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Purification and Detection of a PDGA Depolymerase from Pusillimonas noertemannii
纯化和检测诺尔曼氏极小单孢菌中的PDGA解聚酶   

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Abstract

The purification of a target protein from a complex mixture of proteins is a challenging undertaking. If the target protein has been previously characterised, then information such as subcellular location, function, molecular weight and pI can be used for the design of a purification strategy. However, if the target protein is uncharacterised or little information regarding its characteristics is available, a generic purification protocol can be employed that is optimised as additional characteristics of the target protein are determined during subsequent purification steps. Herein, we describe the protocol for the purification and detection of a poly-γ-D-glutamic acid (PDGA) depolymerase from a consortium culture of two Gram-negative bacteria using a combination of chromatography, 2D-electrophoresis and zymography.

Materials and Reagents

  1. Pusillimonas noertemannii BS8 (isolated by soil enrichment)
  2. Pseudomonas fluorescens BS2 (isolated by soil enrichment)
  3. Poly-γ-D-glutamic acid >250 kDa [prepared in-house; protocol for production can be found in Negus and Taylor (2014)]
  4. Protein assay dye reagent (Bio-Rad Laboratories, catalog number: 500-0006 )
  5. 1.5 ml Microcentrifuge tubes (Eppendorf, catalog number: 0030120086 )
  6. Laemmli sample buffer (Sigma-Aldrich, catalog number: S3401-10VL )
  7. IPG strips (immobilized pH 3-10, 7 cm) (Bio-Rad Laboratories, catalog number: 163-2000 )
  8. Mineral oil (Bio-Rad Laboratories, catalog number: 163-2129 )
  9. Rehydration trays (Bio-Rad Laboratories, catalog number: 165-4035 )
  10. Acrylamide solution (Bio-Rad Laboratories, catalog number 161-0156 )
  11. TEMED (Sigma-Aldrich, catalog number T9281 )
  12. Agarose overlay solution (Bio-Rad Laboratories, catalog number 163-2111 )
  13. Zymogram renaturation buffer (Bio-Rad Laboratories, catalog number: 161-0765 )
  14. Zymogram development buffer (Bio-Rad Laboratories, catalog number: 161-0766 )
  15. Molecular biology grade water (Merck KGaA, catalog number: H20MB05010 )
  16. 30% ethanol
  17. 50% ethanol
  18. Minimal salts medium broth (see Recipes)
  19. Anion exchange binding buffer (see Recipes)
  20. Anion exchange elution buffer (see Recipes)
  21. Equilibration buffer (see Recipes)
  22. Non-reducing sample buffer (see Recipes)
  23. SDS buffer (see Recipes)
  24. Resolving gel buffer (see Recipes)
  25. Stacking gel buffer (see Recipes)
  26. 10% SDS (see Recipes)
  27. 10% APS (see Recipes)
  28. 10x TGE running buffer (see Recipes)
  29. Coomassie-based dye (see Recipes)
  30. Coomassie destain solution (see Recipes)
  31. Methylene blue stain (see Recipes)
  32. 70% ethanol (see Recipes)

Equipment

  1. Universal tube (Sterilin®, catalog number: 128B/P )
  2. Erlenmeyer conical flask narrow neck 2 L (Thermo Fisher Scientific, catalog number: 11577422 )
  3. Cuvettes (1.6 ml semi-micro) (Thermo Fisher Scientific, catalog number: 10594175 )
  4. Syringe driven filters (0.22 µm, polyethersulfone) (Millex®, catalog number: SLGP033RS )
  5. Electrode paper wicks (Bio-Rad Laboratories, catalog number: 165-4071 )
  6. Shaking incubator (Infors AG, model: Multitron standard )
  7. Spectrophotometer (PerkinElmer, model: Lambda 25 )
  8. Benchtop centrifuge (Sigma-Aldrich, model: 4-16K )
  9. Sonicator (MSE, model: Soniprep 150 )
  10. Exponential sonicator probe (MSE, catalog number: 38121-114A )
  11. Centrifugal protein concentrators (Sartorious, model: Vivaspin 20, 3 kDa MWCO, catalog number: VS2092 )
  12. Centrifugal protein concentrators (Sartorious, model: Vivaspin 6, 3 kDa MWCO, catalog number: VS0692 )
  13. Static incubator (Unitemps, discontinued) (B&T)
  14. ÄKTAprime plus chromatography system (GE Healthcare, Prime plus, catalog number: 11-0013-13 )
  15. Anion-exchange chromatography column (GE Healthcare, model: Hi-Trap Q XL, catalog number: 17-5158-01 )
  16. Orbital rocking platform (Stuart, model: SSM1 )
  17. Thermal cycler (TECHNE, discontinued, model: FTGENE 2D )
  18. Electrophoresis cell (Bio-Rad Laboratories, Mini-PROTEAN® Tetra Cell, catalog number: 165-8003 )
  19. Isoelectric focusing cell (Bio-Rad Laboratories, Protean, catalog number: 165-4000 )
  20. Vortex mixer (Appleton Woods, Clifton cyclone, catalog number: AA6500 )

