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Purification of 70S Ribosomes from Bacillus subtilis
从枯草杆菌中纯化70S核糖体   

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参见作者原研究论文

本实验方案简略版
Microbiology
Jun 2014

Abstract

The eubacterial ribosome (70S) is a macromolecular complex that is composed of a small (30S) subunit and a large (50S) subunit. The small subunit comprises the 16S ribosomal RNA (rRNA) and more than 20 ribosomal proteins (r-proteins), whereas the large subunit comprises the 23S and 5S rRNAs and more than 30 r-proteins. Bacillus subtilis (B. subtilis) has 57 r-ribosomal protein genes and three rRNAs (16S, 23S and 5S rRNAs). Among them, we identified 21 r-proteins of the small subunit and 31 r-proteins of the large subunit in B. subtilis (Nanamiya et al., 2004). The functions and roles of individual components of the ribosome have not yet been completely clarified. Herein we describe in detail an ultracentrifugation-based protocol for the preparation of 70S ribosomes from exponentially growing cells of B. subtilis.

Keywords: Bacillus subtilis (枯草芽孢杆菌), Ribosome (核糖体), 70S ribosomes (70S核糖体)

Materials and Reagents

  1. Bacillus subtilis 168
  2. NaCl
  3. BD BactoTM tryptone (Difco, catalog number: 211705 )
  4. BD BactoTM yeast extract (Difco, catalog number: 212750 )
  5. Agar (Nissui, catalog number: 05835 )
  6. Tris (Wako Pure Chemical Industries, catalog number: 512-97505 )
  7. Magnesium acetate (Wako Pure Chemical Industries, catalog number: 139-15335 )
  8. Ammonium acetate (Wako Pure Chemical Industries, catalog number: 019-02835 )
  9. Dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 042-29222 )
  10. Phenylmethylsulphonyl fluoride (PMSF)
  11. Sucrose (Sigma-Aldrich, catalog number: S0389-55G )
  12. Diethylpyrocarbonate (DEPC) treated water
    Note: DEPC was added to distilled water at a final concentration of 1%, and then it stored at room temperature for over two hours. The DEPC-treated distilled water was autoclaved twice (121 °C, 20 min) and stored at room temperature.
  13. LB medium (see Recipes)
  14. Buffer I (see Recipes)
  15. 10-40% sucrose solutions (see Recipes)

Equipment

  1. 2 L flasks with cotton plugs
  2. Innova 4080 bench top incubator shaker (New Brunswick scientific)
  3. French pressure cell press [Aminco International (USA), model: FA-078 ]
  4. Mini cell [Aminco International (USA), model: FA-003 ]
  5. Gradient fractionator (BIOCOMP, catalog number: 152-001 )
  6. Gradient master [Aminco International (USA), catalog number: 107-201M ]
  7. Micro collector (ATTO Technology, catalog number: AC-5700P )
  8. Nano drop 2000 (Thermo Fisher Scientific)
  9. High-speed refrigerated centrifuge (e.g. Hitachi, model: himac CR22GII )
  10. Angle rotor R10A3 (fixed angle, max: 10,000 rpm,18,800 x g, volume: 500 ml x 6) (Hitachi)
  11. Centrifuge MX-300 (TOMY)
  12. TMA-300 rotor (fixed angle, max: 15,000 rpm, 20,380 x g, volume: 2 ml x 24) (TOMY)
  13. TMA-27 rotor (fixed angle, max: 15,000 rpm, 21,130 x g, volume: 15 ml x 4, 50 ml x 4) (TOMY)
  14. Centrifuge tube (Nalgene, catalog number: 3148-0050 )
  15. Ultra-centrifuge (e.g. Hitachi, model: CP-60E )
  16. P55ST2 rotor (swinging bucket rotor, max: 55,000 rpm, 366,000 x g, volume: 5 ml x 6) (Hitachi)
  17. P28S rotor (swinging bucket rotor, max: 28,000 rpm, 141,000 x g, volume: 40 ml x 6) (Hitachi)
  18. P40ST rotor (swinging bucket rotor, max: 40,000 rpm, 284,000 x g, volume: 13 ml x 6) (Hitachi)
  19. Open-top polyclear centrifuge tube for P40ST rotor (Seton Identification Products, part number: 7031 )
  20. Open-top polyclear centrifuge tube for P28S rotor (Seton Identification Products, part number: 7052 )
  21. Centrifuge ware for P55ST2 rotor (Hitachi, part number: 332245A )
    Note: These rotors can substitute for other commercial rotors which can be used by the same centrifugal force as described in procedures.

