搜索

Heterologous Expression and Purification of Catalytic Domain of CESA1 from Arabidopsis thaliana
拟南芥CESA1催化结构域的异源表达和纯化   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Heterologous expression of plant cellulose synthase (CESA) and its purification has remained a challenge for decades impeding detailed biophysical, biochemical and structural characterization of this key enzyme. An in-depth knowledge of structure and function of CESA proteins would enable us to better understand the hierarchical structure of the plant cell wall. Here, we report a detailed, and reproducible method of purification of catalytic domain of CESA1 from Arabidopsis thaliana that was recombinantly expressed in Escherichia coli. The method relies on a two stage purification procedure to obtain the catalytic domain in monomer and trimer forms. The biochemical and biophysical data including low resolution structures of the protein have been published (Vandavasi et al., 2016). Currently the crystallization studies of this protein are underway.

[Background] Cellulose is the most important structural component of plant cell walls and constitutes the Earth’s largest source of biorenewable material, yet the mechanism of its synthesis by plants is poorly understood. The plant cellulose synthesis complex (CSC), also called a ‘rosette’ because of its hexameric appearance in electron microscope images, is a large multi-subunit transmembrane protein complex responsible for synthesis of cellulose chains and their assembly into microfibrils. The number of cellulose synthase (CESA) proteins in the CSC and the number of cellulose chains in a microfibril have been debated for many years. Structural information about CESA proteins from plants is crucial to provide answers to some of the basic questions regarding the mechanism of cellulose synthesis. However, elucidation of the structure of CESA proteins has proved difficult because they are multi-domain proteins comprised of disordered, globular, and membrane associated domains. As an alternative to pursuing structural studies of CESA holoproteins, we are developing approaches for recombinant expression of individual CESA domains (e.g., N-terminal domain, central-cytosolic domain, C-terminal transmembrane domain) in large quantities suitable for structural studies. The current protocol has been optimized for isolation of the catalytic domain of A. thaliana CESA1 as reported (Vandavasi et al., 2016). Using this protocol, it is possible to control the oligomerization state of the protein enabling structural studies of the monomer and the trimeric form of the protein. The approach described may be broadly applicable to other systems.

Keywords: Cellulose synthesis(纤维素的合成), CESA protein(CESA蛋白), Heterologous expression(异源表达), N-Lauroylsarcosine(N-月桂酰肌氨酸)

Materials and Reagents

  1. 0.2 µm filter (VWR, catalog number: 28145-501 )
  2. E. coli BL21-RIL (Agilent Technologies, catalog number: 230280 )
  3. Deionized water
  4. Ampicillin (VWR, catalog number: 97061-442 )
  5. Chloramphenicol (VWR, catalog number: EM-3130 )
  6. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  7. Luria broth (EMD Millipore, catalog number: 71751 )
  8. Sorbitol (Sigma-Aldrich, catalog number: S1876 )
  9. IPTG (Teknova, catalog number: I3325 )
  10. Tris buffer (Sigma-Aldrich, catalog number: 252859 )
  11. Sodium deoxycholic acid (Geno Technology, catalog number: DG090 )
  12. Nonidet (Sigma-Aldrich, catalog number: I8896 )
  13. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  14. Lysozyme (VWR, catalog number: CA-EM5960 )
  15. β-mercaptoethanol (BME) (Sigma-Aldrich, catalog number: M6250 )
  16. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  17. CAPS buffer (Sigma-Aldrich, catalog number: C2632 )
  18. Sodium lauroyl sarcosine (Sigma-Aldrich, catalog number: L9150 )
  19. Glucose (Sigma-Aldrich, catalog number: G5767 )
  20. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  21. HEPES buffer (Sigma-Aldrich, catalog number: H3375 )
  22. LB media supplemented with 0.25 M sorbitol (see Recipes)
  23. Lysis buffer (see Recipes)
  24. Wash buffer-1 (see Recipes)
  25. Wash buffer-2 (see Recipes)
  26. Solubilization buffer (see Recipes)
  27. Dialysis buffer-1 (see Recipes)
  28. Dialysis buffer-2 (see Recipes)