Procedure

  1. Overnight cultures were prepared by rapidly thawing a cryovial containing 1 ml of the depolymerase producing consortium culture and inoculating into 10 ml of minimal salts medium (MSM) broth at a dilution of 1:100 in a 30 ml universal tube. Inoculated cultures were incubated with orbital shaking (120 rpm) at 28 °C for 16 h.
  2. Overnight cultures were inoculated into 500 ml of MSM broth in a 2 L Erlenmeyer flask at a dilution of 1:100 and incubated with orbital shaking (120 rpm) at 28 °C.
  3. Optical density readings (OD600) were taken by transferring 1 ml aliquots to cuvettes and reading in a spectrophotometer until OD600 reached 0.5.
  4. Cells were pelleted by centrifugation at 10,000 x g for 30 min at 4 °C. The cell pellet was resuspended in 10 ml of distilled water and cells disrupted by sonication on ice using an exponential probe (4 x 30 sec bursts at an amplitude of 15 μm at 30 sec intervals).
  5. Cell debris was removed by centrifugation at 13,000 x g for 10 min at 4 °C and the supernatant passed through a syringe driven filter (0.22 μm pore size) to remove any remaining whole cells.
  6. The filtrate containing intracellular protein was concentrated approximately 30-fold and submitted to buffer exchange with anion-exchange binding buffer at 4 °C by centrifugation (5,000 x g) in a Vivaspin 20 protein concentrator (3 kDa MWCO).
  7. Protein concentration of the sample was determined by creating a standard curve using BSA standards at concentrations of 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml and 0.125 mg/ml. Aliquots (20 µl) of protein standard or sample were combined with 1 ml of protein assay dye in a cuvette in duplicate and read at OD595 in a spectrophotometer according to manufacturer’s instruction. The standard curve was used to calculate the concentration of the sample by drawing a line of best fit with linear regression.
  8. The concentration of the sample was adjusted to 2 mg/ml with anion exchange binding buffer in preparation for anion exchange chromatography.
  9. A 1 ml Hi-Trap Q XL column was attached to an ÄKTAprime plus fast protein liquid chromatography system and equilibrated at a flow rate of 1 ml/min with 5 column volumes of anion exchange binding buffer, followed by 5 column volumes of anion exchange elution buffer and finally, 10 column volumes of anion exchange binding buffer.
  10. Intracellular protein (10 mg) in anion exchange binding buffer was bound to the equilibrated column by addition through the injection port of the system in a total volume of 5 ml at a flow rate of 1 ml/min.
  11. Bound proteins were eluted with a 20 ml linear gradient of NaCl ranging from 0 to 1 M in anion exchange elution buffer at a flow rate of 1 ml/min. Linear gradient is created automatically when using the “ion exchange” application template from the ÄKTAprime menu. The gradient was then held at a NaCl concentration of 1 M for a volume of 17 ml to ensure all bound proteins were eluted.
  12. Eluting fractions (1 ml/fraction) were collected in 1.5 ml microcentrifuge tubes over the duration of the NaCl gradient and the chromatogram monitored by absorption at 280 nm.
  13. Individual 1 ml fractions were concentrated approximately 10-fold and subjected to buffer exchange with PBS at 4 °C by centrifugation (5,000 x g) in a Vivaspin 6 protein concentrator. The protein concentration of the concentrated fractions was determined as described in step 7.
  14. Concentrated fractions were examined for depolymerase activity by incubation with PDGA and analysis of polymer degradation products by SDS-PAGE. Reaction mixtures (20 μl) contained 8 μg of PDGA, 0.5 μg of protein and PBS to volume. Reactions were incubated at 37 °C for 16 h in a static incubator and terminated by combining with combined with an equal volume of 2x Laemmli sample buffer heating at 95 °C for 10 min in a thermal cycler. PDGA degradation products were examined by SDS-PAGE.
  15. Acrylamide gels were prepared using the Bio-Rad Mini-PROTEAN® Tetra Cell system with a gel thickness of 1 mm using the following formulation:


