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This protocol was developed in a study aimed to determine the cellular localization of the lysin of pneumococcal phage SV1 (Frias et al., 2013). We obtained proteins from the surface of Streptococcus pneumoniae by elution with choline or those secreted to the medium. The analysis by Western blot of these fractions allowed us to demonstrate that the phage lysin localizes to the cell wall, associating with choline residues in the teichoic acids. Hence, protein extracts can be used to determine the localization of uncharacterized proteins and can also be useful for other biochemical analyses such as protein identification. This protocol can be easily adapted to different pneumococcal strains and growth conditions and it is well suited to isolate other proteins of interest.

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Preparation of Pneumococcal Proteins for Western Blot Analysis
制备肺炎球菌蛋白用于蛋白印迹检测

微生物学 > 微生物生物化学 > 蛋白质 > 免疫检测
作者: Maria João Frias
Maria João FriasAffiliation: Instituto de Microbiologia, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
For correspondence: mfrias@fm.ul.pt
Bio-protocol author page: a654
José Melo-Cristino
José Melo-CristinoAffiliation: Instituto de Microbiologia, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
Bio-protocol author page: a656
 and Mário Ramirez
Mário RamirezAffiliation: Instituto de Microbiologia, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
For correspondence: ramirez@fm.ul.pt
Bio-protocol author page: a505
Vol 3, Iss 13, 7/5/2013, 3196 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.807

[Abstract] This protocol was developed in a study aimed to determine the cellular localization of the lysin of pneumococcal phage SV1 (Frias et al., 2013). We obtained proteins from the surface of Streptococcus pneumoniae by elution with choline or those secreted to the medium. The analysis by Western blot of these fractions allowed us to demonstrate that the phage lysin localizes to the cell wall, associating with choline residues in the teichoic acids. Hence, protein extracts can be used to determine the localization of uncharacterized proteins and can also be useful for other biochemical analyses such as protein identification. This protocol can be easily adapted to different pneumococcal strains and growth conditions and it is well suited to isolate other proteins of interest.

[Abstract]

Materials and Reagents

  1. Pneumococcal cells
  2. Mitomycin C (MitC) (0.1 μg/ml) (Sigma-Aldrich, catalog number: M0503 )
  3. 1x PBS (10x PBS pH 7.2) (Life Technologies, Gibco®/Invitrogen®, catalog number: 70013-016 )
  4. 50 mM Tris pH 7.5
  5. 2% (w/v) Choline chloride in 1x PBS (Sigma-Aldrich, catalog number: C7527 )
  6. NaCl (AppliChem, catalog number: A46615000 )
  7. Tris-HCl (Bio-Rad Laboratories, catalog number: 161-0799 )
  8. Glycerol (AppliChem, catalog number: A2364, 5000 )
  9. SDS (Bio-Rad Laboratories, catalog number: 161-0416 )
  10. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  11. Bromophenol blue (Bio-Rad Laboratories, catalog number: 161-0404 )
  12. Casamino acids
  13. L-Tryptophan
  14. L-Cysteine.HCl
  15. Glutamine
  16. Adenosine
  17. Uridine
  18. Nicotinic acid
  19. Pyridoxine
  20. Ca-pantothenate
  21. Thiamine-HCl
  22. Riboflavin
  23. Biotin
  24. Asparagine
  25. Casein-based semisynthetic medium C+Y (Lacks and Hotchkiss, 1960) (see Recipes)
  26. Loading buffer 5x (see Recipes)
  27. C+Y with 2% choline chloride (see Recipes)

Equipment

  1. Water bath at 37 °C to grow bacterial cultures
  2. Cell density meter (Biochrom WPA CO8000 Cell Density Meter) (Biochrom, catalog number: 80-3000-45 )
  3. Centrifuge
  4. 0.2 μm low-binding-protein membrane (DISMIC-03CP) (Advantec, catalog number: 03CP020AS )
  5. 0.22 μm membrane filter (Frilabo, catalog number: 1520012 )
  6. Amicon Ultra-15 centrifugal filter unit, cut-off 10 kDa (Merck Millipore, catalog number: UFC901024 )
  7. Western blot equipment

