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Jun 2017

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A General Method for Intracellular Protein Delivery through ‘E-tag’ Protein Engineering and Arginine Functionalized Gold Nanoparticles
通过'E标签'蛋白质工程和精氨酸功能化金纳米粒子进行细胞内蛋白质递送的通用方法   

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Abstract

In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and nanoparticles were mixed, they formed nanoassemblies, which got fused to cell membrane upon incubation and directly released the E-tagged protein into cell cytosol. We used this method to deliver a wide variety of proteins with different sizes, charges, and functions in various cell lines (Mout et al., 2017).

To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant E-tagged proteins). Laboratory-synthesis of gold nanoparticles has been previously described (Yang et al., 2011). Desired E-tagged proteins can be cloned from the corresponding genes, and expressed and purified using standard laboratory procedures. We will use E-tagged green fluorescent protein (GFP) as a reference protein here. Users can simply insert an E-tag into their protein of interest, at either terminus. To achieve maximum delivery efficiency, we suggest users testing different length of E-tags. For example, we inserted E = 0 to 20 (E0 means no E-tag insertion, and E20 means 20 glutamic acids insertion in a row) to most of the proteins we tested, and screened for optimal E-tagged length for highest delivery efficiency. E10-tagged proteins gave us the highest delivery efficiency for most of the proteins (except for Cas9, where E20 tag showed highest delivery efficiency).

Once these materials are ready, it takes about ~10 min to make the E-tagged protein and nanoparticle nanoassemblies, which are immediately used for delivery. Complete delivery (~100% for GFP-E10) is achieved in less than 3 h.

Keywords: Intracellular protein delivery (细胞内蛋白质递送), Direct cytosolic protein delivery (直接胞质蛋白递送), E-tagged protein (E标签蛋白), Nanoparticles (纳米粒子)

Background

Intracellular delivery of exogenous proteins into cells is crucial for cellular imaging and diagnosis, therapeutic development, genome engineering and synthetic biology applications (Fu et al., 2014). Many of the applications in cellular engineering and imaging (such as genome editing, cellular imaging) require delivering exogenous proteins, as mammalian cells do not have genes for those proteins. However, access to the whole cytoplasm by the delivered protein remains elusive. A major hurdle in cytoplasmic protein delivery is the endosomal entrapment of the delivered cargo: nanocarrier-based delivery methods result in only a fraction of the entrapped cargo (often ~1%) escaping into the cytosol (Stewart et al., 2016). Additionally, protease-mediated degradation and exocytosis of the remaining entrapped cargo proteins make these strategies ultimately inefficient. Delivery through membrane disruption methods can provide efficient cytosolic protein delivery; however, these methods generally require additional osmolytic surfactants (Erazo-Oliveras et al., 2014), hypertonic agents (D’Astolfo et al., 2015), or mechanical distortion techniques (Han et al., 2015) that may be harmful for the cells. Our protocol provides an approach for direct cytosolic delivery of a desired protein for applications including cellular imaging and basic cell biology research (Mout et al., 2017).

Materials and Reagents

  1. Round bottom 35 mm confocal dish (MATTEK, catalog number: P35G-0-14-C )
  2. 24-well plates (Corning, Costar®, catalog number: 3524 )
  3. Sterile tubes (0.6, 1.5, and/or 2.0 ml) (Fisherbrand)
  4. Sterile pipette tips (white, yellow and blue tips)
  5. Cell lines (i.e., HeLa)
  6. 1x Phosphate buffered saline (PBS) (GE Healthcare, HyCloneTM, catalog number: SH30028.02 )
  7. Plain DMEM media (No serum and antibiotics) (Thermo Fisher Scientific, GibcoTM, catalog number: 10567014 )
  8. Appropriate media for cell culture (i.e., HeLa cells, DMEM, Thermo Fisher Scientific, GibcoTM, catalog number: 10567014 )
  9. Alexa Fluor 488 NHS Ester (Thermo Fisher Scientific, InvitrogenTM, catalog number: A20000 )
  10. Stock solution of ArgNP gold nanoparticles (~50 μM in water, laboratory synthesized)
  11. E-tagged proteins (laboratory purified)

