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Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry
使用荧光光谱法测定蛋白质聚集抑制实验中分子伴侣的活性   

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参见作者原研究论文

本实验方案简略版
mBio
Mar/Apr 2016

Abstract

The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway (Figure 1). Usually molecular chaperones exhibit differential affinities with different conformations of the substrate. With the exception of the sHsp family of molecular chaperones, the shift from a high-affinity binding state to the low-affinity release state is triggered by ATP binding and hydrolysis (Haselback and Buchner, 2015). Aggregation prevention assay is a simple, yet definitive assay to determine the chaperone activity of heat labile proteins such as Maltodextrin glucosidase (MalZ), Citrate Synthase (CS) and NdeI. This is based on the premise that proteins with chaperone like activity should prevent protein substrates (MalZ, CS and NdeI) from thermal aggregation. Here, we describe a detailed protocol for aggregation prevention assay using two different chaperone proteins, resistin and MoxR1, identified from our lab. Resistin, a human protein (hRes) and MoxR1 a Mycobacterium tuberculosis protein were analysed for their effect on prevention of MalZ/Citrate Synthase (CS)/NdeI aggregation.


Figure 1. Mechanism of action of molecular chaperones. Citrate synthase folds via increasingly structured intermediates (I1, I2) from the unfolded state (U) to the folded state (N). Under heat shock conditions, this process is reversed.

Keywords: Molecular chaperone (分子伴侣), Thermal aggregation prevention (热凝聚预防), ATPase activity assay (ATP酶活性测定), GroEL (GroEL), MoxR1 (MoxR1), Resistin proteins (抵抗素蛋白), Refolding of Enzymes & Proteins (酶和蛋白质的再折叠)

Background

To elucidate the chaperone activity of a protein in vitro, several methods have been developed. Primarily these methods are based on examining the enzyme function and the ability of the chaperone to refold and protect enzyme activity under heat or other stress conditions. Other method to identify and study chaperones includes in silico analysis, or co-purification with other proteins. The limitations with such methods are less reproducibility or inherent high chances of false positive results. In the current method the use of light scattering to detect prevention of protein aggregation relies on thermal stabilization of protein only from denatured state, and in the presence of a chaperone. In contrast, if aggregate is formed from native or intermediate state of the protein the amount of aggregation might increase thereby decreasing the chance of false positive. Furthermore, this method uses the purified recombinant protein for the assay and therefore, can also be used to study other chaperone proteins from other bacterial sources.

Materials and Reagents

  1. Pipette tips  
  2. Microcentrifuge tube  
  3. Parafilm
  4. E. coli BL21 (DE3) cells
  5. Citrate synthase ammonium sulfate suspension from porcine heart (Sigma-Aldrich, catalog number: C3260 )
  6. Ammonium sulphate
  7. Tris (pH 8.0) (AMRESCO, catalog number: 0497 )
  8. Luria Bertani agar plates
  9. Ampicillin (Sigma- Aldrich, catalog number: A9518 )
  10. Isopropyl β-D-1-thiogalactopyranoside, IPTG (Sigma-Aldrich, catalog number: I6758 )
  11. DNAse I (Sigma-Aldrich, catalog number: AMPD1 )
  12. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
  13. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 7626 )
  14. Bradford reagent (Bio-Rad Laboratories, catalog number: 5000006 )
  15. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  16. Recombinant protein MoxR1 and hRes (1 mg/ml) (Purified in the laboratory)
  17. Purified recombinant protein GroEL (0.5 mg/ml) (Purified in the laboratory)
  18. Lysozyme (1 mg/ml) (Sigma-Aldrich, catalog number: 4919 )
  19. Milli-Q water
  20. NdeI (20,000 U/ml) (New England BioLab, catalog number: R0111 )
  21. Maltodextrin glucosidase protein (1 mg/ml) (Purified in the laboratory)
  22. EDTA (Sigma-Aldrich, catalog number: E9884 )
  23. Sodium phosphate (dibasic) heptahydrate (Na2HPO4·7H2O) (AMRESCO, catalog number: 0348 )
  24. Sodium phosphate (monobasic) anhydrous (NaH2PO4) (AMRESCO, catalog number: 0571 )
  25. Sodium chloride (NaCl) (AMRESCO, catalog number: 0241 )
  26. Imidazole (AMRESCO, catalog number: 0527 )
  27. Glycerol (AMRESCO, catalog number: 0854 )
  28. TE buffer (see Recipes)
  29. 20 mM sodium phosphate buffer pH 7.4 (see Recipes)
  30. Binding buffer (see Recipes)
  31. Washing buffer (see Recipes)
  32. Elution buffer (see Recipes)
  33. Dialysis buffer (see Recipes)

