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Protein-peptide Interaction by Surface Plasmon Resonance
采用表面等离子体共振检测肽段间的相互作用   

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Abstract

This protocol measures the protein-peptide interaction by surface plasmon resonance (SPR) using Biacore X100 (GE Healthcare). The Biacore system can monitor the direct interaction between biomolecules. There are several methods of immobilizing a ligand to the sensor chip. The optimal immobilization method for each experiment needs to be selected. In this protocol, we employed amine coupling to immobilize the protein to the carboxyl-type sensor chip. The procedure generally follows the “Instrument Handbook” of Biacore X100.

Keywords: Signal(信号), Ligand(配体), Histidine kinase(组氨酸激酶), Sensor(传感器)

Materials and Reagents

  1. Purified protein as ligand
  2. Synthetic peptide dissolved in DMSO as analyte
  3. NaCl
  4. NaOH
  5. HEPES
  6. Tween-20
  7. 50 mM NaOH
  8. Amine coupling kit {750 mg EDC (N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride), 115 mg NHS (N-hydroxysuccinimide), 10.5 ml ethanolamine-HCl; GE Healthcare, catalog number: BR-10000-50 }
  9. 10 mM Acetate buffer pH 4 to 5.5 (GE Healthcare, catalog number: BR-1003-49 to 52 )
  10. DMSO (high grade, such as Sigma-Aldrich, catalog number: 276855 , or Sigma-Aldrich, catalog number: D-1435 )
  11. MilliQ Water
  12. Reagents for immobilization (see Recipes)
  13. Reagents for binding assay (see Recipes)
  14. Ligand (see Recipes)
  15. Analyte (see Recipes)

Equipment

  1. CM5 sensor chip (research grade; GE Healthcare, catalog number: BR-1003-99 )
  2. Biacore X100 Evaluation Software (GE Healthcare)

Procedure

  1. pH scouting (determination of the optimal pH for the ligand)
    The positively charged ligand is electrostatically coupled to the negatively charged surface of the sensor chip, leading to ligand concentration. pH scouting is performed so as to determine the pH range that concentrates the ligand.

    Dilute ligand to a final concentration of 5-200 μg/ml (higher protein concentration is necessary for ligands that are difficult to concentrate) in 10 mM acetate buffer of different pH. A regeneration step using 50 mM NaOH is performed after each pH scouting step.
    Below is an example of pH scouting of a ligand to a sensor chip.



  2. Immobilization
    Prepare the ligand at optimal pH and concentration. The optimal pH is the highest pH that allows ligand concentration in pH scouting. For example, pH5 is the optimal pH for the ligand shown in Figure 1.


    Figure 1. pH scouting
    Below is an example of immobilization of a ligand to a sensor chip.



    * EDC: N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride
    NHS: N-hydroxysuccinimide
    These two and ethanolamine are provided in the Amine Coupling Kit
    ** Flow cell 1 serves as a reference cell.

    After immobilization, run 1x running buffer for immobilization (1x RB-i) at 10 μl/min until the baseline becomes stable. A typical readout of immobilization is show in Figure 2.


    Figure 2. Immobilization

  3. Analyte preparation
    Precise sample preparation is required for accurate measurement, particularly when the analyte is dissolved in organic solvent. Even a subtle difference of organic solvent concentration between the sample and running buffer leads to inaccuracy.
    Below is how we matched the DMSO concentration of our samples.
    1. Dilute 10x HBS buffer with MilliQ water and Tween-20 to 1.1x HBS buffer with 0.11% Tween-20.
    2. Add 25 ml of DMSO and 25 ml of MilliQ water to 450 ml of 1.1x HBS buffer with 0.11% Tween-20 to make 1x running buffer for binding assay (1x RB-b).
    3. Dilute 10 mM peptide in DMSO to 5 mM with MilliQ water, and further dilute to 500 μM with 1.1x HBS buffer with 0.11% Tween20 (this makes 500 μM peptide in 1x RB-b).
    4. Dilute 500 μM peptide mixture to 12.5 μM with 1x RB-b, followed by two-fold dilution to 0.39 μM with 1x RB-b.
  4. Regeneration condition
    For analytes with slow dissociation (when the sensorgram does not go back to the background level at the end of the dissociation step), a regeneration step must be performed after each binding assay. Determine an optimum regeneration condition for each analyte. The regeneration should not change the activity of the immobilized ligand (that is, the responses obtained from the binding assays before and after regeneration should be the same).
  5. Binding assay
    Perform the binding assay was with 1x RB-b.
    Below is an example of a binding assay.