Equipment

  1. Pipettes (P2, P10, P20, P200, P1000)
  2. BSL-2 safety cabinet for cell culture
  3. Cell culture incubator at 5% CO2 and 37 °C
  4. Confocal microscope (any brand)
  5. Flow cytometer (i.e., BD, model: LSR II )

Procedure

There are two key steps for successful high efficient cytosolic protein delivery. First, the length of E-tag determines the delivery efficiency. For each protein, the length may be different. Therefore, users are suggested to make their protein of interest with at least few different E-tags (of different E-tag length, for example, E5, E10, E15, and E20). For most average molecular weight proteins (MW < 50 kDa), E10 tag gives high efficient delivery, however, for large proteins such as Cas9 (MW = 160 kDa) E20 tag gives best delivery. Second, the molar ratio of ArgNPs to E-tagged protein determines the delivery efficiency. Again, for each protein, this ratio may be different. Therefore, users are also suggested to test few different molar ratios of ArgNPs/E-tagged protein of their interest to find out the best delivery. From our experience, for most E-tagged proteins, one of the following molar ratios gave highest delivery efficiency: [ArgNP]/[E-tagged protein] = 1:0.5; 1:1; 1:2; 1:3. However, users are suggested to test any other ratios as they may think appropriate.
In the following protocol (Figure 1), we use GFP-E10 to demonstrate the assembly formation and delivery process. However, as mentioned above, users are requested to identify the optimum E-tag length and ‘working molar ratio’ for their protein of interest using the same procedure as described for GFP-E10. Additionally, E-tagged proteins should be fluorophore-labeled to assess delivery efficiency by confocal microscopy imaging, if the protein is a non-fluorescent protein. [For Alexa Fluor 488 NHS Ester, a labeling protocol can be found on manufacturer’s website.]
Final working nanoassembly concentration is 250 nM of ArgNPs and 750 nM of GFP-E10, which is at 1:3 molar ratio of ArgNPs/GFP-E10. The total volume of the nanoassembly samples required for delivery depends on the kind of cell culture plate used. We generally use 1 ml for round bottom 35 mm confocal dish, 500 μl for 24-well plates, per well. Therefore, the nanoassemblies should be prepared and scaled up according to users need. The following calculation is for one sample in 24-well plate (i.e., 500 μl total volume). Additionally, the following protocol is for HeLa cells only. Nevertheless, we have used a wide variety of cell lines to demonstrate our delivery platform. Some cell lines that we successfully tested are: human embryonic kidney cells (HEK), mammary epithelial cells (MCF-7), mouse macrophage (RAW 264.7), human ovarian cancer cells (SKOV-3), and T-lymphocyte cells (Jurkat).


Figure 1. Schematic overview of the protocol. Step 1: Formation of ArgNPs/E-tagged protein nanoassembly (takes 10 min), and Step 2: E-tagged protein delivery (takes up to 3 h).

Day 1
Seed cells on 24-well plate at a cell density of 0.8-1 x 105 cells/well (For confocal dish, seed 2.4 x 105 cells per dish). Let the cells grow for 24 h at 5% CO2 and 37 °C in a cell culture incubator.