    *Resolving gel buffer: 1.5 M Tris-HCl (pH 8.8)
    Stacking gel buffer: 0.5 M Tris-HCl (pH 6.8)

  16. Samples of reaction mixture (20 µl) were loaded to the gel and electrophoresed at 150 V in SDS buffer until the bromophenol blue dye reached the bottom of the gel.
  17. Degradation products were visualised by staining gels with methylene blue for 15 min with gentle agitation on an orbital rocking platform. Gels were destained in 30% v/v ethanol (15 min) 30% v/v ethanol (15 min) followed by several changes of distilled water until the background was removed (see Figure 1 for representative result).


    Figure 1. Depolymerase activity of individual fractions separated by anion exchange chromatography. Protein (0.5 µg) from concentrated individual fractions separated by anion exchange chromatography were incubated with PDGA (8 µg) at 37 °C for 16 h in a 20 µl reaction. Degradation products were visualized by SDS-PAGE stained with methylene blue.

  18. Fractions exhibiting depolymerase activity were pooled and concentrated approximately 30-fold at 4 °C by centrifugation (5,000 x g) in a Vivaspin 6 protein concentrator. The protein concentration of the pooled material was determined as described in step 7.
  19. For the detection of proteins with depolymerase activity, 2D-zymography was employed. The protein concentration of the partially purified depolymerase preparation (pooled and concentrated fractions) was adjusted to 2 mg/ml with PBS and aliquots containing 20 μg of total protein were combined with 105 μl of non-reducing sample buffer. Samples were mixed vigorously by vortexing and applied immediately with a pipette to a 7 cm IPG strip (pH 3-10) in a rehydration tray for isoelectric focusing.
  20. To prevent evaporation of the sample, mineral oil (approximately 1 ml) was used to overlay the strips. The rehydration tray was covered with a lid and the strips left to absorb the sample overnight at room temperature.
  21. Electrode paper wicks were moistened with 8 μl of molecular biology grade water and positioned on the electrodes of the focusing tray.
  22. IPG strips were removed from the rehydration tray with a pair of forceps, placed on dry tissue paper with the gel side facing up and covered with wet tissue paper for 30 sec in order to remove any excess mineral oil from the surface of the strip.
  23. IPG strips were then positioned on the focusing tray with the gel side facing down. Approximately 2 ml of mineral oil was layered over the IPG strips, completely covering them to prevent sample evaporation during focusing.
  24. The focusing tray was positioned in a PROTEAN IEF cell and the cover closed. Strips were then focused using the following conditions:
    Conditioning step: 250 V for 15 min, rapid ramp
    Linear ramp: 250 V to reach 4,000 V for 2 h
    Final focusing: 4,000 V for 10 kVh
    Hold step: 500 V indefinitely
    N.B: temperature of the focusing cell was maintained at 20 °C
    Maximum current: 50 μA/IPG strip
  25. Strips were immediately removed from the tray after completion of the focusing step, and transferred to dry tissue paper with the gel side facing up. Excess mineral oil was removed from the surface of the gel by covering the strips with wet tissue paper for 30 sec. IPG strips were then transferred to a clean rehydration tray with the gel side facing up for equilibration before focusing in the second dimension.
  26. IPG strips were covered with 2 ml of equilibration buffer and gently agitated on an orbital shaker for 15 min and the buffer decanted.
  27. IPG strips were removed from the equilibration tray using forceps and placed into the IPG well on a 10% acrylamide gel copolymerised with PDGA at a final concentration of 0.05% w/v. Gels were prepared as described in step 15 with the addition of lyophilised PDGA to the gel mix.
  28. Agarose overlay solution was layered into the IPG well to secure the IPG strip in place and was allowed to solidify at room temperature. Gels were placed in a Bio-Rad Mini-PROTEAN® Tetra Cell reservoir filled with TGE running buffer and electrophoresed at 125 V until the bromophenol blue dye reached the bottom of the gel.
  29. After electrophoresis, duplicate gels were either stained with Coomassie-based dye for the visualisation of protein spots or analysed by zymography.
  30. For the visualisation of protein spots, gels were covered with Coomassie dye for 1 h with gentle agitation on a rocking platform.
  31. The stain was decanted and the gel was washed with several changes of destain solution on a rocking platform until the background had been removed.
  32. Gels developed as zymograms were incubated in Bio-Rad zymogram renaturation buffer for 30 min at room temperature with gentle agitation.
  33. The buffer was decanted and the gel equilibrated in Bio-Rad zymogram development buffer at room temperature for 30 min with gentle agitation.
  34. A final incubation was performed in fresh Bio-Rad zymogram development buffer at 37 °C for 16 h.
  35. The gel was washed with distilled water and stained for detection visualisation of copolymerised PDGA with methylene blue for 15 min.
  36. The gel was then destained in 30% v/v ethanol (15 min) 50% v/v ethanol (15 min) followed by several changes of distilled water.
  37. Areas of depolymerase activity were identified as clear spots against a dark background where the enzyme had hydrolysed the copolymerised PDGA substrate (see Figure 2 for representative result).


