTT (equivalent to 2 mM sulfhydryls); therefore, only 1 mM iodoacetamide will be in excess.

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

Question 53

Are there any chaperone proteins added to the Expressway system extracts for protein folding?

Accepted Answer

E. coli cells do indeed contain some chaperone proteins used for protein folding. However, extra chaperone proteins were not added to the extract.

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

Question 54

Are there other unnatural amino acids that I can use in my Expressway reaction in place of methionine?

Accepted Answer

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.

Question 55

What can I do to maximize my protein yield when using the Expressway system?

Accepted Answer

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.

Question 56

What temperature should I use for the protein synthesis reaction when using the Expressway system?

Accepted Answer

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.

Question 57

I only have a stationary incubator. Can I use this for my Expressway system experiments?

Accepted Answer

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.

Question 58

Can the reaction volume be scaled up or down using the Expressway system?

Accepted Answer

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.

Question 59

Can the reaction time go beyond 2 hours for protein synthesis in the Expressway system?

Accepted Answer

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.

Question 60

Can the proteins made in the Expressway system be glycosylated?

Accepted Answer

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

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

Question 61

Do the Invitrogen pET vectors work in the Expressway system? If not, what vectors do you suggest?

Accepted Answer

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.

Question 62

Is the Expressway S30 extract prepared from a supF or supE E. coli strain? Can the Expressway system be used with modified amino acids?

Accepted Answer

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.

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

Question 63

Should a high copy number plasmid be used with the Expressway system?

Accepted Answer

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.

Question 64

Does the Expressway system help to increase the solubility of proteins?

Accepted Answer

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.

Expressway™ Maxi Cell-Free E. coli Expression System - FAQs

我不小心将我的大肠杆菌slyD-Extract，大肠杆菌反应缓冲液（E. coli Reaction Buffer）（-A.A.）和补料缓冲液（feed buffer）保存在室温下。我还能继续使用它么？

我的T7 RNA聚合酶已用完。你们建议在后续使用什么产品？

我的凝胶中的样品出现弥散的情况。这可能是由什么原因所导致的？

使用Expressway系统反应后，在跑胶时我观察到小分子量产物形成了一条梯带。怎么会发生此种情况？

我获得的蛋白得率不错，但其生物活性很低。我该如何处理？

我的对照组反应不成功。我该如何处理？

我表达大分子量蛋白的得率很低。我应如何提升蛋白得率？

我的蛋白形成了聚集物。我该如何处理？

我的蛋白得率很低或完全不表达，但我的对照组成功生成了蛋白。我该如何处理？

在使用Expressway表达系统时，我应如何在蛋白合成反应结束后对样本进行分析？

我担心蛋白发生降解。我能使用蛋白酶抑制剂么？

我希望获得糖基化的蛋白。使用Expressway系统能实现么？

富含GC的蛋白包含如此多的精氨酸，Expressway系统表达这些蛋白时有困难么？加入额外的精氨酸会对反应有帮助么？

我能对Expressway系统中生成的蛋白进行纯化操作么？

Expressway系统中生成的蛋白中会形成二硫键么？

是否需要向Expressway系统抽提物中添加伴侣蛋白以辅助蛋白正确折叠？

在Expressway反应中，我能使用其他类型的非天然氨基酸替代甲硫氨酸么？

在使用Expressway系统的过程中，我怎样做才能获得最高的蛋白得率？

在使用Expressway系统的过程中，我应以何种温度进行蛋白合成反应？

我只有一台静止的孵箱。它适用于开展Expressway系统实验么？

在使用Expressway系统的过程中，反应体积能够按比例扩大或缩小么？

Expressway系统中的蛋白合成反应时间能延长至2小时以上么？

Expressway系统所生成的蛋白具有糖基化修饰么？

Invitrogen pET载体适用于Expressway系统么？如果不兼容，你们推荐使用何种载体？

Expressway S30抽提物是源自supF或supE大肠杆菌菌株么？Expressway系统是否能够兼容经修饰的氨基酸？

Expressway系统中可使用高拷贝数的质粒么？

Expressway系统对于提升蛋白的溶解性有帮助么？

在使用Expressway系统的过程中，我是否应在蛋白合成反应之前对我的DNA模板进行凝胶纯化？

您应在Expressway系统中使用何种表达模板？

在使用Expressway系统设计载体的过程中，有哪些需要考虑在内的注意事项？

体外蛋白表达系统可使用何种裂解系统？

为何Expressway系统所使用的大肠杆菌抽提物标记了“sly D-”的字样？

Expressway无细胞表达系统与Expressway Lumio无细胞大肠杆菌表达系统之间的区别是什么？

Expressway小量无细胞表达系统（Mini Cell-Free Expression System，货号K990100）与Expressway大量无细胞表达系统，货号K990096/K990097）之间的区别是什么？