Equipment

  1. Glassware
  2. Autoclave (Panasonic, model: MLS-3781L )
  3. Filtering devices for sterilization (Thermo Fisher Scientific, Fisher Scientific, catalog number: 596-4520 )
  4. Bio hood (Labconco, model: 3440009 )
    Note: This product has been discontinued.
  5. Refrigerator or cold room
  6. Temperature controlled shaker incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: MxQTM 6000 )
  7. Magnetic stirring plate (VWR, catalog number: 97042-626 )
  8. UV/Vis spectrophotometer (Thermo Fisher Scientific, Thermo Scientific, model: NanoDrop-2000 )
  9. Apparatus for SDS-PAGE (Mini-PROTEAN® Tetra vertical electrophoresis cell) (Bio-Rad Laboratories, catalog number: 1658004 )
  10. 400 Watt sonicator (All-Spec, BRANSON, model: S450D )
  11. Refrigerated centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: RC 6 Plus )
  12. 10 kDa MWCO PES concentrators (Vivaproducts, model: Vivaspin® 20 )
  13. Superdex 200 size exclusion chromatographic column, 120 ml capacity (GE Healthcare, catalog number: 17-1069-01 )
    Note: This product has been discontinued.
  14. Akta FPLC system (or similar liquid chromatography system)
  15. Dialysis cassettes (Slide-A-LyzerTM dialysis cassette G2, 10 kDa MWCO) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 87730 )

Procedure

  1. Cell culture and protein expression
    The details of cloning can be found in Vandavasi et al. (2016). A plasmid (pET300NT) containing the gene to express the catalytic domain of CESA1 (amino acids 341-845; MW: 59.7 kDa) from A. thaliana (AtCESA1catD) was transformed into E. coli BL21-RIL strain with ampicillin and chloramphenicol as markers for selection and preserved as a 30% glycerol stock at -80 °C. The plasmid DNA is available on request from Dr. Hugh O’Neill at the Oak Ridge National Laboratory.
    1. Inoculate a starter culture of 50 ml of LB media supplemented with ampicillin (100 µg/ml) and chloramphenicol (35 µg/ml) with a dab of the frozen glycerol stock and grow overnight at 37 °C in a shaking incubator with a shaking speed of 250 rpm.
    2. Transfer 25 ml of starter culture to 1 L of LB media supplemented with 0.25 M sorbitol, ampicillin (100 µg/ml) and chloramphenicol (35 µg/ml).
    3. Grow the 1 L culture at 37 °C until the optical density measured at 600 nm in a spectrophotometer (OD600nm) reaches 0.3-0.4 (approximately 2.5 h of growth).
    4. Change the temperature of incubator to 16 °C and continue cell growth for 15-20 min and monitor the OD600nm. When the OD600nm reaches 0.5-0.6 remove 1 ml of culture (pre-induction sample) for SDS-PAGE analysis.
    5. When OD600nm is 0.5-0.6 add IPTG to the culture to a final concentration of 1 mM to induce the expression of the AtCESA1catD.
    6. Incubate the culture at 16 °C for 10-12 h. Remove 0.25 ml of cells (post induction sample) for SDS-PAGE analysis.
    7. Harvest the cells by centrifuging at 4 °C and 7,000 x g for 15 min and store the cell pellet at -80 °C.
    8. Analyze the expression levels of AtCESA1catD in pre and post induction samples using SDS-PAGE (Figure 1).


      Figure 1. SDS-PAGE analysis of pre and post induction. Lane 1: pre-induced whole cells; Lanes 2, 3: post-induced whole cells; Lanes 4, 5: soluble fraction from post-induced cells; Lane 6: Mw markers. The intense bands at approximately 59 kDa marked with an arrow in lanes 2 and 3 compared to the lane 1 represent induction of the AtCESA1catD protein.

  2. Lysis, extraction and purification of inclusion bodies
    1. Resuspend the cell pellet in lysis buffer. 5 ml of lysis buffer per gram of cells (wet weight) worked best for us.
    2. Incubate the cells in lysis buffer for 30 min on ice. Mix the contents using a pipet occasionally.
    3. Sonicate the cell suspension. We applied 6 pulses, each for 30 sec, using a 1.3 cm (diameter) horn and 400 Watt sonicator at 50% power. A cooling period of 60 sec was included between consecutive pulses to avoid heating of the sample.
    4. Centrifuge the cell lysate at 36,000 x g for 30 min at 4 °C to separate the supernatant from the insoluble fraction.
    5. Discard the supernatant and wash the insoluble fraction with 20 ml of wash buffer-1. The washing involves sequential resuspension of the insoluble fraction using a pipette in 20 ml of wash buffer-1 followed by centrifugation at 36,000 x g for 30 min. Repeat the wash for 3 times.
    6. Now wash the insoluble fraction with wash buffer-2 for three times.
      Note: This step is very important and can be optimized by monitoring the removal of impurities in the insoluble fraction using SDS-PAGE.
    7. At this stage the insoluble fraction contains inclusion bodies that can be dissolved in the solubilization buffer or can be stored at -80 °C for long term storage. The inclusion bodies contain approximately 100 mg of protein per liter of culture and so it is suggested to divide the protein into smaller aliquots before storing.