Equipment

  1. Pipette (Gilson, model: Pipetman Neo®)
  2. Centrifuge (Hermle Labor Technik, model: Z 326 K )
  3. PerkinElmer spectrofluorometer (PerkinElmer, model: LS55 ) with controlled temperature peltier block
  4. Incubator (Eppendorf, BrunswickTM, model: 44/44R )
  5. Quartz cuvette (PerkinElmer, Suprasil®, catalog number: B0631071 )
  6. pH meter (Thermo Fisher Scientific, Thermo Scientific, model: CyberScan Ph 510 )
  7. Fluorescence spectrophotometer (Agilent Technologies, model: Cary Eclipse )

Software

  1. Microsoft Excel
  2. Graphpad Prism 5

Procedure

  1. Preparation of citrate synthase and maltodextrin glucosidase
    1. Commercially available citrate synthase is supplied in ammonium sulphate suspension to keep the enzyme in an inactive state. The suspension is then pipetted into a fresh microcentrifuge tube and centrifuge at 15,000 x g, for about 10 min at 4 °C. The enzyme CS is present in a solid state and therefore will be collected in the pellet fraction.
    2. Discard the supernatant and dissolve the pellet containing enzyme CS in sterile water at a concentration of 1 mg/ml.
    3. The CS should be further purified by size exclusion chromatography and dialysed against 50 mM Tris (pH 8.0). For refolding assays, CS stock solutions at concentration of 150 μM is prepared in TE buffer by concentrating CS using Amicon Ultra microconcentrator with 30 kDa cutoff before use.
    4. The expression plasmids pCS19MalZ containing (His)6 tagged coding region of malz gene are transformed into E. coli BL21 (DE3) competent cells and plating is performed on Luria Bertani agar plates containing 100 µg/ml ampicillin for selecting pCS19MalZ.
    5. A single colony from the transformed bacterial cells grown on Luria Bertani agar plate containing 100 µg/ml ampicillin is picked and grown in the Luria Bertani broth containing 100 µg/ml ampicillin.
    6. The tube is incubated for overnight at 37 °C with constant shaking at a rate of 150 rpm.
    7. About 2% of the overnight culture is inoculated in fresh Luria Bertani broth containing ampicillin 100 µg/ml and then incubate it for 2 h at 37 °C with constant shaking at a rate of 150 rpm.
    8. The expression of MalZ protein is then induced with 100 µM IPTG and left for 12 h.
    9. The IPTG induced culture is pelleted through centrifugation at 11,510 x g for 50 min. The supernatant is discarded, wash the cell pellet with binding buffer and resuspend the cells in binding buffer, along with DNAse I (1 µl/ml of cell lysate), 0.5 mM MgCl2 and 1 mM PMSF (a serine protease inhibitor).
    10. The cells are disrupted through sonication on ice, and the supernatant is collected after centrifugation. (Sonication was performed for 5 min at 1 sec on and 2 sec off cycle and at 30% amplitude)
    11. The protein is further purified by Ni-NTA affinity chromatography. The column is washed with 5 column volumes of washing buffer and the protein is eluted with 5 column volumes of elution buffer (Goyal et al., 2014).
      Note: Nickel nitrilotriacetic [Ni-NTA] chromatography is used for purification of 6x His-tagged recombinant proteins overproduced in bacteria. The Ni-NTA agarose resins possess high affinity and specificity for 6x His-tagged recombinant fusion proteins. The proteins bound to the resin are eluted by competition with imidazole.
    12. The concentration of the protein is determined using Bradford reagent. The standard curve is plotted using different concentration of bovine serum albumin by determining the Optical Density using spectrophotometer and plotting the 595 nm values (y-axis) versus their concentration in µg/ml (x-axis).The concentration of dialyzed protein is then determined using the standard curve.