    Figure 2. Pooled fractions (20 μg protein) exhibiting depolymerase activity were subjected to 2D-electrophoresis and 2D-zymography in gels containing co-polymerized 0.05% PDGA under non-reducing conditions. In zymograms, depolymerase activity appeared as a bright spot against a dark background (white arrow); corresponding protein spot on Coomassie blue-stained gel indicated by black arrow.

Recipes

  1. Minimal salts medium broth
    4.3 g K2HPO4
    3.4 g KH2PO4
    2.0 g (NH4)2SO4
    0.34 g MgCl2.6H2O
    0.026 g CaCl2.2H2O
    0.2% w/v PDGA
    Distilled H2O to 1,000 ml, sterlise by autoclaving
  2. Anion exchange binding buffer
    20 mM Tris-HCl
    Adjust pH to 9.0 with HCl
  3. Anion exchange elution buffer
    1 M NaCl
    20 mM Tris-HCl
    Adjust to pH 9.0 with HCl
  4. Equilibration buffer
    2% SDS
    0.05 M Tris-HCl (pH 8.8)
    30% glycerol
    Make up to 100 ml dH2O
  5. Non-reducing sample buffer
    4 M urea
    4% CHAPS
    0.2% carrier ampholytes
    0.002% bromophenol blue
  6. SDS buffer
    2% SDS
    0.05 M Tris-HCl
    30% glycerol
    Adjust pH to 8.8 with HCl
  7. Resolving gel buffer
    1.5 M Tris-HCl
    Adjust pH to 8.8 with HCl
  8. Stacking gel buffer
    0.5M Tris-HCl
    Adjust pH to 6.8 with HCl
  9. 10% SDS
    10 g SDS
    Distilled H2O to 1 L
  10. 10% APS
    100 mg APS
    1 ml of distilled H2O
  11. 10x TGE running buffer
    30.0 g Tris-HCl
    144.0 g glycine
    10.0 g SDS
    Adjust volume to 1,000 ml with distilled H2O
  12. Coomassie-based dye
    1 g Coomassie brilliant blue
    500 ml methanol
    100 ml glacial acetic acid
    Distilled H2O to 1,000 ml
  13. Coomassie destain solution
    100 ml glacial acetic acid
    200 ml methanol
    Distilled H2O to 1,000 ml
  14. Methylene blue stain
    2 g methylene blue
    0.01 g KOH
    220 ml ethanol
    Distilled H2O to 1,000 ml
  15. 70% ethanol
    700 ml ethanol
    300 ml distilled

Acknowledgments

This work was supported by a Medical Research Council Capacity Building Studentship award.