  3. Solubilization of inclusion bodies and size exclusion chromatography at 4 °C
    1. Solubilize the inclusion bodies obtained from 1 L culture in 10 ml of ice cold solubilization buffer by gentle pipetting and incubation for 20-30 min on ice with gentle mixing.
    2. After 30 min centrifuge the entire contents at 16,000 x g for 45 min to remove insoluble impurities. Collect the supernatant and check the absorbance at 280 nm to estimate the protein concentration. The extinction coefficient of AtCESA1catD is 80,510 M-1 cm-1.
    3. The final protein concentration of the solubilized protein should be in the range of 15-20 mg/ml. A high concentration of sample is a prerequisite to achieve an efficient separation using size exclusion chromatography. Concentrate the sample to 15-20 mg/ml if necessary using 10 kDa MWCO PES concentrators.
    4. An Akta FPLC chromatography system was used.
    5. Equilibrate a Superdex 200 (120 ml) column in solubilization buffer.
    6. Filter the solubilized sample through 0.2 µm membrane filters and load 1-2 ml of 15-20 mg/ml of sample on to the Superdex 200 (120 ml) column. Set the flow rate at 1 ml/min. In size exclusion chromatography, the sample volume is critical for optimizing the separation of proteins. For a 120 ml column, the optimal sample volume is in the range of 1-2 ml. The combination of a relatively high concentration of sample (15-20 mg/ml) and a small sample volume resulted in a resolved peak of the target protein at a relatively high concentration.
    7. Collect the fractions (1 ml) for analysis using SDS-PAGE to judge the purity (see Figures 2 and 3). Pool the fractions with pure AtCESA1and concentrate to 10-15 mg/ml using a 10 KDa MWCO PES concentrator. For example, Lanes 3-7 of the SDS-PAGE gel in Figure 3 represent the main peak fractions of the elution profile in Figure 2. These are the fractions that were pooled for further study.
    8. Dialyze the concentrated and pure sample (1-5 ml) against 1 L of dialysis buffer-1 for 24 h to obtain the pure catalytic domain in monomeric form.


      Figure 2. Representative elution profile of AtCESA1catD through a Superdex 200 (120 ml) chromatographic column. 1.5 ml of sample was loaded. Fractions were of 1 ml and the flow rate was set at 1 ml/min. Absorbance is expressed in milli-absorbance units.


      Figure 3. Fractions from Superdex 200 (120 ml) size exclusion chromatography. Lane 1: Marker; Lanes 2-8: Fractions 53 to 59 from size exclusion chromatography.

  4. Oligomerization of monomers into trimers at 4 °C
    1. The monomeric protein self-assembles into trimer by gradual removal of sodium lauroyl sarcosine during dialysis. Typically, 1-2 ml of monomeric protein at a protein concentration of 10-15 mg/ml is dialyzed against 1 L of dialysis buffer-2 for 48 h at 4 °C. The oligomerization of the monomeric protein into a trimer can be monitored by measuring the hydrodynamic radius of the sample using dynamic light scattering (DLS) (Vandavasi et al., 2016).

Data analysis

No special data processing or analysis was required for this procedure.

Notes

  1. IPTG induction: When OD600nm is 0.5-0.6 add IPTG to the culture to a final concentration of 1 mM to induce the expression of the AtCESA1catD. Based on our experience, this step is very important because we have found that the protein expression is not induced if the OD600nm exceeds 0.8.
  2. Protein concentration: We suggest using concentrators with PES (polyether sulfone) membranes to concentrate the CESA protein because carbohydrate binding proteins can bind to concentrators that have regenerated cellulose membranes.