Procedure


I.   Extraction of choline-binding proteins by choline wash

  1. Grow lysogenic cells without holin activity, hence incapable of lysis, in C+Y at 37 °C until OD600 nm of approximately 0.9 is reached (overnight culture). We used a lysogenic strain since this protocol was developed to determine the localization of the phage lysin. Moreover, since holin function activates phage-mediated lysis, we eliminated holin activity in this strain to avoid possible phage lysin escape (Frias et al., 2013).
  2. Dilute 1:100 in 7 ml of fresh C+Y and continue incubation at 37 °C to an OD600 nm of 0.2-0.25, which takes approximately 2 h. Then, induce the phage by treating with Mitomycin C (MitC) at a final concentration of 0.1 μg/ml or left untreated as control.
  3. Take samples (7 ml) at different time points, for instance in 20 min-intervals, after MitC treatment. In the case of untreated cultures, collect the samples at the same time points after the culture reached OD600 nm 0.2-0.25.
  4. Harvest the cells by centrifugation (3,200 x g for 10 min at 4 °C).
  5. Wash the cells once with 0.5 culture volumes (3.5 ml) of cold 1x PBS.
  6. To obtain the total cell pellet fraction, suspended the cells in 200 μl of 50 mM Tris pH 7.5 and store at -20 °C.
  7. For choline wash, gently suspend the PBS washed cells (in step 5) in 200 μl of 2% choline chloride (w/v) prepared in 1x PBS and incubate 30 min at 4 °C without agitation to elute the choline binding proteins (avoiding cell lysis).
  8. As control for the specificity of the choline wash in removing only choline binding proteins, incubate cells in the same conditions with 1x PBS or 2% (w/v) NaCl prepared in 1x PBS.
  9. Collect bacteria by centrifugation (3,200 x g for 15 min at 4 °C). To obtain the cell pellet fraction after choline extraction, wash the pellet once with 0.5 volumes of cold 1x PBS, suspended in 200 μl of 50 mM Tris pH 7.5 and store at -20 °C.
  10. Filter the supernatant, which corresponds to the choline wash fraction, through a 0.2 μm low-binding-protein membrane to ensure the removal of all bacteria. Store at -20 °C.
  11. Separate proteins on SDS-PAGE: Boil 5-15 μl of the pellet fractions and 45 μl of the supernatant fractions for 5 min with 1x loading buffer and load onto the gel. You will need the antibody for your protein to visualize the protein on Western blot and it is important to control for possible cell lysis using an antibody for a known cytoplasmic protein (Figure 1). When comparing the amount of the protein of interest between samples, do not forget to normalize by a loading protein control. As an alternative to loading equal sample volumes followed by normalization, determine the protein concentration of each sample and load the same amount of total protein in each lane.


    Figure 1. Choline extracts increasing amounts of phage lysin (a choline-binding protein) from the cell surface. Equal aliquots were taken at the indicated times from MitC-treated lysogenic cultures (without holin activity). Cells were harvested by centrifugation and directly suspended in Tris buffer (cell pellet fraction, P) or choline washed (choline wash fraction, Scholine). As control, cells collected at 60 and 80 min were washed with PBS (SPBS). All fractions were tested by Western blotting for the phage lysin Svl (37 kDa) presence with the appropriate antibody. P and S fractions were also tested for the cytoplasmic elongation factor Ts (43 kDa) to control for cell lysis (Frias et al., 2013).