Day 2

  1. Step 1: ArgNPs/E-tagged protein nanoassembly preparation
    1. This step (Step 1) should be performed 10 min before the delivery experiment (Step 2).
    2. Add 100 μl of 1x PBS into a sterile 1.5 ml tube.
    3. Add 2.5 μl of ArgNPs (50 μM stock) into it.
      Note: The final working concentration of ArgNPs is 250 nM, and the total working sample volume is 500 μl. If the stock concentration of ArgNPs is different, please adjust the amount (volume) of nanoparticle to be added. Also, scale up or down based on the total working volume of the assembly sample.
    4. Then add 7.5 μl of GFP-E10 (50 μM stock) into the ArgNPs solution and mix well. Here, the final working concentration of GFP-E10 is 750 nM at a [ArgNPs]/[GFP-E10] molar ratio = 1:3 (250:750 nM). Again, adjust the volume of GFP-E10 or E-tagged protein of interest based on stock solution concentration and required molar ratio.
      Note: At this step, users can choose their protein of interest with different E-tag length and a few [ArgNPs]/[E-tagged protein] molar ratios to find out maximum cytosolic delivery.
    5. Incubate the complex at room temperature for 10 min.
    6. The nanoassembly is ready for the next step, i.e., delivery.
  2. Step 2: Delivery
    1. While the nanoassembly was incubated for 10 min, users should prepare the cells for delivery in the meantime.
      Note: Don’t incubate for more than 10 min, as this may cause aggregate formation instead of well-defined assemblies.
    2. Wash the cells with 1x PBS, twice.
    3. Add ~400 μl of DMEM plain media (or any media of interest) into the nanoassembly solution in the tube. Mix well by gently pipetting up and down.
      Note: Media with serum should be strictly avoided at this step; media with serum may require further optimization such as the length of E-tag, the ratio of nanoparticles to E-tagged protein, and incubation time.
    4. Add the whole (500 μl) sample into the washed-cells.
    5. Incubate the cells in appropriate condition (usually 5% CO2 and 37 °C) for 3 h. Complete protein delivery should be achieved after 3 h of incubation.
    6. Wash away the media after 3 h, and replace with fresh desired media (DMEM with 10% serum and 1% antibiotics, and any supplements if needed).

Data analysis

  1. Two types of data assessments are performed to evaluate efficient cytosolic protein delivery. First, thorough distribution of the protein in the cytoplasm (using confocal microscopy imaging) would indicate effective delivery, whereas, punctate distribution would mean endosomal entrapment of proteins (Figures 2A and 2B). Second, the percentage of transduced cells can be assessed by using either confocal microscopy imaging or flow cytometry analysis (Figure 2C) (For detailed procedure for confocal microscopy and flow cytometry, please see Mout et al., 2017). Note that, the protein of interest must be fluorophore-labeled, or a fused fluorescent protein be used for the above imaging and quality assessment purposes.
  2. Following are a few representative results showing effective cytosolic delivery of GFP-E10. To compare the effectiveness of our strategy with other methods that suffer from endosomal entrapment of the delivered protein, we also provided an image showing (+36)-GFP delivery that predominately gets trapped in the endosomes (Cronican et al., 2010). Likewise, percentage GFP delivery was compared among GFP-E0, GFP-E5, and GFP-E10 using flow cytometry analysis. The result shows highest delivery efficiency for GFP-E10. Please see Mout et al., 2017 for more details.


    Figure 2. Representative data showing efficient protein delivery vs. ineffective delivery. Confocal microscopy images showing examples of effective cytosolic delivery of GFP-E10 (A), versus endosomal delivery of (+36)-GFP in HeLa cells (B). Note that, GFP-E10 was delivered using above protocol at a molar ratio of [ArgNPs]/[GFP-E10] = 1:3 (250:750 nM). For comparison purpose, we delivered (+36) GFP following a reported method (Cronican et al., 2010). C. Flow cytometric determination of delivery efficiency of E-tagged GFP with different tag length. As shown in the figure, GFP-E10 exhibited highest deliver efficiency.

Notes

Every batch of ArgNPs can be slightly deferent in terms of surface ligand coverage and therefore users are suggested to test different molar ratio of ArgNPs/E-tagged protein to find out optimum ratio for maximum delivery.

Acknowledgments

This research was supported by the NIH (GM077173), and NSF (CHE-1307021). This protocol was adapted from Mout et al., 2017.
The authors declare the following competing financial interest(s): V.M.R and R.M. submitted a non-provisional patent to USPTO (Application number PCT/US2016/015711) on the invention.