Recipes

  1. LB media supplemented with 0.25 M sorbitol
    Dissolve 20 g of LB (peptone from casein: 10 g; yeast extract: 5 g; NaCl: 5 g) in 750 ml of ultrapure water and sterilize by autoclaving.
    Prepare 250 ml of 1 M sorbitol and sterilize by filtering through a 0.2 µm filter.
    Mix both LB and sorbitol under aseptic conditions.
  2. Lysis buffer
    20 mM Tris-HCl, pH 8.0
    200 mM NaCl
    0.2 mg/ml lysozyme
  3. Wash buffer-1
    20 mM Tris, pH 8.0
    1% sodium deoxycholic acid
    1% Nonidet
    10 mM BME
  4. Wash buffer-2
    20 mM Tris, pH 8.0
    1% Triton X-100
  5. Solubilization buffer
    50 mM CAPS (pH 10.5)
    0.2% (w/v) sodium lauroyl sarcosine
    0.2 M glucose
    3% (v/v) glycerol
    5 mM DTT
    Note: The pH of the solubilization buffer should be 10.5 measured at room temperature (25 °C). We found that at pH < 10.5 the protein is not readily soluble.
  6. Dialysis buffer-1
    50 mM CAPS, pH 8.0
    0.2% (w/v) sodium lauroyl sarcosine
    0.2 M glucose
    3% (v/v) glycerol
    5 mM DTT
  7. Dialysis buffer-2
    20 mM HEPES, pH 8.0
    50 mM NaCl
    10% glycerol
    5 mM DTT

Notes:
a. NaOH was used to adjust the pH of all the buffers used in this protocol.
b. All the buffers were sterile filtered using 0.2 µm filters.

Acknowledgments

This work was supported by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (ORNL), managed by UT-Batelle, LLC, for the U. S. Department of Energy (DOE) under contract No.DE-AC05-00OR22725 and by the Center for Lignocellulose Structure and formation (CLSF), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences.
This manuscript has been authored by UT-Battelle, LLC under Contract No.DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

References

  1. Vandavasi, V. G., Putnam, D. K., Zhang, Q., Petridis, L., Heller, W. T., Nixon, B. T., Haigler, C. H., Kalluri, U., Coates, L., Langan, P., Smith, J. C., Meiler, J. and O'Neill, H. (2016). A structural study of CESA1 catalytic domain of Arabidopsis cellulose synthesis complex: evidence for CESA trimers. Plant Physiol 170(1): 123-135.

简介

植物纤维素合酶(CESA)的异源表达及其纯化在数十年来一直是阻碍该关键酶的详细生物物理,生物化学和结构表征的挑战。对CESA蛋白的结构和功能的深入了解将使我们能够更好地理解植物细胞壁的层次结构。在这里,我们报告了在大肠杆菌中重组表达的来自拟南芥的CESA1的催化结构域的详细的和可重复的纯化方法。该方法依赖于两阶段纯化方法以获得单体和三聚体形式的催化结构域。已经公开了包括蛋白质的低分辨率结构的生物化学和生物物理数据(Vandavasi等人,2016)。目前,这种蛋白质的结晶研究正在进行中。

[Backg 圆形] 纤维素是植物细胞壁最重要的结构组分,地球最大的生物可再生材料来源,但它的植物合成机制了解很少。植物纤维素合成复合物(CSC),也称为"玫瑰花结",因为其在电子显微镜图像中的六聚体外观,是一个大的多亚基跨膜蛋白复合物负责纤维素链的合成和它们组装成微原纤维。 CSC中的纤维素合酶(CESA)蛋白的数目和微原纤维中的纤维素链的数目已经辩论了许多年。来自植物的CESA蛋白的结构信息对于提供关于纤维素合成机制的一些基本问题的答案是至关重要的。然而,阐明CESA蛋白的结构被证明是困难的,因为它们是由无序,球状和膜相关结构域组成的多结构域蛋白。作为对CESA环蛋白的结构研究的替代方案,我们正在开发用于单个CESA结构域(例如N端结构域,中央胞质结构域,C端跨膜结构域)的重组表达的方法大量适合结构研究。已经针对α的催化结构域的分离优化了目前的方案。 thaliana CESA1(Vandavasi ,2016)。使用该方案,可以控制蛋白质的寡聚化状态,使得能够结构研究单体和三聚体形式的蛋白质。所描述的方法可以广泛地适用于其他系统。