  2. Thermally induced aggregation assay 
    1. To elucidate the chaperonic function of hRes protein on the thermal aggregation of CS, recombinant GroEL protein is used as a positive control and lysozyme as a negative control.
      Note: Chaperone to substrate ratio is critical and initially different ratios should be used to determine the optimal ratio for the effect of chaperone activity.
    2. We maintained 1:1 ratio between hRes (chaperone) vs. CS (Suragani et al., 2013). The proteins are used at 0.15 µM concentration.
    3. Similarly, to analyze the chaperone function of MoxR1 protein on thermal aggregation of MalZ, GroEL is used as a positive control and lysozyme as a negative control. GroEL and lysozyme are used at 2 µM concentration. In this case, we maintained the initial 12:1 ratio between MoxR1 (chaperone) and MalZ (Bhuwan et al., 2016) (see Note 1).
    4. Stirred 0.75 ml quartz cuvettes are to be used in a fluorescence spectrophotometer. The stirring is achieved by removing the cuvettes out of the spectrophotometer and sealing it by Parafilm and mixing the solution by see-saw movement during the time interval of each data point. The emission and excitation slits should be set to 2-5 nm.
    5. Pre-equilibrate 50 mM Tris (pH 8.0) with and without chaperones at 45-47 °C for 50 min. The chaperone dialysis buffer without chaperones is used as a control which is included in the assay to rule out the possibility of stabilizing effects of buffer.
      Note: After each sample analysis the quartz cuvettes should be thoroughly washed with a strong jerk using MilliQ water to remove the traces of previous protein samples and any film formed on the side of the cuvettes and after washing, the cuvette should be dried before using it at the next step.
    6. The initial light scattering signals are monitored at a constant wavelength of 320 nm for CS and NdeI. Here, the data points should be taken at every 5 min and the kinetics are constantly measured for 45 min at 45 °C. The increase in light scattering is observed to visualize the thermal aggregation of CS.
    7. The light scattering signal for MalZ is to be recorded at 500 nm wavelength.
      Note: The range of absorbance for dynamic light scattering to detect aggregation is 300 nm to 600 nm and can also be optimized for different chaperone analysis.
    8. Take the data at 5 min interval and the kinetics is monitored for 15 min at 47 °C (see Note 2).
    9. The signal prior to CS or NdeI and MalZ addition is set to 0 (= no aggregation).
    10. The readings are exported in a Microsoft Excel format and graph.
    11. Plot the data for Optical Density on x-axis vs. Time on y-axis using Origin or Graphpad Prism 5 software to obtain a graph.
    12. The resulting sigmoidal graphs will allow identifying the chaperonic function of a protein by preventing thermal aggregation of CS or NdeI and MalZ.

Data analysis

The experimental data showing recombinant hRes and MoxR1 proteins mediated prevention of thermal aggregation of CS or NdeI and MalZ proteins are depicted in Figures 2 and 3.


Figure 2. rhRes, proteins prevent thermal aggregation of citrate synthase (CS) and NdeI proteins. Light scattering assay for aggregation prevention for (A) rhRes, F49YrhRes, and CS at 45 °C for 45 min. B. Resistin prevents thermal aggregation of NdeI and C. Citrate synthase. Each experiment should be performed in triplicates (Suragani et al., 2013). GroEL served as positive control while lysozyme was used as a negative control. F49 amino acid of hRes predicted to be an important residue to exhibit chaperonic activity was changed to tyrosine by site directed mutagenesis.
Note: Proteins such as CS consist of polypeptide chains that are affected by multiple parameters such as temperature, pH and chemical environment. Further, preparation method, storage conditions or buffer varies from batch to batch; these can influence the scattering pattern of the proteins in a sample.


Figure 3. MoxR1 and GroEL proteins prevent thermal aggregation of MalZ protein. MoxR1 and GroEL were used as positive control while lysozyme was used as a negative control. Each experiment should be performed in triplicate (Bhuwan et al., 2016).

Notes

  1. To check the activity of chaperones on the inactivation of CS or other heat liable proteins such as MalZ, a different range of stoichiometric ratios up to 32:1 (chaperone to CS) can be used (Haselback and Buchner, 2015)
  2. Since the slope at the beginning of the reaction is used for comparison, it is important that both pipetting as well as sample processing is always performed simultaneously.

Recipes

Note: Buffer composition for MalZ protein purification.