References

  1. Negus, D. and Taylor, P. W. (2014). A poly-gamma-(D)-glutamic acid depolymerase that degrades the protective capsule of Bacillus anthracis. Mol Microbiol 91(6): 1136-1147.

简介

从蛋白质的复杂混合物中纯化靶蛋白是一项具有挑战性的任务。 如果靶蛋白以前已被表征,则诸如亚细胞定位,功能,分子量和pI的信息可用于设计纯化策略。 然而,如果目标蛋白质是未表征的或者关于其特征的很少的信息可用,则可以使用通用的纯化方案,其随着在随后的纯化步骤期间测定靶标蛋白质的附加特征而被优化。 在这里,我们描述了从两个革兰氏阴性细菌的联合培养物使用色谱,2D电泳和酶谱的组合纯化和检测聚-γ-D-谷氨酸(PDGA)解聚酶的协议。

材料和试剂

  1. Pusillimonas noertemannii BS8(通过土壤富集隔离)
  2. 荧光假单胞菌 BS2(通过土壤富集分离)
  3. 聚-γ-D-谷氨酸> 250kDa [室内制备; 生产协议可以在Negus和Taylor(2014)中找到]
  4. 蛋白质测定染料试剂(Bio-Rad Laboratories,目录号:500-0006)
  5. 1.5ml微量离心管(Eppendorf,目录号:0030120086)
  6. Laemmli样品缓冲液(Sigma-Aldrich,目录号:S3401-10VL)
  7. IPG条(固定化pH 3-10,7cm)(Bio-Rad Laboratories,目录号:163-2000)
  8. 矿物油(Bio-Rad Laboratories,目录号:163-2129)
  9. 再水合盘(Bio-Rad Laboratories,目录号:165-4035)
  10. 丙烯酰胺溶液(Bio-Rad Laboratories,目录号161-0156)
  11. TEMED(Sigma-Aldrich,目录号T9281)
  12. 琼脂糖覆盖溶液(Bio-Rad Laboratories,目录号163-2111)
  13. Zymogram复性缓冲液(Bio-Rad Laboratories,目录号:161-0765)
  14. Zymogram显影缓冲液(Bio-Rad Laboratories,目录号:161-0766)
  15. 分子生物学级水(Merck KGaA,目录号:H20MB05010)
  16. 30%乙醇
  17. 50%乙醇
  18. 最小盐培养基(见配方)
  19. 阴离子交换结合缓冲液(参见配方)
  20. 阴离子交换洗脱缓冲液(参见配方)
  21. 平衡缓冲液(参见配方)
  22. 非还原样品缓冲液(参见配方)
  23. SDS缓冲液(参见配方)
  24. 解决凝胶缓冲液(参见配方)
  25. 堆叠凝胶缓冲液(参见配方)
  26. 10%SDS(见配方)
  27. 10%APS(参见配方)
  28. 10x TGE运行缓冲区(参见配方)
  29. 考马斯染料(参见配方)
  30. 考马斯脱色溶液(见配方)
  31. 亚甲蓝染料(见配方)
  32. 70%乙醇(见配方)

设备

  1. 通用管(Sterilin ,目录号:128B/P)
  2. 锥形瓶2L(Thermo Fisher Scientific,目录号:11577422)
  3. 比色杯(1.6ml半微量)(Thermo Fisher Scientific,目录号:10594175)
  4. 注射器驱动的过滤器(0.22μm,聚醚砜)(Millex ,目录号:SLGP033RS)
  5. 电极纸灯芯(Bio-Rad Laboratories,目录号:165-4071)
  6. 摇匀培养箱(Infors AG,型号:Multitron标准)
  7. 分光光度计(PerkinElmer,型号:Lambda 25)
  8. 台式离心机(Sigma-Aldrich,型号:4-16K)
  9. 超声波仪(MSE,型号:Soniprep 150)
  10. 指数声波探测器(MSE,目录号:38121-114A)
  11. 离心蛋白浓缩器(Sartorious,型号:Vivaspin 20,3kDa MWCO,目录号:VS2092)
  12. 离心蛋白浓缩器(Sartorious,型号:Vivaspin 6,3kDa MWCO,目录号:VS0692)
  13. 静态培养箱(单位,停止)(B& T)
  14. ÄKTAprimeplus色谱系统(GE Healthcare,Prime plus,目录号:11-0013-13)
  15. 阴离子交换色谱柱(GE Healthcare,型号:Hi-Trap Q XL,目录号:17-5158-01)
  16. 轨道摇摆平台(Stuart,型号:SSM1)
  17. 热循环仪(TECHNE,停产,型号:FTGENE 2D)
  18. 电泳细胞(Bio-Rad Laboratories,Mini-PROTEAN Tetra Cell,目录号:165-8003)
  19. 等电聚焦电池(Bio-Rad Laboratories,Protean,目录号:165-4000)
  20. 涡旋混合器(Appleton Woods,Clifton cyclone,目录号:AA6500)