References

  1. Cronican, J. J., Thompson, D. B., Beier, K. T., McNaughton, B. R., Cepko, C. L. and Liu, D. R. (2010). Potent delivery of functional proteins into Mammalian cells in vitro and in vivo using a supercharged protein. ACS Chem Biol 5(8): 747-752.
  2. D’Astolfo, D. S., Pagliero, R. J., Pras, A., Karthaus, W. R., Clevers, H., Prasad, V., Lebbink, R. J., Rehmann, H. and Geijsen, N. (2015). Efficient intracellular delivery of native proteins. Cell 161(3): 674-690.
  3. Erazo-Oliveras, A., Najjar, K., Dayani, L., Wang, T. Y., Johnson, G. A. and Pellois, J. P. (2014). Protein delivery into live cells by incubation with an endosomolytic agent. Nat Methods 11(8): 861-867.
  4. Fu, A., Tang, R., Hardie, J., Farkas, M. E. and Rotello, V. M. (2014). Promises and pitfalls of intracellular delivery of proteins. Bioconjug Chem 25(9): 1602-1608.
  5. Han, X., Liu, Z., Jo, M. C., Zhang, K., Li, Y., Zeng, Z., Li, N., Zu, Y. and Qin, L. (2015). CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation. Sci Adv 1(7): e1500454.
  6. Mout, R., Ray, M., Tay, T., Sasaki, K., Yesilbag Tonga, G. and Rotello, V. M. (2017). General strategy for direct cytosolic protein delivery via protein-nanoparticle co-engineering. ACS Nano 11(6): 6416-6421.
  7. Stewart, M. P., Sharei, A., Ding, X., Sahay, G., Langer, R. and Jensen, K. F. (2016). In vitro and ex vivo strategies for intracellular delivery. Nature 538(7624): 183-192.
  8. Yang, X. C., Samanta, B., Agasti, S. S., Jeong, Y., Zhu, Z. J., Rana, S., Miranda, O. R. and Rotello, V. M. (2011). Drug delivery using nanoparticle-stabilized nanocapsules. Angew Chem Int Ed Engl 50(2): 477-481.

简介

在这个协议中,我们描述了直接胞质蛋白递送的方法,其避免了递送的蛋白质的内体截留(endosomal embrapment)。我们通过用寡聚谷氨酸标签(E-tag)标记期望的蛋白质,随后使用载体金纳米颗粒来递送这些E标记的蛋白质来实现这一点。当电子标记的蛋白质和纳米粒子混合时,它们形成了纳米组装体,在孵育后融合到细胞膜上,并直接将电子标签蛋白质释放到细胞胞液中。我们使用这种方法在各种细胞系中提供各种不同大小,电荷和功能的蛋白质(Mout et al。,2017)。

要使用此协议,第一步是生成所需的材料(金纳米粒子,重组E标记蛋白质)。先前已经描述了金纳米颗粒的实验室合成(Yang等人,2011)。所需的电子标记的蛋白质可以从相应的基因克隆,并使用标准的实验室程序表达和纯化。我们将在这里使用电子标记的绿色荧光蛋白(GFP)作为参考蛋白。用户可以简单地在任意一端插入一个电子标签到他们感兴趣的蛋白质中。为了达到最大的传送效率,我们建议用户测试不同长度的电子标签。例如,对于我们测试的大多数蛋白质,我们插入了E = 0到20(E0表示没有E标签插入,E20表示20个谷氨酸插入一行),并筛选最佳的E标记长度以获得最高的递送效率。对于大多数蛋白质,E10标记的蛋白质给予我们最高的递送效率(除了Cas9,其中E20标签显示最高的递送效率)。