  1. TE buffer
    50 mM Tris pH 8.0
    2 mM EDTA pH 8.0
    Autoclaved at 121 °C for 20 min
  2. 200 mM sodium phosphate buffer pH 7.4 (50 ml)
    Mix 40.5 ml of 0.2 M Na2HPO4·2H2O and 9.5 ml of 0.2 M NaH2PO4·H2O
    0.2 M Na2HPO4·2H2O (35.6 g/L)
    0.2 M NaH2PO4·H2O (27.6 g/L)
  3. Binding buffer
    20 mM sodium phosphate, containing 500 mM NaCl, at pH 7.4
  4. Washing buffer
    20 mM sodium phosphate, containing 500 mM NaCl and 10 mM imidazole, at pH 7.4
  5. Elution buffer
    20 mM sodium phosphate, containing 500 mM NaCl and 500 mM imidazole, at pH 7.4
  6. Dialysis buffer
    20 mM sodium phosphate, containing 500 mM NaCl and 10% glycerol, at pH 7.4

Acknowledgments

This protocol was adapted from our earlier publications (Suragani et al., 2013 and Bhuwan et al., 2016). The work was supported by a Centre of Excellence Phase 2 Grant (BT/R12817/COE/34/23/2015) from the Department of Biotechnology, Ministry of Science & Technology, Government of India to S.E.H. and N.Z.E.

References

  1. Bhuwan, M., Arora, N., Sharma, A., Khubaib, M., Pandey, S., Chaudhuri, T. K., Hasnain, S. E. and Ehtesham, N. Z. (2016). Interaction of Mycobacterium tuberculosis virulence factor RipA with chaperone MoxR1 is required for transport through the TAT secretion system. mBio 7(2): e02259.
  2. Goyal, M., Chaudhuri, T. K. and Kuwajima, K. (2014). Irreversible denaturation of maltodextrin glucosidase studied by differential scanning calorimetry, circular dichroism, and turbidity measurements. PLoS One 9(12): e115877.
  3. Haslbeck, M. and Buchner, J. (2015). Assays to characterize molecular chaperone function in vitro. Methods Mol Biol 1292: 39-51.
  4. Suragani, M., Aadinarayana, V. D., Pinjari, A. B., Tanneeru, K., Guruprasad, L., Banerjee, S., Pandey, S., Chaudhuri, T. K. and Ehtesham, N. Z. (2013). Human resistin, a proinflammatory cytokine, shows chaperone-like activity. Proc Natl Acad Sci U S A 110(51): 20467-20472.

简介

稳定其他蛋白质抵抗热聚集的能力是伴侣蛋白质的主要特征之一。分子伴侣与蛋白质的非构象构象结合。通过动态关联和解离循环来触发底物的折叠,这些循环使底物蛋白保持在折叠通路上(图1)。通常分子伴侣表现出与底物不同构象的差异亲和力。除了分子伴侣的sHsp家族之外,从高亲和力结合状态向低亲和力释放状态的转变由ATP结合和水解引发(Haselback和Buchner,2015)。聚集预防测定是一种简单但尚未确定的测定法,用于确定热不稳定蛋白质如麦芽糖糊精葡糖苷酶(MalZ),柠檬酸盐合酶(CS)和Nde I的伴侣活性。这是基于具有伴侣相似活性的蛋白质预防来自热聚集的蛋白质底物(MalZ,CS和Nde I)的前提。在这里,我们描述了使用我们实验室鉴定的两种不同伴侣蛋白,抵抗素和MoxR1的聚合预防测定的详细方案。分析抗性蛋白(hRes)和MoxR1 a结核分枝杆菌蛋白质对预防MalZ /柠檬酸合酶(CS)/ Nde I聚集的影响。

背景
图1.分子伴侣的作用机制。柠檬酸合酶通过越来越多的结构化中间体(I <1>,2 )从展开状态折叠( U)到折叠状态(N)。在热休克条件下,这个过程是相反的。

为了阐明体外蛋白质的伴侣活性,已经开发了几种方法。主要是这些方法基于检查酶功能和伴侣在热或其他胁迫条件下重折叠和保护酶活性的能力。识别和研究伴侣的其他方法包括电脑分析或与其他蛋白质的共同纯化。这种方法的局限性是较少的重现性或固有的假阳性结果的机会。在目前的方法中,使用光散射来检测蛋白质聚集的预防依赖于仅来自变性状态的蛋白质的热稳定性,并且在分子伴侣的存在下。相比之下,如果聚集体由蛋白质的天然或中间状态形成,则聚集的量可能增加,从而减少假阳性的几率。此外,该方法使用纯化的重组蛋白进行测定,因此也可用于从其他细菌来源研究其他伴侣蛋白。