Expressway无细胞大肠杆菌表达试剂盒（Cell-Free E. coli Expression Kit）能够克服哪些体内表达系统的缺点？

无细胞表达系统拥有哪些优势？

你们能提供一个关于Expressway系统的概述么？

I accidentally stored my E. coli slyD-Extract, E. coli Reaction Buffer (-A.A.), and 2X feed buffer at room temperature. Can I still use them?

I've run out of the T7 RNA polymerase for my cell-free expression. What do you suggest I use?

I'm getting smearing after running my cell-free expression reaction on a gel.. What could be the cause of this?

I'm seeing a ladder of small-sized products after running my reaction on a gel when using the Expressway system. Why is this?

With a cell-free expression system, I'm getting good protein yield, but it has low biological activity. What can I do?

I am using using a cell-free expression system and my control reaction is not working. What should I do?

I am using a cell-free expression system and I'm getting low protein yield with my large protein. Is there anything I can do to increase my yield?

I am using a cell-free expression system and my protein is forming aggregates. What should I do?

I'm trying to express my protein in vitro and am getting low to no protein yield, whereas my control reaction is producing protein. What should I do?

How can I analyze my samples after the protein synthesis reaction when using the Expressway expression system?

I am using the Expressway system and I'm worried about protein degradation. Can I use a protease inhibitor?

I want my proteins to be glycosylated. Can I use the Expressway system?

Since so many amino acids will code for Arg, will this make it difficult to express GC-rich proteins using the Expressway system? Will the addition of extra Arg to the reaction be helpful?

Can I purify proteins produced from the Expressway system?

Will proteins made in the Expressway system form disulfide bonds?

Are there any chaperone proteins added to the Expressway system extracts for protein folding?

Are there other unnatural amino acids that I can use in my Expressway reaction in place of methionine?

What can I do to maximize my protein yield when using the Expressway system?

What temperature should I use for the protein synthesis reaction when using the Expressway system?

I only have a stationary incubator. Can I use this for my Expressway system experiments?

Can the reaction volume be scaled up or down using the Expressway system?

Can the reaction time go beyond 2 hours for protein synthesis in the Expressway system?

Can the proteins made in the Expressway system be glycosylated?

Do the Invitrogen pET vectors work in the Expressway system? If not, what vectors do you suggest?

Is the Expressway S30 extract prepared from a supF or supE E. coli strain? Can the Expressway system be used with modified amino acids?

Should a high copy number plasmid be used with the Expressway system?

Does the Expressway system help to increase the solubility of proteins?

Should I gel purify my DNA template before using it in my protein synthesis reaction using the Expressway system?

What template should I use for expression using the Expressway system?

Are there considerations I need to take into account for my vector design when using the Expressway system?

What lysate systems can be used for in vitro protein expression?

Why is the E. coli extract used in the Expressway system labeled as "sly D"?

What is the difference between the Expressway Cell-Free and Expressway Lumio Cell-Free E. coli Expression Systems?

What is the difference between the Expressway Mini Cell-Free Expression System (Cat. No. K990100) and the Expressway Maxi Cell-Free Expression System (Cat. Nos. K990096/K990097)?

What are some disadvantages of an in vivo expression system that can be overcome with the Expressway Cell-Free E. coli Expression Kit?

What are the advantages of using a cell-free expression system?

Can you give me an overview of the Expressway system?

Because so many amino acids will code for Arg, will this make it be difficult to express GC-rich proteins using the Expressway System? Will the addition of extra Arg to reaction be helpful in this case?

Where can I purchase a Thermomixer?

Is the Expressway S30 extract prepared from a supF or supE E. coli strain? Can the Expressway system be used with modified amino acids?

Should a high copy number plasmid be used with Expressway system?

Can the reaction volume be scaled up using the Expressway System?