一旦这些材料准备好了,大约需要10分钟才能制作立即用于递送的E标记蛋白质和纳米颗粒纳米组装体。完成交付(GFP-E10〜100%)在不到3小时内完成。

【背景】外源蛋白质细胞内递送至细胞对于细胞成像和诊断,治疗发展,基因组工程和合成生物学应用是至关重要的(Fu等人,2014)。细胞工程和成像中的许多应用(例如基因组编辑,细胞成像)需要递送外源蛋白质,因为哺乳动物细胞不具有那些蛋白质的基因。然而,通过递送的蛋白质进入整个细胞质仍然难以捉摸。细胞质蛋白质递送的主要障碍是递送货物的内体截留:基于纳米载体的递送方法仅导致一部分截留的货物(通常约1%)逃逸到胞质溶胶中(Stewart等人 >,2016)。另外,蛋白酶介导的对剩余捕获的货物蛋白质的降解和胞吐作用使得这些策略最终效率低下。通过膜破坏方法递送可以提供有效的胞质蛋白质递送;然而,这些方法通常需要额外的渗透性表面活性剂(Erazo-Oliveras等人,2014),高渗剂(D'Astolfo等人,2015)或机械扭曲技术(Han等人,2015),可能对细胞有害。我们的方案为细胞成像和基础细胞生物学研究(Mout等人,2017)的应用提供了直接胞质溶胶递送期望的蛋白质的方法。

关键字:细胞内蛋白质递送, 直接胞质蛋白递送, E标签蛋白, 纳米粒子

材料和试剂

  1. 圆底35mm共焦盘(MATTEK,目录号:P35G-0-14-C)
  2. 24孔板(Corning,Costar ®,产品目录号:3524)
  3. 无菌管(0.6,1.5和/或2.0毫升)(Fisherbrand)
  4. 无菌吸头(白色,黄色和蓝色的提示)
  5. 细胞系(即。,HeLa)
  6. 1x磷酸盐缓冲盐水(PBS)(GE Healthcare,HyClone TM,目录号:SH30028.02)
  7. 纯DMEM培养基(无血清和抗生素)(Thermo Fisher Scientific,Gibco TM,产品目录号:10567014)
  8. 用于细胞培养的合适培养基(即,HeLa细胞,DMEM,Thermo Fisher Scientific,Gibco TM,产品目录号:10567014)
  9. Alexa Fluor 488 NHS酯(Thermo Fisher Scientific,Invitrogen TM,目录号:A20000)
  10. ArgNP金纳米颗粒的储备溶液(约50μM在水中,实验室合成)
  11. 电子标记的蛋白质(实验室纯化)

设备

  1. 移液器(P2,P10,P20,P200,P1000)
  2. BSL-2细胞培养安全柜
  3. 在5%CO 2和37℃下的细胞培养孵育器
  4. 共聚焦显微镜(任何品牌)
  5. 流式细胞仪(即em,即BD,型号:LSR II)

程序

有两个关键的步骤成功的高效胞质蛋白递送。首先,E-tag的长度决定了传递效率。对于每种蛋白质,长度可能不同。因此,建议用户用至少几个不同的E标签(具有不同的E标签长度,例如E5,E10,E15和E20)制造他们感兴趣的蛋白质。对于大多数平均分子量的蛋白质(MW <50kDa),E10标签提供高效递送,然而,对于大蛋白质如Cas9(MW = 160kDa),E20标签产生最佳递送。其次,ArgNPs与E标记蛋白质的摩尔比决定了递送效率。再次,对于每种蛋白质,这个比率可能不同。因此,也建议使用者测试几种不同的他们感兴趣的ArgNP / E标记蛋白质的摩尔比以找出最佳递送。根据我们的经验,对于大多数电子标记的蛋白质,以下摩尔比之一给出最高的递送效率:[ArgNP] / [E标记的蛋白质] = 1:0.5; 1:1; 1:2; 1:3。不过,建议用户测试任何其他比例,因为他们认为是适当的。
在下面的协议(图1)中,我们使用GFP-E10来演示组件的形成和交付过程。然而,如上所述,要求用户使用与GFP-E10所述相同的程序来鉴定其感兴趣的蛋白质的最佳E-标签长度和“工作摩尔比”。此外,如果蛋白质是非荧光蛋白,则应用荧光标记的电子标记的蛋白质以通过共焦显微成像评估递送效率。 [对于Alexa Fluor 488 NHS酯,标签协议可以在制造商的网站上找到。]
最终工作纳米组装浓度是250nM的ArgNP和750nM的GFP-E10,其与ArgNPs / GFP-E10的摩尔比为1:3。交付所需的纳米组装样品的总体积取决于所使用的细胞培养板的种类。我们通常使用1毫升圆底35毫米共聚焦盘,每孔500微升的24孔板。因此,应该根据用户需要准备和扩大纳米装配。以下计算是针对24孔板中的一个样品(即,总体积为500μl)。此外,以下协议仅适用于HeLa细胞。尽管如此,我们已经使用了各种细胞系来展示我们的交付平台。我们成功测试的一些细胞系是:人胚胎肾细胞(HEK),乳腺上皮细胞(MCF-7),小鼠巨噬细胞(RAW 264.7),人卵巢癌细胞(SKOV-3)和T淋巴细胞(Jurkat )。