关键字:分子伴侣, 热凝聚预防, ATP酶活性测定, GroEL, MoxR1, 抵抗素蛋白, 酶和蛋白质的再折叠

材料和试剂

  1. 移液器提示
  2. 微量离心管
  3. 石蜡膜
  4. E。大肠杆菌BL21(DE3)细胞
  5. 来自猪心脏的柠檬酸盐合酶硫酸铵悬浮液(Sigma-Aldrich,目录号:C3260)
  6. 硫酸铵
  7. Tris(pH 8.0)(AMRESCO,目录号:0497)
  8. Luria Bertani琼脂板
  9. 氨苄青霉素(Sigma-Aldrich,目录号:A9518)
  10. 异丙基β-D-1-硫代吡喃半乳糖苷,IPTG(Sigma-Aldrich,目录号:I6758)
  11. DNAse I(Sigma-Aldrich,目录号:AMPD1)
  12. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670)
  13. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:7626)
  14. Bradford试剂(Bio-Rad Laboratories,目录号:5000006)
  15. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  16. 重组蛋白MoxR1和hRes(1 mg/ml)(在实验室中纯化)
  17. 纯化的重组蛋白GroEL(0.5mg/ml)(在实验室中纯化)
  18. 溶菌酶(1mg/ml)(Sigma-Aldrich,目录号:4919)
  19. Milli-Q水
  20. I(20,000U/ml)(New England BioLab,目录号:R0111)
  21. 麦芽糊精葡萄糖苷酶蛋白(1mg/ml)(在实验室中纯化)
  22. EDTA(Sigma-Aldrich,目录号:E9884)
  23. 磷酸二钠(二元)七水合物(Na 2 HPO 4·7H 2 O)(AMRESCO,目录号:0348)
  24. 无水磷酸钠(单碱性)(NaH 2 PO 4)(AMRESCO,目录号:0571)
  25. 氯化钠(NaCl)(AMRESCO,目录号:0241)
  26. 咪唑(AMRESCO,目录号:0527)
  27. 甘油(AMRESCO,目录号:0854)
  28. TE缓冲(见配方)
  29. 20 mM磷酸钠缓冲液pH 7.4(见配方)
  30. 绑定缓冲区(见配方)
  31. 洗涤缓冲液(见配方)
  32. 洗脱缓冲液(见配方)
  33. 透析缓冲液(见配方)

设备

  1. 移液器(Gilson,型号:Pipetman Neo )
  2. 离心机(Hermle Labor Technik,型号:Z 326 K)
  3. PerkinElmer分光荧光计(PerkinElmer,型号:LS55),带有受控温度的peltier块
  4. 孵化器(Eppendorf,Brunswick TM ,型号:44/44R)
  5. 石英比色杯(PerkinElmer,Suprasil ®,目录号:B0631071)
  6. pH计(Thermo Fisher Scientific,Thermo Scientific,型号:CyberScan Ph 510)
  7. 荧光分光光度计(Agilent Technologies,型号:Cary Eclipse)

