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Dec 2013

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Fluorometric Estimation of Viral Thermal Stability
荧光法预测病毒热稳定性   

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Abstract

Differential Scanning Fluorimetry (DSF) is a rapid, economical, and a straightforward technique for estimating the thermal stability of proteins. The principle involves the binding of a fluorescent dye to thermally exposed hydrophobic pockets of a protein. The dyes used in this technique are highly fluorescent in a non-polar environment and are quenched when exposed to aqueous solution. The change in fluorescence can be used to follow unfolding of proteins induced by temperature, pH, or chaotropic agents. The method is well characterized for monomeric proteins. Here, we extend the application to supramolecular protein and nucleo-protein complexes using virus particles as an example. SYPRO-orange™ dye is the dye of choice because it is matched for use with q-PCR instruments and the fluorescence response is stable across a wide range of pH and temperatures. Advantages of this technique over standard biophysical methods include the ability for high-throughput screening of biological and technical replicates and the high sensitivity.

Keywords: Biophysics (生物物理学), Virology (病毒学), Fluorescence (荧光), Hydrophobic patches (疏水补丁), Subunit interactions (亚基的相互作用)

Materials and Reagents

  1. Purified Virus Like Particles (VLPs) of Adeno-associated virus serotypes 1, 2, 5, 8
  2. Purified recombinant AAV (rAAV) capsids packaging a green fluorescent protein gene (GFP), rAAV1-GFP, rAAV5-GFP, rAAV8-GFP
  3. PBS (pH 7.0) at 25 °C
  4. Citric acid (Thermo Fisher Scientific, catalog number: BP339-500 )
  5. Disodium phosphate (VWR International, catalog number: BDH4538-1KGP )
  6. 5,000x SYPRO Orange (Life Technologies, catalog number: S6651 )
  7. Sodium hydroxide (Thermo Fisher Scientific, catalog number: BP359-500 )
  8. 12 N hydrochloric acid (EMD Millipore, catalog number: HX0603-3 )
  9. Sodium chloride (VWR International, catalog number: BDH4534-5KGP )
  10. Magnesium chloride (Thermo Fisher Scientific, catalog number : BP214-500 )
  11. Molecular biology grade water
  12. Citrate-Phosphate buffer (see Recipes)

Equipment

  1. Rotor Gene-3000, qPCR instrument with 36-well rotor (QIAGEN)
  2. 0.2 ml PCR tubes (BioExpress, catalog number: T-3013-1FC )
  3. Aluminum foil
  4. Pipette tips (10 µl, 200 µl) (VWR International, catalog numbers: 37001-162 and 53508-810 )
  5. Pipetts (200 µl, 0.1-2.5 µl) (Eppendorf, BioExpress, catalog numbers: P-3015-6 and P-3015-1 )
  6. pH meter (Accument Basic AB 15 pH meter) (Thermo Fisher Scientific)
  7. Milli-Q purification system (EMD Millipore)

Software

  1. Rotor gene software (Version 1.7, Build 94)
  2. Microsoft Excel 2007 on Windows 7

Procedure

  1. Each of the VLP and recombinant virus samples were diluted into PBS or the citrate-phosphate buffer to obtain concentrations between 0.1-0.24 mg/ml. A sample of lysozyme, at a concentration of 0.24 mg/ml, was included in each run as a positive control. The concentrations of the samples were varied to obtain similar fluorescence intensities on the instrument.
  2. Aliquots of the concentrated 5,000x SYPRO Orange was stored at -20 °C and gently and completely thawed out to room temperature before every reaction setup.
  3. 1 µl of the SYPRO Orange was diluted into 100 µl of distilled water to obtain a 1% 50x working stock. As SYPRO Orange is light sensitive, the working stock was covered with aluminum foil and kept in the dark till right before distribution.
  4. 2.5 µl of the working stock was added to each of the above samples. The remaining working stock, if any, was discarded if not used after 4 h.
  5. The samples were kept in an aluminum foil covered box to prevent exposure to light till they were transferred into the qPCR machine.
  6. The assays were conducted with a temperature ramping from 30 to 99 °C, increasing 1 degree every 1 min.
  7. The data was exported into a text (.csv) file and transferred into Microsoft Excel 2007 for data analysis.