Procedure

  1. Crude preparation of 70S ribosomes
    1. B. subtilis is streaked out for single colonies and pre-incubated on LB agar plates at 30 °C for 14-16 h.
    2. Cells are taken from single colonies on the plates and suspended in approximately 10 ml of LB medium with high turbidity. The cell suspension was diluted into 3 L of LB medium at an OD600 of 0.04, and distributed equally among six 2-liter flasks, each containing 500 ml of the cell culture. The flasks are incubated at 37 °C with shaking at 250 rpm.
      Note: B. subtilis is routinely cultured in volumes that do not exceed one quarter of the volume of the flask used.
    3. Cells are harvested by centrifugation, when the OD600 of the culture reaches 0.2, using six 500-ml centrifuge tubes (7,000 x g; 5 min, 4 °C). Cell pellets are resuspended with small volume of the culture supernatant, transferred from a 500-ml centrifugation tube to six 15-ml sample tubes and pellets are collected (3,500 x g, 5 min, 4 °C). The cell pellets are frozen by liquid N2.
    4. Each cell pellet is suspended in 3.5 ml of Buffer I pre-chilled on ice.
    5. Cells are disrupted using a French press (8,000 p.s.i.) passing the sample through three times.
    6. Cell debris is removed by centrifugation (11,000 x g, 30 min, 4 °C).
    7. The supernatant (cell lysate) is centrifuged at 30,000 x g for 30 min at 4 °C, for example in a HITACHI P55ST2 rotor at 18,000 rpm.
    8. The resulting supernatant (S30 supernatant) is centrifuged at 200,000 x g for 100 min at 4 °C (e.g. in a HITACHI P55ST2 rotor at 45,000 rpm).
    9. Each pellet is resuspended with 200 μl of Buffer I and the suspension stored at -80 °C until required (S100 pellet fraction).
      Note that the S100 fraction can be seen as amber pellet at bottom of the tube. Using a glass rod may be preferable to resuspend the pellet because resuspension of the pellet by pipetting is not easy.
      Note: Care is taken to avoid generating foam while resuspending the pellet.

  2. Purification of 70S ribosomes
    1. To further purify the 70S ribosomes, prepare a 10-40% sucrose gradient in SW28 centrifuge tubes using a Gradient Master gradient mixer.
    2. 10% and 40% sucrose solutions are prepared in Buffer I.
    3. The 10 to 40% sucrose gradient is generated in a SW28 centrifuge tube using a Gradient Master gradient mixer.
    4. The S100 pellet fraction is applied on the top of the 10 to 40% sucrose gradient and centrifuged at 67,000 x g for 17 h at 4 °C (e.g. in a HITACHI P28S rotor at 22,500 rpm, using 270 A260 units per centrifuge tube).
    5. Samples are taken by Piston Gradient Fractionator and Micro collector. Absorbance profiles at 260 nm are determined using a Nano drop 2000.
      Note: An example of the ribosome profile from crude cell extracts is shown in Figure 1.
    6. Collected fractions containing 70S ribosomes are identified as a peak with an absorbance at 260 nm.
    7. The ribosomes are recovered from the pooled fractions, which are diluted with Buffer I (1:2 dilutions) and centrifuged at 64,000 x g for 17 h at 4 °C. (e.g. in a HITACHI P40ST rotor at 22,500 rpm).
    8. The supernatant is removed completely from the centrifuge tubes.
    9. The resulting 70S ribosome pellets are covered with 300 μl of Buffer I and left overnight at 4 °C. The pellet is then gently resuspended in Buffer I using a glass rod and stored at -80 °C until required.
      Note: Care is taken to avoid generating foam while resuspending the pellet.

Representative data



Figure 1. Profile of ribosome from crude cell extract by 10-40% sucrose gradient sedimentation. The crude cell extract prepared from the log phase cells (OD600 = 0.2) of wild type strain (168 trpC2) was applied on the top of the 10 to 40% sucrose gradient and centrifuged at 55,000 x g for 17.5 h at 4 °C (e.g. in a HITACHI P40ST rotor at 21,000 rpm, using 10 A260 units per centrifuge tube). The A260 measurement shows as the same profile pattern of A420 measurement, and the peak of 70S ribosome can be determined. When the S100 fraction was subjected to the 10 to 40% sucrose gradient and centrifugation, the peak smaller than 30S was not observed.