软件

  1. Microsoft Excel
  2. Graphpad棱镜5

程序

  1. 制备柠檬酸合酶和麦芽糖糊精糖苷酶
    1. 在硫酸铵悬浮液中提供市售的柠檬酸合成酶以保持酶处于非活性状态。然后将悬浮液移液到新鲜的微量离心管中,并在4℃下以15,000×g离心约10分钟。酶CS以固态存在,因此将被收集在颗粒级分中。
    2. 弃去上清液,将含有酶的CS溶解在无菌水中,浓度为1 mg/ml。
    3. 应通过尺寸排阻色谱进一步纯化CS,并对50mM Tris(pH 8.0)透析。对于重折叠测定,浓度为150μM的CS储备溶液在TE缓冲液中通过在使用前使用具有30kDa截止值的Amicon Ultra微量浓缩器浓缩CS来制备。
    4. 将含有(His)6标记的编码区域的表达质粒pCS19MalZ转化到E.大肠杆菌BL21(DE3)感受态细胞,并在含有100μg/ml氨苄青霉素的Luria Bertani琼脂平板上进行电镀以选择pCS19MalZ。
    5. 从含有100μg/ml氨苄青霉素的Luria Bertani琼脂平板上生长的转化的细菌细胞的单个菌落被挑选并在含有100μg/ml氨苄青霉素的Luria Bertani肉汤中生长。
    6. 将管在37℃下以150rpm的速率恒定振荡孵育过夜。
    7. 将约2%的过夜培养物接种在含有氨苄青霉素100μg/ml的新鲜的Luria Bertani肉汤中,然后在37℃下以150rpm的速率恒定振荡孵育2小时。
    8. 然后用100μMIPTG诱导MalZ蛋白的表达,并保留12小时。
    9. 将IPTG诱导的培养物以11,510×g离心分离50分钟。弃去上清液,用结合缓冲液洗涤细胞沉淀,并将细胞与DNAse I(1μl/ml细胞裂解物),0.5mM MgCl 2和1mM PMSF(丝氨酸蛋白酶抑制剂)。
    10. 细胞通过冰上超声处理破碎,离心后收集上清液。 (超声处理在1秒钟和2秒关闭周期和30%振幅下进行5分钟)
    11. 蛋白质通过Ni-NTA亲和层析进一步纯化。将柱用5倍柱体积的洗涤缓冲液洗涤,并用5倍柱体积的洗脱缓冲液(Goyal等人,2014)洗脱该蛋白质, 。
      注意:次氮基三乙酸镍[Ni-NTA]色谱用于纯化在细菌中过量产生的6x His标记的重组蛋白。 Ni-NTA琼脂糖树脂对6x His标记的重组融合蛋白具有高亲和力和特异性。结合树脂的蛋白质通过与咪唑竞争而被洗脱。
    12. 使用Bradford试剂测定蛋白质的浓度。使用不同浓度的牛血清白蛋白通过使用分光光度计测定光密度并绘制595nm值(y轴)与其浓度(μg/ml(x轴))的标准曲线。然后透析蛋白质的浓度使用标准曲线确定。

  2. 热诱导聚集分析
    1. 为了阐明hRes蛋白在CS聚集上的分子功能,将重组GroEL蛋白用作阳性对照和溶菌酶作为阴性对照。
      注意:分子伴侣与底物的比例至关重要,最初应使用不同的比例来确定伴侣活动影响的最佳比例。
    2. 我们在hRes(伴侣)与CS(Suragani等人,2013年)之间保持1:1的比例。蛋白质的浓度为0.15μM
    3. 类似地,为了分析MoxR1蛋白在MalZ热聚集上的伴侣功能,GroEL用作阳性对照和溶菌酶作为阴性对照。 GroEL和溶菌酶以2μM浓度使用。在这种情况下,我们保持了MoxR1(伴侣)和MalZ(Bhuwan等人,2016)之间的初始比例为12:1(见注1)。
    4. 在荧光分光光度计中使用搅拌的0.75毫升石英比色皿。通过从分光光度计中除去比色杯并通过Parafilm密封并在每个数据点的时间间隔期间通过跷跷板移动混合溶液来实现搅拌。发射和激发狭缝应设置为2-5nm。
    5. 在45-47℃下使用和不用伴侣预先平衡50mM Tris(pH 8.0)50分钟。没有伴侣的伴侣透析缓冲液用作测定中包含的对照,以排除稳定缓冲液效果的可能性。
      注意:每次样品分析后,应使用MilliQ水彻底冲洗石英比色杯,以除去以前的蛋白质样品和形成在比色杯一侧的任何膜的痕迹,洗涤后,将试管干燥在下一步使用它之前。
    6. 初始光散射信号以320nm的恒定波长监测CS和NdeI。这里,每5分钟取数据点,45℃下不间断地测量动力学45分钟。观察到光散射的增加可视化CS的热聚集。
    7. 用于MalZ的光散射信号将以500nm波长记录。
      注意:动态光散射检测聚集的吸光度范围为300 nm至600 nm,也可针对不同的伴侣分析进行优化。
    8. 以5分钟的间隔取数据,并在47℃监测动力学15分钟(见注2)。
    9. CS或者Nde I和MalZ添加之前的信号设置为0(=无聚合)。
    10. 读数以Microsoft Excel格式和图形导出。
    11. 使用Origin或Graphpad Prism 5软件在y轴上使用x轴与时间绘制光密度数据,以获得图形。
    12. 所得到的S形图将允许通过阻止CS或NdeI和MalZ的热聚集来鉴定蛋白质的伴侣功能。