II.  Preparation of culture medium fractions in the presence of 2% choline

  1. Grow lysogenic cells without holin activity in C+Y at 37 °C until OD600 nm of approximately 0.9 is reached (overnight culture).
  2. Dilute 1:100 in 7 ml of fresh C+Y and continue incubation at 37 °C to an OD600 nm of 0.2-0.25.
  3. Collect cells by centrifugation (3,200 x g for 10 min at 4 °C) and discard the supernatant.
  4. Suspend lysogens in 7 ml of C+Y with 2% choline chloride, treat the cells with MitC (0.1 μg/ml) to induce the phage and continue the incubation (alternatively, bacteria can be grown in the absence of choline).
  5. Take samples (7 ml) at different time points after MitC treatment.
  6. Harvest the cells by centrifugation (3,200 x g for 10 min at 4 °C). To obtain the cell pellet fraction, wash the cells once with 0.5 volumes of cold 1x PBS, suspend in 200 μl of 50 mM Tris pH 7.5 and store at -20 °C.
  7. Collect the supernatant, which corresponds to the culture medium fraction, and filter through a 0.22 μm membrane filter. Besides the secreted proteins, this fraction also includes the choline binding proteins which are eluted from the pneumococcal surface in the presence of 2% choline chloride in the growth medium. Note that the choline binding proteins can be extracted using procedure described in Section I.
  8. Concentrate the supernatant 35-fold (final volume of 200 μl) by centrifugation (3,200 x g at 4 °C for approximately 15 min) on an Amicon Ultra-15 centrifugal filter unit (cut-off 10 kDa). It is recommended that the molecular weight cut off of the membrane is at least 3 times smaller than the molecular weight of the protein being retained. Since we wanted to study the 37 kDa phage lysin, we selected a 10 kDa cut off.
  9. Note that this experiment also allows to test for the membrane permeabilizing effect of a specific compound since in this case one expects the release of cytoplasmic proteins into the culture medium. If this is the goal of the experiment, after challenging the (MitC-treated) cultures with the compound of interest in step 4, the samples are processed as described in the following steps.
  10. Separate proteins on SDS-PAGE: Boil 5-15 μl of the pellet fractions and 45 μl of the supernatant fractions for 5 min with 1x loading buffer and load onto the gels. You will need the antibody for your protein to visualize the protein on Western blot and it is important to control for eventual cell lysis using an antibody for a known cytoplasmic protein.

Recipes

  1. C+Y (pH 8), 463 ml
    400 ml of PreC (A)
    13 ml of Supplement (B)
    10 ml of 1 mg/ml (w/v) glutamine in water
    10 ml of Adams III (C)
    5 ml of 2% (w/v) pyruvate in water
    15 ml of 1 M potassium phosphate (KPO4) buffer pH 8
    10 ml of 5% (w/v) yeast extract in water.
    (A) PreC, 2,000 ml
    2.42 g of sodium acetate anhydrous
    10 g of casamino acids
    0.01 g of L-Tryptophan
    0.1 g of L-Cysteine.HCl
    Add dH2O to 2,000 ml
    Adjust to pH 7.4-7.6
    Autoclave
    Store at room temperature.
    (B) Supplement, 213 ml
    30 ml of 3 in 1 Salts (D)
    60 ml of 20% (w/v) glucose in water
    3 ml of 50% (w/v) sucrose in water
    60 ml of 2 mg/ml (w/v) adenosine in water
    60 ml of 2 mg/ml (w/v) uridine in water
    Filter sterilize (0.22 μm)
    Store at 4 °C
    (C) Adams III, 400 ml
    0.8 g of asparagine
    0.08 g of choline
    0.64 ml of 1% (w/v) CaCl2 in water
    64 ml of Adams I (E)
    16 ml of Adams II (F)
    Add dH2O to 400 ml
    Filter sterilize (0.22 μm)
    Store in the dark at 4 °C
    (D) 3 in 1 salts, 100 ml
    10 g of MgCl2.6H2O
    0.05 g of CaCl2 anhydrous
    0.02 ml of 0.1 M MnSO4.4H2O
    Add dH2O to 100 ml
    Autoclave
    Store at room temperature
    (E) Adams I, 200 ml
    0.03 g of nicotinic acid
    0.035 g of pyridoxine
    0.12 g of Ca-pantothenate
    0.032 g of thiamine-HCl
    0.014 g of riboflavin
    0.06 ml of 0.5 mg/ml (w/v) biotin in water
    Add dH2O to 200 ml
    Filter sterilize (0.22 μm)
    Store in the dark at 4 °C
    (F) Adams II, 100 ml
    0.05 g of FeSO4.7H2O
    0.05 g of CuSO4.5H2O
    0.05 g of ZnSO4.7H2O
    0.02 g of MnCl2.4H2O
    1 ml of HCl concentrated
    Add dH2O to 100 ml
    Autoclave
    Store at room temperature
  2. 5x loading buffer (62.5 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 10% β-mercaptoethanol), 8 ml
    1 ml of 0.5 M Tris-HCl (pH 6.8) 
    1.6 ml of Glycerol
    1.6 ml of 10% SDS
    0.8 ml of β-mercaptoethanol
    0.4 ml of 0.5% (w/v) bromophenol blue in water
    Add dH2O to 8 ml
    Store at -20 °C
  3. C+Y with 2% choline chloride, 100 ml
    2 g of choline chloride
    Add C+Y to 100 ml
    Filter sterilize (0.22 μm)
    Store at 4 °C