Representative data

  1. The raw intensity data from all the samples was normalized to an upper limit of 100% by dividing all values by the largest recorded intensity in that sample. The data was plotted with temperature on the X-axis and the normalized intensity on the Y-axis.
  2. The raw data was also used to calculate the change in fluorescence per change in temperature (dF/dT). The maximum value obtained from this calculation was defined as the melting temperature (Tm). This can be accomplished by taking the difference between sequential fluorescent readings divided by the change in temperature between measurements. The change in intensity (dF) can then be graphed vs temperature. The largest value will can then be visualized or selected from the list. As described here, the temperature step-size is 1 degree.


    Figure 1. Showing a representative image of two Adeno-associated virus samples and a Lysozyme positive control. The x-axis shows temperature and y-axis shows the fluorescence intensity normalized to a 100%.

Notes

  1. SYPRO Orange can be excited with UV light at 300 nm or using visible light at 472 nm. The emission is measured 570 nm.
  2. The qPCR machine had a limited set of filters for SYPRO Orange. Though the excitation was possible at 470 nm, the spectra were collected at an emission wavelength of 550 nm. Although this was 20 nm lesser than the optimal emission wavelength, the peak intensity observed was ~50% of that observed at 570 nm. More importantly, the signal to noise ratio was very high thereby implying high quality data.
  3. The complementary data obtained from Differential Scanning Calorimetry (data not shown) also correlated accurately with that obtained from our method thereby further enhancing the value of our data (Rayaprolu et al., 2013).
  4. There are 36 vacant positions available on the rotor of the machine. A PCR tube that contains only buffer solution and the dye is used as a negative control. For this sample to be recognized as the negative control, this PCR tube needs to be placed in the first position in the rotor. The second position is occupied by a tube containing only buffer with no protein or dye added. This lets us determine if the buffer solution is contributing to the fluorescence intensity. The third position contains a lysozyme reaction aliquot (as described in the Procedure section). This acts as a positive control. Lysozyme’s melting temperature has been well documented in the literature. We compare the results we got from the DSF with the previously documented ones. More than one concentration of lysozyme was initially tested to confirm the melting temperature and the fluorescence linearity.

Recipes

  1. Citrate-Phosphate buffer
    43.6% 0.2 M disodium phosphate
    6.4% of 0.1 M citric acid
    50 mM sodium chloride
    Check to see if the pH is 7.0, if not, adjust with hydrochloric acid or sodium hydroxide to attain pH 7.0.
    Make up to the required volume with water

Acknowledgments

The work was funded by the grant R01 AI081961-01A1 from National Institutes of Health (NIH). This protocol was adapted from Rayaprolu et al. (2012).

References

  1. Rayaprolu, V., Kruse, S., Kant, R., Venkatakrishnan, B., Movahed, N., Brooke, D., Lins, B., Bennett, A., Potter, T., McKenna, R., Agbandje-McKenna, M. and Bothner, B. (2013). Comparative analysis of adeno-associated virus capsid stability and dynamics. J Virol 87(24): 13150-13160.
  2. Vedadi, M., Niesen, F. H., Allali-Hassani, A., Fedorov, O. Y., Finerty, P. J., Jr., Wasney, G. A., Yeung, R., Arrowsmith, C., Ball, L. J., Berglund, H., Hui, R., Marsden, B. D., Nordlund, P., Sundstrom, M., Weigelt, J. and Edwards, A. M. (2006). Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc Natl Acad Sci U S A 103(43): 15835-15840.
  3. Yeh, A. P., McMillan, A. and Stowell, M. H. (2006). Rapid and simple protein-stability screens: application to membrane proteins. Acta Crystallogr D Biol Crystallogr 62(Pt 4): 451-457.
  4. http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence- spectraviewer.html#product=S6650.

简介

差示扫描荧光测定(DSF)是一种快速,经济,直接的技术,用于估计蛋白质的热稳定性。该原理涉及荧光染料与蛋白质的热暴露的疏水口袋的结合。在该技术中使用的染料在非极性环境中是高度荧光的,并且当暴露于水溶液时淬灭。荧光的变化可用于跟踪由温度,pH或离液剂诱导的蛋白质的解折叠。该方法充分表征了单体蛋白质。在这里,我们将应用程序扩展到超分子蛋白和核蛋白复合物使用病毒颗粒为例。 SYPRO-orange™染料是首选的染料,因为它与q-PCR仪器配合使用,荧光响应在宽的pH和温度范围内是稳定的。该技术相对于标准生物物理方法的优点包括对生物和技术重复的高通量筛选的能力和高灵敏度。