步骤1:形成ArgNPs / E标记的蛋白质纳米组装体(需要10分钟),和步骤2:电子标记的蛋白质递送(需要3小时)。图1:方案的示意性概述 。

第1天
在24孔板上的种子细胞,细胞密度为0.8-1×10 5个细胞/孔(对于共聚焦培养皿,种子每个培养皿2.4×10 5个细胞)。让细胞在5%CO 2和37℃的细胞培养箱中生长24小时。

第2天

  1. 步骤1:ArgNPs / E标记蛋白质纳米装配制备
    1. 这个步骤(步骤1)应该在交付实验(步骤2)之前10分钟进行。
    2. 添加100微升的1x PBS到无菌1.5毫升管。
    3. 添加2.5μL的ArgNPs(50μM库存)。
      注意:ArgNPs的最终工作浓度是250 nM,总的工作样品体积是500μl。如果ArgNPs的库存浓度不同,请调整要添加的纳米粒子的量(体积)。另外,根据装配样品的总工作量进行放大或缩小。
    4. 然后添加7.5μLGFP-E10(50μM储备)到ArgNPs溶液中并充分混合。此处,[ArgNPs] / [GFP-E10]摩尔比= 1:3(250:750nM)时,GFP-E10的最终工作浓度为750nM。再次,根据原液浓度和所需的摩尔比调整感兴趣的GFP-E10或E标签蛋白的体积。
      注意:在这一步,用户可以选择不同的E标签长度和几个[ArgNPs] / [E标签蛋白]摩尔比的感兴趣的蛋白质,以找出最大的胞质输送。 />

    5. 在室温下孵育复合物10分钟
    6. 纳米装配已准备好进行下一步,即交付。
  2. 第2步:交付
    1. 当纳米组装被孵化10分钟,用户应准备细胞交付在此期间。
      注意:不要孵育超过10分钟,因为这可能会导致聚集体形成,而不是定义明确的组装。
    2. 用1x PBS清洗细胞两次。
    3. 加入〜400μL的DMEM普通培养基(或任何感兴趣的媒体)到管中的纳米组装溶液中。轻轻地上下吹打混匀。
      注意:这一步应严格避免血清培养基;血清培养基可能需要进一步优化,如E标签的长度,纳米颗粒与E标记蛋白质的比例以及孵育时间。
    4. 将全部(500μl)样品加入洗涤的细胞中。
    5. 孵育细胞在适当的条件下(通常5%CO 2和37℃)3小时。温育3小时后应完成蛋白质递送。
    6. 3小时后将培养基冲洗干净,更换为新鲜的所需培养基(含10%血清和1%抗生素的DMEM,以及任何补充剂,如果需要的话)。

数据分析

  1. 进行两类数据评估以评估有效的胞质蛋白递送。首先,蛋白质在细胞质中的彻底分布(使用共聚焦显微镜成像)将指示有效递送,而点状分布将意味着蛋白质的内体截留(图2A和2B)。其次,转导细胞的百分比可以通过使用共聚焦显微镜成像或流式细胞仪分析来评估(图2C)(关于共聚焦显微术和流式细胞术的详细程序,请参见Mout等人,2017年)。请注意,感兴趣的蛋白质必须荧光标记,或融合荧光蛋白用于上述成像和质量评估目的。
  2. 以下是一些有代表性的结果,显示GFP-E10有效的胞质输送。为了比较我们的策略与遭受递送蛋白的内体截留的其他方法的有效性,我们还提供了显示(+36)-GFP递送的图像,其主要被捕获在内体中(Cronican等人,2010)。同样地,使用流式细胞术分析比较GFP-E0,GFP-E5和GFP-E10之间的GFP递送百分比。结果显示GFP-E10的最高输送效率。请参阅Mout et al 。,2017了解更多详情。