数据分析

显示重组hRes和MoxR1蛋白介导的CS或NdeI和MalZ蛋白的热聚集预防的实验数据如图2和3所示。


图2. rhRes,蛋白质阻止柠檬酸合酶(CS)和NdeI蛋白的热聚集。(A)rhRes,F49YrhRes和CS的聚集预防的光散射测定在45℃45分钟。 B.抗性阻止了Nde I和C.柠檬酸合酶的热聚集。每个实验应一式三份进行(Suragani等人,2013)。 GroEL作为阳性对照,溶菌酶用作阴性对照。通过定点诱变将hRes的F49氨基酸预测为显示伴侣活性的重要残基变为酪氨酸。
注意:蛋白质如CS由受多个参数(如温度,pH和化学环境)影响的多肽链组成。此外,制备方法,储存条件或缓冲剂因批次而异;这些可以影响样品中蛋白质的散射图。


图3.MoxR1和GroEL蛋白质可防止MalZ蛋白的热聚集。使用MoxR1和GroEL作为阳性对照,而溶菌酶用作阴性对照。每个实验应一式三份进行(Bhuwan等人,2016)。

笔记

  1. 为了检查分子伴侣对灭活CS或其他易发热的蛋白质如MalZ的活性,可以使用不同范围的化学计量比高达32:1(伴侣到CS)(Haselback和Buchner,2015)
  2. 由于反应开始时的斜率用于比较,重要的是同时进行移液和样品处理。

食谱

注意:用于MalZ蛋白纯化的缓冲液组合物

  1. TE缓冲区
    50mM Tris pH 8.0
    2mM EDTA pH 8.0
    在121℃高压灭菌20分钟
  2. 200mM磷酸钠缓冲液pH 7.4(50ml) 混合40.5ml的0.2M Na 2 HPO 4·2H 2 O和9.5ml的0.2M NaH 2 O 3, PO 4 H 2 O
    0.2M Na 2 HPO 4/2H 2 O(35.6g/L)
    0.2M NaH 2 PO 4·H 2 O(27.6g/L)
  3. 绑定缓冲区
    20mM磷酸钠,含有500mM NaCl,pH7.4
  4. 洗涤缓冲液
    20mM磷酸钠,含有500mM NaCl和10mM咪唑,pH7.4
  5. 洗脱缓冲液
    20mM磷酸钠,含有500mM NaCl和500mM咪唑,pH7.4
  6. 透析缓冲液
    20mM磷酸钠,含有500mM NaCl和10%甘油,pH 7.4

致谢

该协议已经从我们早期的出版物(Suragani等人,2013和Bhuwan等人,2016)中改编而成。这项工作得到了科学和技术部生物技术系的卓越中心2期授权(BT/R12817/COE/34/23/2015)的支持。技术,印度政府向S.E.H.和N.Z.E.

参考文献

  1. Baowan,M.,Arora,N.,Sharma,A.,Khubaib,M.,Pandey,S.,Chaudhuri,TK,Hasnain,SE and Ehtesham,NZ(2016)。< a class ="ke-insertfile "href ="http://www.ncbi.nlm.nih.gov/pubmed/26933057"target ="_ blank">结核分枝杆菌的相互作用毒力因子RipA与伴侣MoxR1是运输通过TAT分泌系统。 mBio 7(2):e02259。
  2. Goyal,M.,Chaudhuri,TK and Kuwajima,K.(2014)。  通过差示扫描量热法,圆二色性和浊度测量研究的麦芽糖糊精葡糖苷酶的不可逆变性 9(12):e115877。
  3. Haslbeck,M。和Buchner,J。(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25804746"target ="_ blank" >用于表征分子伴侣功能的测定方法。方法Mol Biol 1292:39-51。
  4. Suragani,M.,Aadinarayana,VD,Pinjari,AB,Tanneeru,K.,Guruprasad,L.,Banerjee,S.,Pandey,S.,Chaudhuri,TK and Ehtesham,NZ(2013)。人类抵抗素,一种促炎细胞因子,显示伴侣样活性。 Proc Natl Acad Sci USA 110/51(51):20467-20472。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bhuwan, M., Suragani, M., Ehtesham, N. Z. and Hasnain, S. E. (2017). Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry. Bio-protocol 7(2): e2107. DOI: 10.21769/BioProtoc.2107.
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