    Data analysis is performed with the Biacore X100 Evaluation Software (GE Healthcare).
    A sensorgram of protein-peptide interaction is shown in Figure 3 and also in the reference (Eguchi et al., 2012).


    Figure 3. Binding assay

Recipes

The buffers used for immobilization and binding must be optimized for every ligand and analyte. The following recipe worked with our protein and peptide samples.

  1. Reagents for immobilization
    1x running buffer for immobilization (1x RB-i)
    10 mM HEPES (pH 7.5)
    150 mM NaCl
    0.05% Tween-20
  2. Reagents for binding assay
    10x HBS buffer [100 mM HEPES (pH7.5), 1.5 M NaCl]
    1x running buffer for binding assay (1x RB-b)
    10 mM HEPES (pH7.5)
    150 mM NaCl
    0.1% Tween-20
    5% DMSO
  3. Ligand
    Purified protein (purity > 90%) equilibrated in 1x RB-i.
  4. Analyte
    Synthetic peptide dissolved in DMSO.

Acknowledgments

This protocol was adapted from Eguchi et al. (2012), and generally follows the “Instrument Handbook” of Biacore X100 (GE Healthcare). This work was supported by a Grant-in-Aid for Scientific Research (A, 20248012) from the Japan Society for the Promotion of Science (JSPS), the Research and Development Program for New Bio-Industry Initiatives (2006–2010) of Bio-Oriented Technology Research Advancement Institution (BRAIN), Japan, MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2011-2015 (S1101035), Sasakawa Scientific Research Grant from The Japan Science Society, and the Institute for Fermentation, Osaka.

References

  1. Eguchi, Y., Ishii, E., Yamane, M. and Utsumi, R. (2012). The connector SafA interacts with the multi-sensing domain of PhoQ in Escherichia coli. Mol Microbiol 85(2): 299-313.

简介

该协议使用Biacore X100(GE Healthcare)通过表面等离子体共振(SPR)测量蛋白质 - 肽相互作用。 Biacore系统可以监测生物分子之间的直接相互作用。 存在将配体固定到传感器芯片的几种方法。 需要选择每个实验的最佳固定方法。 在该协议中,我们使用胺偶联固定蛋白质到羧基型传感器芯片。 该程序通常遵循Biacore X100的"仪器手册"。

关键字:信号, 配体, 组氨酸激酶, 传感器

材料和试剂

  1. 纯化蛋白作为配体
  2. 合成肽溶于DMSO作为分析物
  3. NaCl
  4. NaOH
  5. HEPES
  6. 吐温-20
  7. 50 mM NaOH
  8. 胺偶联试剂盒{750mg EDC(N-乙基-N' - (3-二甲基氨基丙基)碳二亚胺盐酸盐),115mg NHS(N-羟基琥珀酰亚胺),10.5ml乙醇胺-HCl; GE Healthcare,目录号:BR-10000-50}
  9. 10mM乙酸盐缓冲液pH4至5.5(GE Healthcare,目录号:BR-1003-49至52)
  10. DMSO(高级,如Sigma-Aldrich,目录号:276855或Sigma-Aldrich,目录号:D-1435)
  11. MilliQ水
  12. 固定试剂(见配方)
  13. 结合测定试剂(参见配方)
  14. 配体(见配方)
  15. 分析物(参见配方)

设备

  1. CM5传感器芯片(研究级; GE Healthcare,目录号:BR-1003-99)
  2. Biacore X100评估软件(GE Healthcare)

程序

  1. pH检测(测定配体的最佳pH)
    带正电荷的配体静电耦合到传感器芯片的带负电荷的表面,导致配体浓度。 进行pH研究以确定浓集配体的pH范围
    在不同pH的10mM乙酸盐缓冲液中稀释配体至终浓度为5-200μg/ml(对于难以浓缩的配体,更高的蛋白质浓度是必需的)。 在每个pH研究步骤之后进行使用50mM NaOH的再生步骤 下面是配体到传感器芯片的pH研究的实例