程序

  1. 通过快速解冻含有1ml产生解聚酶的聚生体培养物的冷冻小瓶,并在30ml通用管中以1:100的稀释度接种到10ml最小盐培养基(MSM)肉汤中来制备过夜培养物。将接种的培养物在28℃下轨道振荡(120rpm)孵育16小时
  2. 将过夜培养物以1:100的稀释度接种在2L锥形瓶中的500ml MSM肉汤中,并在28℃下进行轨道振荡(120rpm)孵育。
  3. 通过将1ml等分试样转移至比色杯并在分光光度计中读数直至OD 600达到0.5来获取光密度读数(OD 600)。
  4. 通过在4℃下以10,000×g离心30分钟使细胞沉淀。将细胞沉淀重悬于10ml蒸馏水中,并通过使用指数探针在冰上超声处理破碎细胞(4×30秒爆发,振幅为15μm,间隔30秒)。
  5. 通过在4℃下以13,000xg离心10分钟除去细胞碎片,并使上清液通过注射器驱动的过滤器(0.22μm孔径)以除去任何剩余的全细胞。
  6. 将含有细胞内蛋白质的滤液浓缩约30倍,并通过在Vivaspin 20蛋白质浓缩器(3kDa MWCO)中离心(5000×g)在4℃下与阴离子交换结合缓冲液进行缓冲液交换, 。
  7. 通过使用BSA标准品以1mg/ml,0.5mg/ml,0.25mg/ml和0.125mg/ml的浓度产生标准曲线来确定样品的蛋白质浓度。将蛋白质标准品或样品的等分试样(20μl)与1ml蛋白质测定染料在小杯中合并,一式两份,并根据制造商的说明在分光光度计中在OD 595处读取。使用标准曲线通过用线性回归绘制最佳拟合线来计算样品的浓度
  8. 用阴离子交换结合缓冲液将样品的浓度调节至2mg/ml,用于阴离子交换层析。
  9. 将1ml Hi-Trap Q XL柱连接到ÄKTAprimeplus快速蛋白液相色谱系统,并以1ml/min的流速用5个柱体积的阴离子交换结合缓冲液平衡,然后用5个柱体积的阴离子交换洗脱缓冲液,最后加入10倍柱体积的阴离子交换结合缓冲液
  10. 在阴离子交换结合缓冲液中的细胞内蛋白质(10mg)通过系统的注射端口以5ml /分的总体积以1ml/min的流速加入而结合到平衡的柱上。
  11. 结合的蛋白质用在阴离子交换洗脱缓冲液中的0-1M的NaCl线性梯度洗脱,流速为1ml/min。使用ÄKTAprime菜单中的"离子交换"应用程序模板时,将自动创建线性渐变。然后将梯度保持在NaCl浓度为1M,体积为17ml,以确保所有结合的蛋白质被洗脱
  12. 在NaCl梯度的持续时间内将洗脱级分(1ml /级分)收集在1.5ml微量离心管中,通过在280nm吸收监测色谱。
  13. 将单个1ml级分浓缩约10倍,并通过在Vivaspin 6蛋白质浓缩器中离心(5000×g)在4℃下与PBS进行缓冲液交换。如步骤7所述测定浓缩级分的蛋白质浓度
  14. 通过与PDGA温育和通过SDS-PAGE分析聚合物降解产物来检查浓缩级分的解聚酶活性。反应混合物(20μl)含有8μgPDGA,0.5μg蛋白质和PBS至体积。将反应物在37℃下在静态培养箱中孵育16小时,并通过与等体积的2×Laemmli样品缓冲液合并终止,在95℃下在热循环仪中加热10分钟。通过SDS-PAGE检查PDGA降解产物
  15. 使用具有1mm凝胶厚度的Bio-Rad Mini-PROTEAN Tetra Cell系统,使用以下配方制备丙烯酰胺凝胶:


    *溶解凝胶缓冲液:1.5M Tris-HCl(pH8.8)
    堆叠胶缓冲液:0.5M Tris-HCl(pH 6.8)

  16. 将反应混合物样品(20μl)加载到凝胶中,并在150V在SDS缓冲液中电泳直到溴酚蓝染料到达凝胶底部。
  17. 降解产物通过用亚甲基蓝染色凝胶15分钟,在轨道摇摆平台上轻轻搅拌来显现。凝胶在30%v/v乙醇(15分钟)30%v/v乙醇(15分钟)中脱色,然后几次更换蒸馏水,直到除去背景(参见图1的代表性结果)。


    图1.通过阴离子交换层析分离的单个级分的解聚酶活性。通过阴离子交换层析分离的来自浓缩的单个级分的蛋白质(0.5μg)与PDGA(8μg)在37℃下孵育16小时在20μl反应中。降解产物通过用亚甲基蓝染色的SDS-PAGE显现
  18. 合并显示解聚酶活性的级分,并在Vivaspin 6蛋白质浓缩器中通过离心(5000×g)在4℃浓缩约30倍。如步骤7所述测定合并的材料的蛋白质浓度
  19. 为了检测具有解聚酶活性的蛋白质,使用2D-酶谱。用PBS将部分纯化的解聚酶制剂(合并的和浓缩的级分)的蛋白质浓度调节至2mg/ml,将含有20μg总蛋白质的等分试样与105μl非还原性样品缓冲液合并。通过涡旋剧烈地混合样品,并用移液管立即施加到用于等电聚焦的再水化托盘中的7cm IPG条(pH 3-10)。
  20. 为了防止样品蒸发,使用矿物油(约1ml)覆盖条。用盖子覆盖再水化托盘,并且将条留在室温下吸收样品过夜
  21. 电极纸灯芯用8μl分子生物学级水润湿并定位在聚焦托盘的电极上
  22. 用一对镊子从再水化托盘中取出IPG条,置于干燥的薄纸上,凝胶面朝上并用湿纸巾覆盖30秒,以从条表面除去任何过量的矿物油。 br />
  23. 然后将IPG条带定位在聚焦托盘上,凝胶侧面朝下。将约2ml矿物油层叠在IPG条上,完全覆盖它们以防止样品在聚焦期间蒸发
  24. 聚焦托盘位于PROTEAN IEF单元中,盖子关闭。然后使用以下条件使条带聚焦:
    调节步骤:250 V 15分钟,快速斜坡
    线性斜坡:250 V,达到4,000 V,持续2 h
    最终聚焦:10 kVh时为4,000 V
    保持步长:无限期为500伏
    N.B:聚焦电池的温度保持在20℃ 最大电流:50μA/IPG条
  25. 在聚焦步骤完成后立即从盘中取出条带,并转移到干燥的薄纸上,凝胶面朝上。通过用湿纸巾覆盖条带30秒,从凝胶表面除去过量的矿物油。然后将IPG条转移至干净的再水化托盘,凝胶侧朝上以达到平衡,然后在第二维上聚焦。
  26. IPG条用2ml平衡缓冲液覆盖并在定轨振荡器上轻轻搅拌15分钟,缓冲液倾析。
  27. 使用镊子从平衡盘中取出IPG条,并置于IPG孔中与终浓度为0.05%w/v的PDGA共聚的10%丙烯酰胺凝胶上。如步骤15所述制备凝胶,向凝胶混合物中加入冷冻干燥的PDGA。
  28. 将琼脂糖覆盖溶液分层到IPG孔中以将IPG条固定在适当位置,并使其在室温下固化。将凝胶置于装有TGE运行缓冲液的Bio-Rad Mini-PROTEAN Tetra细胞池中,并在125V电泳,直到溴酚蓝染料到达凝胶底部。
  29. 电泳后,用基于考马斯的染料染色重复的凝胶以观察蛋白质斑点或通过酶谱法分析。
  30. 对于蛋白质斑点的可视化,用考马斯染料在摇摆平台上温和搅拌覆盖凝胶1小时。
  31. 倾析污渍,并在摇摆平台上用几个变化的脱色溶液洗涤凝胶,直到背景被除去。
  32. 作为酶谱形成的凝胶在Bio-Rad酶谱复性缓冲液中在室温下温和搅拌孵育30分钟。
  33. 倾析缓冲液,并在室温下在Bio-Rad酶谱显影缓冲液中平衡凝胶30分钟,同时温和搅拌。
  34. 在新鲜的Bio-Rad酶谱显影缓冲液中在37℃下进行最终温育16小时。
  35. 用蒸馏水洗涤凝胶,并染色以检测用亚甲基蓝共聚的PDGA的显影15分钟
  36. 然后将凝胶在30%v/v乙醇(15分钟),50%v/v乙醇(15分钟)中脱色,随后用几次蒸馏水改变。
  37. 解聚酶活性区域被鉴定为针对黑色背景的澄清斑点,其中酶已经水解共聚的PDGA底物(参见图2的代表性结果)。