    图2.显示有效蛋白质递送与无效递送的代表性数据共聚焦显微镜图像显示GFP-E10的有效胞质递送(A)的实例,与HeLa中(+36)-GFP的内体内递送细胞(B)。注意,使用上述方案以[ArgNPs] / [GFP-E10] = 1:3(250:750nM)的摩尔比递送GFP-E10。为了比较,我们按照报道的方法(Cronican等人,2010)递送(+36)GFP。 C.流式细胞仪测定具有不同标签长度的E标签GFP的递送效率。如图所示,GFP-E10显示出最高的传送效率。

笔记

每一批ArgNPs在表面配体覆盖率方面可能略有不同,因此建议用户测试不同摩尔比的ArgNPs / E标签蛋白,以找出最佳递送比例。

致谢

这项研究得到了NIH(GM077173)和NSF(CHE-1307021)的支持。该协议是从Mout et al。,2017年改编的。
作者声明以下竞争性财务利益:V.M.R和R.M.向USPTO(申请号PCT / US2016 / 015711)提交了非临时专利。

参考

  1. Cronican,J.J.,Thompson,D.B。,Beier,K.T.,McNaughton,B.R。,Cepko,C.L.和Liu,D.R。(2010)。 功能性蛋白质在哺乳动物细胞体内的有效传递和使用超荷电蛋白在体内。 ACS Chem Biol 5(8):747-752。
  2. D'Astolfo,D. S.,Pagliero,R. J.,Pras,A.,Karthaus,W. R.,Clevers,H.,Prasad,V.,Lebbink,R. J.,Rehmann,H.和Geijsen,N.(2015)。 天然蛋白质的高效细胞内递送 161( 3):674-690。
  3. Erazo-Oliveras,A.,Najjar,K.,Dayani,L.,Wang,T.Y.,Johnson,G.A。和Pellois,J.P.(2014)。 蛋白质通过与内体裂解剂孵育而进入活细胞 Nat.Methods 11(8):861-867。
  4. Fu,A.,Tang,R.,Hardie,J.,Farkas,M.E。和Rotello,V.M。(2014)。 蛋白质细胞内递送的承诺和缺陷
  5. Han,X.,Liu,Z.,Jo,M.C.,Zhang,K.,Li,Y.,Zeng,Z.,Li,N.,Zu,Y.and Qin,L.(2015)。 CRISPR-Cas9通过膜变形难以转染细胞 Sci Adv 1(7):e1500454。
  6. Mout,R.,Ray,M.,Tay,T.,Sasaki,K.,Yesilbag Tonga,G.和Rotello,V. M.(2017)。 通过蛋白质纳米颗粒联合工程直接胞质蛋白递送的一般策略 ACS Nano 11(6):6416-6421。
  7. Stewart,M.P.,Sharei,A.,Ding,X.,Sahay,G.,Langer,R.和Jensen,K.F。(2016)。 体外和体外策略细胞内递送。 538(7624):183-192。
  8. Yang,X. C.,Samanta,B.,Agasti,S. S.,Jeong,Y.,Zhu,Z.J.,Rana,S.,Miranda,O.R。和Rotello,V.M。(2011)。 使用纳米颗粒稳定纳米胶囊的药物递送 Angew Chem Int Ed Engl 50(2):477-481。
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引用:Mout, R. and Rotello, V. M. (2017). A General Method for Intracellular Protein Delivery through ‘E-tag’ Protein Engineering and Arginine Functionalized Gold Nanoparticles. Bio-protocol 7(24): e2661. DOI: 10.21769/BioProtoc.2661.
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