References

  1. Frias, M. J., Melo-Cristino, J. and Ramirez, M. (2013). Export of the pneumococcal phage SV1 lysin requires choline-containing teichoic acids and is holin-independent. Mol Microbiol 87(2): 430-445.
  2. Lacks, S. and Hotchkiss, R. D. (1960). A study of the genetic material determining an enzyme in Pneumococcus. Biochim Biophys Acta 39: 508-518.

材料和试剂

1.     肺炎球菌细胞

2.     丝裂霉素CMitC)(0.1μg/ml)(Sigma-Aldrich,目录号:M0503

3.     1×PBS10×PBS pH 7.2)(Life TechnologiesGibco /Invitrogen ,目录号:70013-016

4.     50mM Tris pH7.5

5.     2%(w/v)氯化胆碱的1x PBSSigma-Aldrich,目录号:C7527

6.     NaClAppliChem,目录号:A46615000

7.     Tris-HClBio-Rad Laboratories,目录号:161-0799

8.     甘油(AppliChem,目录号:A2364,5000

9.     SDSBio-Rad Laboratories,目录号:161-0416

10.  β-巯基乙醇(Sigma-Aldrich,目录号:M3148

11.  溴酚蓝(Bio-Rad Laboratories,目录号:161-0404

12.  酪氨酸

13.  L-色氨酸

14.  L-半胱氨酸 HCl

15.  谷氨酰胺

16.  腺苷

17.  尿嘧啶

18.  烟酸

19.  吡哆醇

20.  泛酸钙

21.  硫胺素-HCl

22.  核黄素

23.  生物素

24.  天冬酰胺

25.  酪蛋白基半合成培养基C + YLacksHotchkiss1960)(参见配方)

26.  加载缓冲区5x(请参阅配方)

27.  C + Y2%氯化胆碱(参见配方)

设备

1.     37℃水浴中培养细菌培养物

2.     细胞密度计(Biochrom WPA CO8000细胞密度计)(Biochrom,目录号:80-3000-45

3.     离心机

4.     0.2μm低结合蛋白膜(DISMIC-03CP)(Advantec,目录号:03CP020AS

5.     0.22μm膜过滤器(Frilabo,目录号:1520012

6.     Amicon Ultra-15离心过滤器单元,截止10kDaMerck Millipore,目录号:UFC901024

7.     Western印迹设备

程序


<
- p - >

I.   通过胆碱洗涤提取胆碱结合蛋白< - p - >

1.     生长没有穴蛋白活性的溶源细胞,因此不能 裂解,在37℃的C + Y中,直至达到OD 600nm0.9 (过夜培养)。 我们使用溶源性菌株,因为这个协议是 以确定噬菌体溶素的定位。 此外, 因为穴蛋白功能激活噬菌体介导的溶解,我们消除了holin   活性以避免可能的噬菌体溶素逃逸(Frias等人, al 。,2013)。

2.     稀释11007毫升新鲜的C + Y,并继续 37℃下孵育至0.2-0.25OD 600nm,这需要 2小时。 然后,通过用丝裂霉素C处理诱导噬菌体 MitC),终浓度为0.1μg/ml,或不处理 控制