  2. 固定
    在最佳pH和浓度下制备配体。最佳pH是允许pH监测中的配体浓度的最高pH。例如,pH5是图1所示配体的最佳pH

    图1. pH检测
    下面是配体固定到传感器芯片的实例


    * EDC:N-乙基-N' - (3-二甲基氨基丙基)碳二亚胺盐酸盐 NHS:N-羟基琥珀酰亚胺 这两种和乙醇胺在胺偶联试剂盒
    中提供 **流动池1用作参考池。

    固定后,运行1×运行缓冲液固定(1×RB-i)以10μl/min,直到基线稳定。典型的固定读数如图2所示。


    图2.固定

  3. 分析物准备
    准确的样品制备需要准确的测量,特别是当分析物溶解在有机溶剂中。 即使样品和运行缓冲液之间的有机溶剂浓度的微小差异也导致不准确。
    下面是我们如何匹配我们的样品的DMSO浓度
    1. 用MilliQ水稀释10x HBS缓冲液,用0.11%Tween-20稀释Tween-20至1.1x HBS缓冲液。
    2. 将25ml DMSO和25ml MilliQ水加入到450ml具有0.11%Tween-20的1.1x HBS缓冲液中,以制备用于结合测定的1x运行缓冲液(1×RB-b)。
    3. 用MilliQ水将DMSO中的10mM肽稀释至5mM,并用含有0.11%Tween20的1.1x HBS缓冲液进一步稀释至500μM(这使得1x RB-b中的500μM肽)。
    4. 用1x RB-b稀释500μM肽混合物至12.5μM,然后用1x RB-b两倍稀释至0.39μM。
  4. 再生条件
    对于具有缓慢解离的分析物(当在解离步骤结束时传感图不回到背景水平)时,必须在每次结合测定后进行再生步骤。确定每个分析物的最佳再生条件。再生不应改变固定化配体的活性(即,在再生之前和之后从结合测定获得的反应应该是相同的)。
  5. 结合测定
    用1x RB-b进行结合测定。
    以下是结合测定的实例。




    使用Biacore X100评估软件(GE Healthcare)进行数据分析。
    蛋白质 - 肽相互作用的传感图显示在图3和参考文献中(Eguchi等人,2012)。


    图3.结合测定

食谱

用于固定和结合的缓冲液必须针对每种配体和分析物进行优化。 以下配方与我们的蛋白质和肽样品配合使用。

  1. 用于固定的试剂
    1x运行缓冲液(1x RB-i)
    10mM HEPES(pH7.5) 150mM NaCl 0.05%Tween-20
  2. 用于结合测定的试剂
    10×HBS缓冲液[100mM HEPES(pH7.5),1.5M NaCl] 1×用于结合测定的运行缓冲液(1x RB-b)
    10mM HEPES(pH7.5)
    150mM NaCl 0.1%Tween-20
    5%DMSO
  3. 配体
    用1x RB-i平衡的纯化蛋白(纯度> 90%)
  4. 分析物
    合成肽溶于DMSO

致谢

该方案改编自Eguchi等人(2012),并且通常遵循Biacore X100(GE Healthcare)的"仪器手册"。这项工作得到了日本科学促进会(JSPS),新生物产业发展研究与开发计划(2006-2010)的科学研究助学金(A,20248012)的支持 - 日本技术研究推进机构(BRAIN),2011 - 2015年私立大学战略研究基金会支持计划(S1101035),日本科学协会Sasakawa科学研究资助和大阪发酵研究所。

参考文献

  1. Eguchi,Y.,Ishii,E.,Yamane,M。和Utsumi,R。(2012)。 连接器SafA与大肠杆菌中PhoQ的多重感测结构域相互作用。 em> Mol Microbiol 85(2):299-313。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ishii, E., Eguchi, Y. and Utsumi, R. (2013). Protein-peptide Interaction by Surface Plasmon Resonance. Bio-protocol 3(3): e321. DOI: 10.21769/BioProtoc.321.
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