关键字:纤维素的合成, CESA蛋白, 异源表达, N-月桂酰肌氨酸

材料和试剂

  1. 0.2μm过滤器(VWR,目录号:28145-501)
  2. E。大肠杆菌BL21-RIL(Agilent Technologies,目录号:230280)
  3. 去离子水
  4. 氨苄青霉素(VWR,目录号:97061-442)
  5. 氯霉素(VWR,目录号:EM-3130)
  6. 甘油(Sigma-Aldrich,目录号:G5516)
  7. Luria broth(EMD Millipore,目录号:71751)
  8. 山梨醇(Sigma-Aldrich,目录号:S1876)
  9. IPTG(Teknova,目录号:I3325)
  10. Tris缓冲液(Sigma-Aldrich,目录号:252859)
  11. 脱氧胆酸钠(Geno Technology,目录号:DG090)
  12. Nonidet(Sigma-Aldrich,目录号:I8896)
  13. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  14. 溶菌酶(VWR,目录号:CA-EM5960)
  15. β-巯基乙醇(BME)(Sigma-Aldrich,目录号:M6250)
  16. Triton X-100(Sigma-Aldrich,目录号:T8787)
  17. CAPS缓冲液(Sigma-Aldrich,目录号:C2632)
  18. 月桂酰肌氨酸钠(Sigma-Aldrich,目录号:L9150)
  19. 葡萄糖(Sigma-Aldrich,目录号:G5767)
  20. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  21. HEPES缓冲液(Sigma-Aldrich,目录号:H3375)
  22. 补充有0.25M山梨醇(参见Recipes)的LB培养基
  23. 裂解缓冲液(见配方)
  24. 洗涤缓冲液-1(见配方)
  25. 洗涤缓冲液-2(见配方)
  26. 溶解缓冲液(参见配方)
  27. 透析缓冲液-1(参见配方)
  28. 透析缓冲液-2(参见配方)

设备

  1. 玻璃器皿
  2. 高压灭菌器(Panasonic,型号:MLS-3781L)
  3. 用于灭菌的过滤装置(Thermo Fisher Scientific,Fisher Scientific,目录号:596-4520)
  4. 生物罩(Labconco,型号:3440009)
    注意:此产品已停产。
  5. 冰箱或冷藏室
  6. 温控摇床培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,型号:MxQ TM 6000)
  7. 磁力搅拌板(VWR,目录号:97042-626)
  8. UV/Vis分光光度计(Thermo Fisher Scientific,Thermo Scientific,型号:NanoDrop-2000)
  9. 用于SDS-PAGE(Mini-PROTEAN Tetra TM垂直电泳池)(Bio-Rad Laboratories,目录号:1658004)的装置
  10. 400瓦特超声波仪(All-Spec,BRANSON,型号:S450D)
  11. 冷冻离心机(Thermo Fisher Scientific,Thermo Scientific TM ,型号:RC 6 Plus)
  12. 10kDa MWCO PES聚能器(Vivaproducts,型号:Vivaspin 20)
  13. Superdex 200尺寸排阻色谱柱,120ml容量(GE Healthcare,目录号:17-1069-01) 注意:此产品已停产。
  14. Akta FPLC系统(或类似的液相色谱系统)
  15. 透析盒(Slide-A-Lyzer TM透析盒G2,10kDa MWCO)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:87730)

程序

  1. 细胞培养和蛋白表达
    克隆的细节可以在Vandavasi等人的 中找到。 (2016年)。含有表达来自A的CESA1的催化结构域(氨基酸341-845; MW:59.7kDa)的基因的质粒(pET300NT) thaliana (AtCESA1catD)转化为E。大肠杆菌BL21-RIL菌株用氨苄青霉素和氯霉素作为选择标记并在-80℃保存为30%甘油原液。质粒DNA可以从Oak Ridge国家实验室的Hugh O'Neill博士处索取
    1. 用冷冻甘油储备液的一滴接种补充有氨苄青霉素(100μg/ml)和氯霉素(35μg/ml)的50ml LB培养基的发酵剂培养物,并在振荡培养箱中在37℃下振荡为250rpm。
    2. 将25ml起始培养物转移到补充有0.25M山梨醇,氨苄青霉素(100μg/ml)和氯霉素(35μg/ml)的1L LB培养基中。
    3. 在37℃下生长1L培养物,直到在分光光度计(OD 600nm)中在600nm测量的光密度达到0.3-0.4(约2.5小时的生长)。
    4. 将培养箱的温度改变为16℃,并继续细胞生长15-20分钟,并监测OD 600nm。当OD 600nm达到0.5-0.6时,去除1ml用于SDS-PAGE分析的培养物(预诱导样品)。
    5. 当OD 600nm为0.5-0.6时,向培养物中加入IPTG至终浓度为1mM以诱导AtCESA1catD的表达。
    6. 孵育培养物在16℃下10-12小时。取出0.25ml细胞(诱导后样品)用于SDS-PAGE分析
    7. 通过在4℃和7,000xg离心15分钟收获细胞,并将细胞沉淀物保存在-80℃。
    8. 使用SDS-PAGE分析诱导前和诱导后样品中AtCESA1catD的表达水平(图1)。