Recipes

  1. LB medium
    0.5% NaCl
    1% tryptone
    0.5% yeast extract
    1.5% agar (only use for plates)
  2. Buffer I (200 ml is enough, pre-chilled on ice)
    20 mM
    Tris-HCl (pH 7.6)
    15 mM
    magnesium acetate
    100 mM
    ammonium acetate
    0.1 mM
    DTT
    2 mM
    PMSF
    Buffer I is prepared with DEPC treated water.
    Note: Buffer I can be stored without DTT and PMSF. DTT and PMSF are added to the buffer before use. Buffer I should be pre-chilled throughout the entire procedure.
  3. 10-40% sucrose solutions
    10-40% sucrose solutions are made in Buffer I.

Acknowledgments

This protocol was adapted from Nanamiya et al. (2004) and Suzuki et al. (2014). This work was supported in part by Grants-in-Aid for Scientific Research (C) (G. A.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References

  1. Nanamiya, H., Akanuma, G., Natori, Y., Murayama, R., Kosono, S., Kudo, T., Kobayashi, K., Ogasawara, N., Park, S. M., Ochi, K. and Kawamura, F. (2004). Zinc is a key factor in controlling alternation of two types of L31 protein in the Bacillus subtilis ribosome. Mol Microbiol 52(1): 273-283.
  2. Nanamiya, H., Kawamura, F. and Kosono, S. (2006). Proteomic study of the Bacillus subtilis ribosome: Finding of zinc-dependent replacement for ribosomal protein L31 paralogues. J Gen Appl Microbiol 52(5): 249-258.
  3. Natori, Y., Nanamiya, H., Akanuma, G., Kosono, S., Kudo, T., Ochi, K. and Kawamura, F. (2007). A fail-safe system for the ribosome under zinc-limiting conditions in Bacillus subtilis. Mol Microbiol 63(1): 294-307.
  4. Suzuki, S., Tanigawa, O., Akanuma, G., Nanamiya, H., Kawamura, F., Tagami, K., Nomura, N., Kawabata, T. and Sekine, Y. (2014). Enhanced expression of Bacillus subtilis yaaA can restore both the growth and the sporulation defects caused by mutation of rplB, encoding ribosomal protein L2. Microbiology 160(Pt 6): 1040-1053.
  5. Tagami, K., Nanamiya, H., Kazo, Y., Maehashi, M., Suzuki, S., Namba, E., Hoshiya, M., Hanai, R., Tozawa, Y., Morimoto, T., Ogasawara, N., Kageyama, Y., Ara, K., Ozaki, K., Yoshida, M., Kuroiwa, H., Kuroiwa, T., Ohashi, Y. and Kawamura, F. (2012). Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp(0) mutant of Bacillus subtilis triggers YvyD-dependent dimerization of ribosome. Microbiologyopen 1(2): 115-134.

简介

真细菌核糖体(70S)是由小(30S)亚基和大(50S)亚基组成的大分子复合物。 小亚基包含16S核糖体RNA(rRNA)和超过20个核糖体蛋白(r-蛋白),而大亚基包含23S和5S rRNA和超过30个r蛋白。 枯草芽孢杆菌(<枯草芽孢杆菌)具有57个核糖体蛋白基因和三个rRNA(16S,23S和5S rRNA)。 其中,我们确定21 r蛋白的小亚基和31 r蛋白的大亚基在B。 枯草芽孢杆菌(Nanamiya等人,2004)。 核糖体的各个组分的功能和作用尚未完全澄清。 在本文中,我们详细描述了从B的指数生长细胞制备70S核糖体的基于超速离心的方案。 枯草芽孢杆菌。

关键字:枯草芽孢杆菌, 核糖体, 70S核糖体

Materials and Reagents

  1. Bacillus subtilis 168
  2. NaCl
  3. BD BactoTM tryptone (Difco, catalog number: 211705)
  4. BD BactoTM yeast extract (Difco, catalog number: 212750)
  5. Agar (Nissui, catalog number: 05835)
  6. Tris(Wako Pure Chemical Industries,目录号:512-97505)
  7. 乙酸镁(Wako Pure Chemical Industries,目录号:139-15335)
  8. 乙酸铵(Wako Pure Chemical Industries,目录号:019-02835)
  9. 二硫苏糖醇(DTT)(Wako Pure Chemical Industries,目录号:042-29222)
  10. 苯甲基磺酰氟(PMSF)
  11. 蔗糖(Sigma-Aldrich,目录号:S0389-55G)
  12. 焦碳酸二乙酯(DEPC)处理的水
    注意:将DEPC以1%的终浓度加入蒸馏水中,然后在室温下储存2小时以上。 将DEPC处理的蒸馏水高压灭菌两次(121℃,20分钟),并在室温下贮存。
  13. LB介质(见配方)
  14. 缓冲区I(参见配方)
  15. 10-40%蔗糖溶液(见配方)