3.     在不同的时间点取样品(7ml 实例在20分钟间隔,MitC治疗后。 如果是 未处理的培养物,在相同时间点收集样品 培养物达到OD 600nm 0.2-0.25

4.     通过离心收获细胞(3,200×g,在4℃下10分钟)。

5.     0.5个培养体积(3.5ml)的冷的1x PBS洗涤细胞一次

6.     为了获得总细胞沉淀级分,将细胞悬浮于200μl50mM Tris pH7.5中并储存在-20℃。

7.     对于   胆碱洗涤,轻轻悬浮PBS洗涤的细胞(在步骤5中)在200μl   的在1×PBS中制备的2%氯化胆碱(w/v),并在37℃温育30分钟   4℃,不搅拌以洗脱胆碱结合蛋白(避免 细胞裂解)。

8.     作为胆碱特异性的控制 洗涤只去除胆碱结合蛋白,孵育细胞 相同条件下用1×PBS2%(w/vNaCl1×PBS中制备

9.     搜集   细菌通过离心(3,200×g,在4℃下15分钟)。 获得 胆碱提取后的细胞沉淀级分,洗涤沉淀一次 0.5体积的冷的1x PBS,悬浮在200μl50mM Tris pH 7.5,并储存在-20°C

10.  过滤上清液,其中 对应于胆碱洗级分,通过0.2μm 低结合蛋白膜以确保所有细菌的去除。 储存于-20°C

11.  SDS-PAGE上分离蛋白质:煮沸5-15μl   的沉淀级分和45μl上清液级分 min,用1x加样缓冲液装载到凝胶上。 你将需要 抗体为您的蛋白质可视化蛋白质的蛋白质印迹和 重要的是使用抗体控制可能的细胞裂解   一个已知的细胞质蛋白(图1)。 当比较的数量 在样品之间的感兴趣的蛋白质,不要忘记正常化   加载蛋白质控制。 作为加载相等样品的替代方法 体积,然后归一化,确定蛋白质浓度 并在每个泳道中加入相同量的总蛋白
http://www.bio-protocol.org/attached/image/20141201/20141201233937_8194.png
1.胆碱从细胞表面提取增加量的噬菌体溶素(胆碱结合蛋白)。 在指定时间从MitC处理的溶原培养物(没有穴蛋白活性)取等量等分试样。通过离心收获细胞,并直接悬浮于Tris缓冲液(细胞沉淀级分,P)或胆碱洗涤(胆碱洗涤级分,S胆碱)中。作为对照,在6080分钟收集的细胞用PBSS PBS)洗涤。通过蛋白质印迹法检测噬菌体溶素Sv137kDa)与适当抗体的存在的所有级分。还测试了PS级分的细胞质延伸因子Ts43kDa)以控制细胞裂解(Frias等人,2013)。

II 2%胆碱存在下制备培养基级分< - p - >

1.     37℃下在C + Y中生长没有穴蛋白活性的溶原细胞,直到达到OD 600nm0.9(过夜培养)。

2.     7ml新鲜的C + Y中稀释1100,并继续在37℃下温育至0.2-0.25OD 600nm

3.     通过离心收集细胞(3,200×g,在4℃下10分钟),并弃去上清液。

4.     将溶菌素悬浮在含有2%氯化胆碱的7ml C + Y中,用MitC0.1μg/ml)处理细胞以诱导噬菌体并继续孵育(或者,细菌可在不存在胆碱的情况下生长)。 />

5.     MitC处理后的不同时间点取样品(7ml)。

6.     通过离心收获细胞(3,200×g,在4℃下10分钟)。为了获得细胞沉淀级分,用0.5体积的冷的1x PBS洗涤细胞一次,悬浮在200μl50mM Tris pH7.5中并储存在-20℃。

7.     收集对应于培养基级分的上清液,并通过0.22μm膜过滤器过滤。除了分泌的蛋白质之外,该级分还包括在生长培养基中在2%氯化胆碱存在下从肺炎球菌表面洗脱的胆碱结合蛋白。注意胆碱结合蛋白可以使用第一节中描述的程序提取。