      图1.诱导前和诱导后的SDS-PAGE分析。泳道1:预诱导的全细胞;泳道2,3:后诱导的全细胞;泳道4,5:来自后诱导的细胞的可溶性级分;泳道6:M w w标记。与泳道1相比,在泳道2和泳道3中用箭头标记的约59kDa的强带代表AtCESA1catD蛋白的诱导。

  2. 包涵体的溶解,提取和纯化
    1. 在裂解缓冲液中重悬细胞沉淀。每克细胞(湿重)5ml裂解缓冲液对我们来说效果最好。
    2. 孵育细胞在裂解缓冲液在冰上30分钟。偶尔用移液管混合内容物。
    3. 超声细胞悬浮液。我们使用1.3cm(直径)喇叭和400瓦特超声波仪以50%功率施加6个脉冲,每个脉冲30秒。在连续脉冲之间包括60秒的冷却时间,以避免样品的加热
    4. 将细胞裂解物在36,000×g离心30分钟,在4℃下分离上清液和不溶性部分。
    5. 弃去上清液并用20ml洗涤缓冲液-1洗涤不溶性级分。洗涤包括使用移液管在20ml洗涤缓冲液-1中连续重悬不溶性级分,随后在36,000×g离心30分钟。重复洗涤3次。
    6. 现在用洗涤缓冲液-2洗涤不溶性级分三次。
      注意:此步骤非常重要,可以通过使用SDS-PAGE监测不溶性馏分中杂质的去除来优化。
    7. 在该阶段,不溶性级分包含可以溶解在溶解缓冲液中的包涵体,或者可以在-80℃下储存以长期储存。包涵体每升培养物中含有约100mg蛋白质,因此建议在储存之前将蛋白质分成较小的等分试样。

  3. 在4℃下包涵体的溶解和尺寸排阻色谱法
    1. 通过温和地移液并在轻微混合下在冰上孵育20-30分钟,使通过1L培养物获得的包涵体在10ml冰冷的溶解缓冲液中溶解。
    2. 30分钟后,将全部内容物以16,000×g离心45分钟以除去不溶性杂质。收集上清液并检查280nm处的吸光度以估计蛋白质浓度。 AtCESA1catD的消光系数为80,510M -1 cm -1
    3. 溶解的蛋白质的最终蛋白质浓度应在15-20mg/ml的范围内。高浓度的样品是使用尺寸排阻色谱法实现有效分离的先决条件。如果需要,使用10kDa MWCO PES浓缩器将样品浓缩至15-20mg/ml。
    4. 使用Akta FPLC色谱系统
    5. 在溶解缓冲液中平衡Superdex 200(120ml)柱。
    6. 通过0.2μm膜过滤器过滤溶解的样品,并将1-2ml 15-20mg/ml样品加载到Superdex 200(120ml)柱上。将流速设置为1 ml/min。在尺寸排阻色谱中,样品体积对于优化蛋白质的分离是至关重要的。对于120ml柱,最佳样品体积在1-2ml的范围内。相对高浓度的样品(15-20mg/ml)和小样品体积的组合导致相对高浓度的目标蛋白质的分辨峰。
    7. 收集级分(1ml)用于使用SDS-PAGE分析,以判断纯度(参见图2和图3)。使用10KDa MWCO PES浓缩器将馏分与纯的AtCESA1混合并浓缩至10-15mg/ml。例如,图3中的SDS-PAGE凝胶的泳道3-7代表图2中洗脱曲线的主峰部分。这些是合并用于进一步研究的部分。
    8. 将浓缩的纯样品(1-5ml)对1L透析缓冲液-1透析24小时,得到单体形式的纯催化结构域。