关键字:生物物理学, 病毒学, 荧光, 疏水补丁, 亚基的相互作用

材料和试剂

  1. 腺相关病毒血清型1,2,5,8的纯化病毒样颗粒(VLPs)
  2. 纯化的重组AAV(rAAV)衣壳包装绿色荧光蛋白基因(GFP),rAAV1-GFP,rAAV5-GFP,rAAV8-GFP
  3. PBS(pH7.0),在25℃下
  4. 柠檬酸(Thermo Fisher Scientific,目录号:BP339-500)
  5. 磷酸二钠(VWR International,目录号:BDH4538-1KGP)
  6. 5,000x SYPRO Orange(Life Technologies,目录号:S6651)
  7. 氢氧化钠(Thermo Fisher Scientific,目录号:BP359-500)
  8. 12 N盐酸(EMD Millipore,目录号:HX0603-3)
  9. 氯化钠(VWR International,目录号:BDH4534-5KGP)
  10. 氯化镁(Thermo Fisher Scientific,目录号:BP214-500)
  11. 分子生物学级水
  12. 柠檬酸盐 - 磷酸盐缓冲液(参见配方)

设备

  1. Rotor Gene-3000,具有36孔转子的qPCR仪器(QIAGEN)
  2. 0.2ml PCR管(BioExpress,目录号:T-3013-1FC)
  3. 铝箔
  4. 移液管吸头(10μl,200μl)(VWR International,目录号:37001-162和53508-810)
  5. 将移液管(200μl,0.1-2.5μl)(Eppendorf,BioExpress,目录号:P-3015-6和P-3015-1)
  6. pH计(Accument Basic AB 15 pH计)(Thermo Fisher Scientific)
  7. Milli-Q纯化系统(EMD Millipore)

软件

  1. Rotor基因软件(版本1.7,Build 94)
  2. Windows 7上的Microsoft Excel 2007

程序

  1. 将每个VLP和重组病毒样品稀释到PBS或柠檬酸盐 - 磷酸盐缓冲液中,以获得0.1-0.24mg/ml之间的浓度。在每次运行中包括浓度为0.24mg/ml的溶菌酶样品作为阳性对照。改变样品的浓度以在仪器上获得相似的荧光强度
  2. 将浓缩的5,000x SYPRO Orange的等分试样储存在-20℃,并在每次反应设置之前温和地完全解冻至室温。
  3. 将1μlSYPRO Orange稀释到100μl蒸馏水中,得到1%50x工作液。由于SYPRO Orange是光敏感的,工作材料用铝箔覆盖并保持在黑暗中,直到分发之前。
  4. 将2.5μl工作母液加入到上述每个样品中。如果在4小时后不使用,剩余的工作油料(如果有的话)被丢弃
  5. 将样品保存在铝箔覆盖的盒子中以防止暴露于光,直到它们转移到qPCR机器中
  6. 该测定在温度从30℃升至99℃,每1分钟增加1度进行
  7. 数据导出为文本(.csv)文件,并传输到Microsoft Excel 2007中进行数据分析

代表数据

  1. 通过将所有样品除以该样品中最大记录强度,将来自所有样品的原始强度数据标准化至100%的上限。 数据绘制在X轴上的温度和Y轴上的归一化强度
  2. 原始数据还用于计算荧光每温度变化(dF/dT)的变化。将由该计算得到的最大值定义为熔融温度(Tm)。这可以通过取连续荧光读数之间的差除以测量之间的温度变化来完成。然后可以相对于温度绘制强度变化(dF)。然后可以从列表中可视化或选择最大值。如这里所述,温度步长为1度。