    图2.在非还原条件下,在含有共聚的0.05%PDGA的凝胶中对显示解聚酶活性的合并级分(20μg蛋白质)进行2D电泳和2D-酶谱。 在酶谱中,解聚酶活性表现为对黑暗背景的亮点(白色箭头);考马斯蓝染色凝胶上的相应蛋白斑点由黑色箭头表示

食谱

  1. 最小盐培养基
    4.3克K sub 2 HPO 4 <
    3.4g KH 2 PO 4 sub/
    2.0g(NH 4)2 SO 4 4
    0.34g MgCl 2 6H 2 O
    0.026g CaCl 2 。 2H 2 0.2%w/v PDGA
    蒸馏H 2 O至1000ml,通过高压灭菌
    灭菌
  2. 阴离子交换结合缓冲液
    20mM Tris-HCl
    用HCl
    调节pH至9.0
  3. 阴离子交换洗脱缓冲液
    1 M NaCl
    20mM Tris-HCl
    用HCl
    调节至pH 9.0
  4. 平衡缓冲器
    2%SDS
    0.05 M Tris-HCl(pH 8.8)
    30%甘油 补足100毫升dH 2 O
  5. 非还原样本缓冲区
    4 M尿素
    4%CHAPS
    0.2%载体两性电解质 0.002%溴酚蓝
  6. SDS缓冲区
    2%SDS
    0.05M Tris-HCl
    30%甘油 用HCl
    调节pH至8.8
  7. 解决凝胶缓冲液
    1.5M Tris-HCl
    用HCl
    调节pH至8.8
  8. 堆叠凝胶缓冲液
    0.5M Tris-HCl
    用HCl
    调节pH至6.8
  9. 10%SDS
    10克SDS
    蒸馏H 2 O至1L
  10. 10%APS
    100 mg APS
    1ml蒸馏的H 2 O 2 /
  11. 10x TGE运行缓冲区
    30.0g Tris-HCl
    144.0g甘氨酸
    10.0克SDS
    用蒸馏H 2 O蒸气将体积调节至1000ml
  12. 考马斯染料
    1克考马斯亮蓝色
    500 ml甲醇
    100ml冰乙酸 蒸馏H 2 O至1000ml
  13. 考马斯脱色溶液
    100ml冰乙酸 200 ml甲醇
    蒸馏H 2 O至1000ml
  14. 亚甲蓝染料
    2g亚甲蓝
    0.01克KOH / 220 ml乙醇 蒸馏H 2 O至1000ml
  15. 70%乙醇
    700 ml乙醇 蒸馏出300毫升

致谢

这项工作得到了医学研究委员会能力建设学生奖。

参考文献

  1. Negus,D.和Taylor,P.W。(2014)。 一种聚-γ-(D) - 谷氨酸解聚酶, 炭疽芽孢杆菌。 Mol Microbiol 91(6):1136-1147。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Negus, D. and Taylor, P. W. (2014). Purification and Detection of a PDGA Depolymerase from Pusillimonas noertemannii. Bio-protocol 4(21): e1280. DOI: 10.21769/BioProtoc.1280.
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