      图2.通过Superdex 200(120ml)色谱柱的AtCESA1catD的代表性洗脱曲线。加载1.5ml样品。级分为1ml,流速设为1ml/min。吸光度以毫吸光度单位表示。


      图3.来自Superdex 200(120ml)尺寸排阻色谱的级分。泳道1:标记;泳道2-8:来自尺寸排阻色谱的级分53至59
  4. 在4℃下将单体低聚成三聚体
    1. 单体蛋白通过在透析期间逐渐去除月桂酰肌氨酸钠而自组装成三聚体。通常,在4℃下,将1ml蛋白质浓度为10-15mg/ml的单体蛋白质1-2ml相对于1L透析缓冲液-2透析48小时。可以通过使用动态光散射(DLS)测量样品的流体动力学半径来监测单体蛋白质到三聚体中的寡聚化(Vandavasi等人,2016)。

数据分析

此过程不需要特殊的数据处理或分析。

笔记

  1. IPTG诱导:当OD 600nm为0.5-0.6时,向培养物中加入IPTG至终浓度为1mM,以诱导AtCESA1catD的表达。基于我们的经验,该步骤是非常重要的,因为我们发现如果OD 600nm超过0.8,则不诱导蛋白质表达。
  2. 蛋白质浓度:我们建议使用带有PES(聚醚砜)膜的浓缩器来浓缩CESA蛋白,因为碳水化合物结合蛋白可以与再生纤维素膜的浓缩器结合。

食谱

  1. 补充有0.25M山梨醇的LB培养基 将20g LB(来自酪蛋白的蛋白胨:10g;酵母提取物:5g; NaCl:5g)溶解在750ml超纯水中,并通过高压灭菌进行灭菌。 准备250ml的1M山梨糖醇,并通过0.2μm过滤器过滤灭菌 在无菌条件下混合LB和山梨醇
  2. 裂解缓冲液
    20mM Tris-HCl,pH8.0 200 mM NaCl
    0.2mg/ml溶菌酶
  3. 洗涤缓冲液-1
    20 mM Tris,pH 8.0
    1%脱氧胆酸钠 1%Nonidet
    10mM BME
  4. 洗涤缓冲液2
    20 mM Tris,pH 8.0
    1%Triton X-100
  5. 溶解缓冲液
    50mM CAPS(pH 10.5) 0.2%(w/v)月桂酰肌氨酸钠 0.2 M葡萄糖
    3%(v/v)甘油 5 mM DTT
    注意:溶解缓冲液的pH应在室温(25℃)下测定10.5。我们发现, 10.5蛋白质不容易溶解。
  6. 透析缓冲液-1
    50 mM CAPS,pH 8.0
    0.2%(w/v)月桂酰肌氨酸钠 0.2 M葡萄糖
    3%(v/v)甘油 5 mM DTT
  7. 透析缓冲液-2
    20mM HEPES,pH 8.0
    50mM NaCl 10%甘油 5mM DTT

注意:
a。 使用NaOH调节本协议中使用的所有缓冲液的pH。 b。使用0.2μm过滤器无菌过滤所有缓冲液。

致谢

这项工作得到了由UT-Batelle,LLC管理的Oak Ridge国家实验室(ORNL)的实验室指导研究和开发计划的支持,美国能源部(DOE),合同号为DE-AC05-00OR22725,中心为木质纤维素结构和形成(CLSF),由美国能源部,科学办公室,基础能源科学办公室资助的能源前沿研究中心。
该手稿由UT-Battelle,LLC在与美国能源部签订的合同号为DE-AC05-00OR22725的条款下创作。美国政府保留并且出版商接受该出版物,承认美国政府保留一个非排他性,已缴纳,不可撤销的世界范围的许可证出版或复制这份手稿的出版形式,或允许他人这样做,为美国政府的目的。能源部将根据DOE公共访问计划( http://energy.gov/downloads/doe-public-access-plan )。

参考文献

  1. Vandavasi,VG,Putnam,DK,Zhang,Q.,Petridis,L.,Heller,WT,Nixon,BT,Haigler,CH,Kalluri,U.,Coates,L.,Langan,P.,Smith,JC,Meiler ,J.和O'Neill,H。(2016)。 
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。