设备

  1. 2 L烧瓶,带棉塞
  2. Innova 4080台式孵化器(新不伦瑞克科学)
  3. 法国压力电池压力机[Aminco International(USA),型号:FA-078]
  4. Mini cell [Aminco International(USA),型号:FA-003]
  5. 梯度分馏器(BIOCOMP,目录号:152-001)
  6. Gradient master [Aminco International(USA),目录号:107-201M]
  7. 微收集器(ATTO Technology,目录号:AC-5700P)
  8. Nano drop 2000(Thermo Fisher Scientific)
  9. 高速冷冻离心机(例如,日立,型号:himac CR22GII)
  10. 角转子R10A3(固定角,最大:10,000rpm,18,800 x×g,体积:500ml×6)(日立)
  11. 离心机MX-300(TOMY)
  12. TMA-300转子(固定角,最大:15,000rpm,20,380×g,体积:2ml×24)(TOMY)
  13. TMA-27转子(固定角,最大:15,000rpm,21,130×g,体积:15ml×4,50ml×4)(TOMY)
  14. 离心管(Nalgene,目录号:3148-0050)
  15. 超离心机(例如,日立,型号:CP-60E)
  16. P55ST2转子(旋转叶片转子,最大:55,000rpm,366,000×g ,体积:5ml×6)(日立)
  17. P28S转子(旋转叶片转子,最大:28,000rpm,141,000×g ,体积:40ml×6)(日立)
  18. P40ST转子(摆动转子转子,最大:40,000rpm,284,000×g,体积:13ml×6)(日立)
  19. P40ST转子用敞口型多孔离心管(Seton Identification Products,型号:7031)
  20. 用于P28S转子的敞口型多孔离心管(Seton Identification Products,部件号:7052)
  21. P55ST2转子离心机(日立,部件号:332245A)
    注意:这些转子可以代替其他商业转子,可以按照相同的离心力使用。

程序

  1. 70S核糖体的粗制准备
    1. B。 枯草芽孢杆菌在单菌落中划线并在LB琼脂平板上在30℃下预温育14-16小时。
    2. 细胞取自平板上的单个菌落,并悬浮于其中 约10ml具有高浊度的LB培养基。细胞 悬浮液稀释到OD OD 600为0.04的3L LB培养基中, 在6个2升烧瓶中平均分配,每个烧瓶含有500ml 细胞培养。将烧瓶在37℃下在250℃下振荡孵育 rpm。
      注意:枯草芽孢杆菌常规培养的体积不超过所用瓶子体积的四分之一。
    3. 当培养物的OD大于600时,通过离心收获细胞  达到0.2,使用六个500ml离心管(7,000×g; 5分钟, C)。用少量培养物重悬细胞沉淀 上清液,从500-ml离心管转移到6个15-ml 收集样品管和沉淀(3500xg,5分钟,4℃)。的 细胞沉淀物通过液体N 2 2冷冻
    4. 将每个细胞沉淀悬浮于在冰上预冷的3.5ml缓冲液I中
    5. 使用French压机(8,000p.s.i.)破碎细胞,使样品通过三次。
    6. 通过离心(11,000×g,30分钟,4℃)除去细胞碎片。
    7. 将上清液(细胞裂解物)以30,000×g离心30分钟 min,在4℃下,例如在HITACHI P55ST2转子中以18,000rpm
    8. 将所得上清液(S30上清液)以200,000×离心   在HITACHI P55ST2转子中以45,000rpm在4℃下(例如)进行100分钟。
    9. 每个沉淀用200μl缓冲液I重悬浮 悬浮液保存在-80℃直至需要(S100沉淀部分)。
      请注意,S100馏分可以看作是底部的琥珀色沉淀   管。 使用玻璃棒可优选重悬沉淀 因为通过移液的颗粒的再悬浮不容易。
      注意:注意避免在重悬沉淀时产生泡沫。