    图1.显示两个腺相关病毒样品和溶菌酶阳性对照的代表性图像。 x轴显示温度,y轴显示标准化为100%的荧光强度。 >

笔记

  1. SYPRO橙色可以用300nm的UV光或使用472nm的可见光激发。发射测量为570nm
  2. qPCR机器具有用于SYPRO Orange的有限的一组过滤器。尽管在470nm下可以进行激发,但是在550nm的发射波长下收集光谱。尽管这比最佳发射波长小20nm,但观察到的峰强度为在570nm观察到的峰强度的〜50%。更重要的是,信噪比非常高,从而意味着高质量的数据
  3. 从差示扫描量热法获得的互补数据(数据未显示)也与从我们的方法获得的互补数据精确相关,从而进一步增强我们的数据的价值(Rayaprolu等人,2013)。
  4. 在机器的转子上有36个空位。将仅含有缓冲溶液和染料的PCR管用作阴性对照。对于该样品被识别为阴性对照,该PCR管需要放置在转子中的第一位置。第二位置由仅含有不含蛋白质或染料的缓冲液的管占据。这使我们能够确定缓冲溶液是否有助于荧光强度。第三位置含有溶菌酶反应等分试样(如过程部分中所述)。这作为一个积极的控制。溶菌酶的熔融温度已经在文献中有详细记载。我们将从DSF获得的结果与以前记录的结果进行比较。最初测试了多于一种浓度的溶菌酶以确认解链温度和荧光线性。

食谱

  1. 柠檬酸盐 - 磷酸盐缓冲液
    43.6%0.2M磷酸二钠
    6.4%的0.1M柠檬酸
    50mM氯化钠
    检查pH是否为7.0,如果没有,用盐酸或氢氧化钠调节至pH7.0 用水补足所需体积

致谢

该工作由来自国立卫生研究院(NIH)的授权R01 AI081961-01A1资助。 该协议改编自Rayaprolu等人(2012)。

参考文献

  1. Rayperro,V.,Kruse,S.,Kant,R.,Venkatakrishnan,B.,Movahed,N.,Brooke,D.,Lins,B.,Bennett,A.,Potter,T.,McKenna, Agbandje-McKenna,M.和Bothner,B。(2013)。 腺相关病毒衣壳稳定性和动力学的比较分析 J Virol 87(24):13150-13160。
  2. Vedadi,M.,Niesen,FH,Allali-Hassani,A.,Fedorov,OY,Finerty,PJ,Jr.,Wasney,GA,Yeung,R.,Arrowsmith,C.,Ball,LJ,Berglund, Hui,R.,Marsden,BD,Nordlund,P.,Sundstrom,M.,Weigelt,J。和Edwards,AM(2006)。 化学筛选方法,用于鉴定促进蛋白质稳定性,蛋白质结晶和结构测定的配体。 Proc Natl Acad Sci USA 103(43):15835-15840。
  3. Yeh,A.P.,McMillan,A。和Stowell,M.H。(2006)。 快速简单的蛋白质稳定性筛选:应用于膜蛋白。 Acta Crystallogr D Biol Crystallogr 62(Pt 4):451-457
  4. http ://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence- spectraviewer.html#product = S6650 。
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引用:Rayaprolu, V., Kruse, S., Kant, R., Movahed, N., Brooke, D. and Bothner, B. (2014). Fluorometric Estimation of Viral Thermal Stability. Bio-protocol 4(15): e1199. DOI: 10.21769/BioProtoc.1199.
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Kathrin Schlicht
Universität Bielefeld
Dear Sir or Madam

We have troubles to reach a virus concentration of 0,1 to 0,24 mg/mL. So our question is how you determined the protein concentration.

Yours sincerely

Kathrin Schlicht
2016/3/2 6:16:03 回复
Vamseedhar Rayaprolu
La Jolla Institute for Allergy and Immunology

Hi Kathrin,

We determined concentrations using gel densitometry. You could also use UV 280 to determine the protein concentration but because our virus sample also had DNA, we preferred running the gel and doing the densitometry. You can increase your protein concentration using molecular a weight cut off filter though AAV samples tend to stick to the filters quite a bit. The samples were obtained from our collaborators. For more information on purification of the virus and checking integrity, please refer to http://jvi.asm.org/content/87/24/13150.full

I hope this helps.

2016/3/2 8:25:51 回复


Kathrin Schlicht
Universität Bielefeld

Hi Vamseedhar,

we produce our AAVs by calcium phosphate-based transfection of plasmid DNA into HEK cells. The viral titer we reach is bellow the titer you need to get a protein concentration that high even after concentration by a molecular cut off filter. So we can`t perform DSF. We really like to test this method. Maybe you know how many HEK cells were transfected.

Thank you for your quick response.

2016/3/2 23:42:39 回复


Vamseedhar Rayaprolu
La Jolla Institute for Allergy and Immunology

Hi,

Sorry for the delayed response. Please look at the original reference (I posted the link in the previous post). Also look at references 51-54 in that link. This method was developed by our collaborators in SF9 using a baculoviral expression system. Let me know if you need more information.

2016/3/5 11:16:06 回复