  2. 70S核糖体的纯化
    1. 为了进一步纯化70S核糖体,制备10-40%蔗糖梯度 在SW28离心管中使用梯度Master梯度混合器
    2. 在缓冲液I中制备10%和40%蔗糖溶液
    3. 使用Gradient Master梯度混合器在SW28离心管中产生10至40%蔗糖梯度
    4. 将S100丸粒部分施用在10至40% 蔗糖梯度,并在4℃下以67,000×g离心17小时(例如,   HITACHI P28S转子以22,500rpm,每个离心机使用270A 260个单位   管)
    5. 通过活塞梯度分馏器和 微收集器。 使用a。测定260nm处的吸光度分布 Nano drop 2000.
      注意:粗细胞提取物的核糖体图谱的一个实例如图1所示。
    6. 收集含有70S核糖体的级分被鉴定为在260nm处具有吸光度的峰
    7. 从合并的级分回收核糖体 用缓冲液I(1:2稀释)稀释,并以64,000×g离心 在4℃下17小时。 (例如在HITACHI P40ST转子上,转速为22,500 rpm)。
    8. 从离心管中完全除去上清液
    9. 所得的70S核糖体沉淀用300μl的包被 缓冲液I并在4℃下放置过夜。 然后轻轻地沉淀沉淀 使用玻璃棒重悬浮于缓冲液I中并储存在-80℃直至 必需。
      注意:注意避免在重悬沉淀时产生泡沫。

代表数据



图1.通过10-40%蔗糖梯度沉降从粗细胞提取物得到的核糖体图谱。从对数期细胞(OD 600 = 0.2)制备的粗细胞提取物将野生型菌株(168μgtrpC2)应用于10至40%蔗糖梯度的顶部,并在4℃下以55,000×g离心17.5小时(例如在HITACHI P40ST转子中以21,000rpm,每个离心管使用10安培260单位)。 A 260nm测量显示为A 420测量的相同的图谱,并且可以确定70S核糖体的峰。当S100馏分经受10至40%蔗糖梯度和离心时,未观察到小于30S的峰。

食谱

  1. LB培养基
    0.5%NaCl
    1%胰蛋白酶
    0.5%酵母提取物
    1.5%琼脂(仅用于平板)
  2. 缓冲液I(200ml就足够了,在冰上预冷)
    20 mM
    Tris-HCl(pH 7.6)
    15 mM
    乙酸镁
    100 mM
    乙酸铵
    0.1 mM
    DTT
    2 mM
    PMSF
    缓冲液I用DEPC处理的水制备 注意:缓冲区I可以在没有DTT和PMSF的情况下存储。 DTT和PMSF在使用前加入缓冲液中。 缓冲液I应在整个程序中预冷。
  3. 10-40%蔗糖溶液 在缓冲液I中制备10-40%蔗糖溶液

确认

该协议改编自Nanamiya等人(2004)和Suzuki等人(2014)。 这项工作得到日本教育,文化,体育,科学和技术部的科学研究助理(C)(G.A.)的部分支持。

参考文献

  1. Nanamiya,H.,Akanuma,G.,Natori,Y.,Murayama,R.,Kosono,S.,Kudo,T.,Kobayashi,K.,Ogasawara,N.,Park,SM,Ochi,K.and Kawamura ,F。(2004)。 锌是控制枯草芽孢杆菌中两种类型L31蛋白交替的关键因素核糖体。 Mol Microbiol 52(1):273-283。
  2. Nanamiya,H.,Kawamura,F.and Kosono,S。(2006)。 枯草芽孢杆菌核糖体的蛋白质组学研究:锌依赖性替代的发现对于核糖体蛋白L31旁系同源物。 J Gen Appl Microbiol 52(5):249-258。
  3. Natori,Y.,Nanamiya,H.,Akanuma,G.,Kosono,S.,Kudo,T.,Ochi,K.and Kawamura,F。(2007)。 在锌限制性条件下在枯草芽孢杆菌中的核糖体的故障安全系统 Mol Microbiol 63(1):294-307。
  4. Suzuki,S。,Tanigawa,O.,Akanuma,G.,Nanamiya,H.,Kawamura,F.,Tagami,K.,Nomura,N.,Kawabata,T.and Sekine, 增强的 枯草芽孢杆菌 yaaA可以恢复由编码核糖体蛋白L2的rplB突变引起的生长和孢子形成缺陷。 160(Pt 6):1040-
  5. Tagami,K.,Nanamiya,H.,Kazo,Y.,Maehashi,M.,Suzuki,S。,Namba,E.,Hoshiya,M.,Hanai,R.,Tozawa,Y.,Morimoto, Ogasawara,N.,Kageyama,Y.,Ara,K.,Ozaki,K.,Yoshida,M.,Kuroiwa,H.,Kuroiwa,T.,Ohashi,Y.and Kawamura, 在(p)ppGpp(0)中表达小(p)ppGpp合成酶YwaC, 突变体触发核糖体的YvyD依赖性二聚化。 1(2):115-134。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Suzuki, S., Akanuma, G. and Kawamura, F. (2015). Purification of 70S Ribosomes from Bacillus subtilis. Bio-protocol 5(7): e1432. DOI: 10.21769/BioProtoc.1432.
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