8.     通过在Amicon Ultra-15离心过滤装置(截止10kDa)上离心(3,200×g4℃,约15分钟)浓缩上清液35倍(终体积200μl 。建议膜的截留分子量比保留的蛋白质的分子量小至少3倍。由于我们想研究37kDa噬菌体溶素,我们选择了10kDa截断

9.     注意,该实验还允许测试特定化合物的膜渗透作用,因为在这种情况下人们期望细胞质蛋白质释放到培养基中。如果这是实验的目标,则在步骤4中用目标化合物挑战(MitC处理的)培养物后,按照以下步骤处理样品。

10.  SDS-PAGE上分离蛋白质:用上样缓冲液将5-15μl沉淀部分和45μl上清液部分煮沸5分钟,并加载到凝胶上。您将需要您的蛋白质的抗体在蛋白质印迹上显现蛋白质,并且使用已知胞质蛋白的抗体控制最终细胞裂解是重要的。

食谱

1.     C + YpH 8),463ml
400ml PreC
A
13
毫升补充(B
10ml
在水中的1mg/mlw/v)谷氨酰胺
10ml
亚当斯IIIC
5ml
在水中的2%(w/v)丙酮酸盐 15ml pH 81M磷酸钾(KPO 4)缓冲液
10ml
在水中的5%(w/v)酵母提取物 APreC2000ml
2.42g
无水乙酸钠
10g
酪蛋白氨基酸 0.01L-色氨酸 0.1g L-半胱氨酸。 HCl
dH <2> O添加至2,000 ml
调整至pH 7.4-7.6
高压灭菌器
在室温下储存。
B)补充,213 ml
30ml 3
1盐(D
60ml 20
%(w/v)葡萄糖的水溶液 3ml 50%(w/v)蔗糖的水溶液 60ml 2mg/mlw/v)腺苷的水溶液
60ml 2mg/ml
w/v)尿苷的水溶液
过滤灭菌(0.22μm
存储在4°C
CAdams III400ml
0.8
克天冬酰胺 0.08克胆碱
0.64ml
1%(w/vCaCl 2水溶液
64ml Adams I
E
16ml Adams II
F
dH <2> O加到400ml
过滤灭菌(0.22μm
4℃下避光保存
D31盐,100ml
10g MgCl 2
6H <2> 0.05g CaCl 2水溶液
0.02ml
0.1M MnSO 4·6H 2 O·4H 2 O·h / dH <2> O添加到100 ml
高压灭菌器
在室温下贮存
EAdams I200ml
0.03
克烟酸
0.035
克吡哆醇 0.12克泛酸钙 0.032克硫胺素-HCl 0.014g核黄素
0.06ml 0.5mg/ml
w/v)生物素的水溶液
dH 2 O加到200ml
过滤灭菌(0.22μm
4℃下避光保存
FAdams II100ml
0.05g
FeSO 4 7H 2 O 0.05gCuSO 4 5H O 0.05gZnSO 4 7H 2 O 0.02gMnCl 2 4H 2 O 1ml HCl浓缩
dH <2> O添加到100 ml
高压灭菌器
在室温下贮存

2.     5x上样缓冲液(62.5mM Tris-HClpH6.8,20%甘油,2SDS10β-巯基乙醇),8ml
1 ml 0.5 M Tris-HCl
pH 6.8
1.6ml
甘油
1.6ml 10
SDS 0.8mlβ-巯基乙醇 0.4ml 0.5%(w/v)溴酚蓝的水溶液
dH <2> O加到8ml
储存于-20°C

3.     C + Y2%氯化胆碱,100ml
2
克氯化胆碱
C + Y添加到100 ml
过滤灭菌(0.22μm
存储在4°C

参考文献

1.     FriasM.J.Melo-CristinoJ.and RamirezM。(2013)。 导出肺炎球菌噬菌体SV1溶素需要含胆碱的磷壁酸和 是不依赖于孔的。 Mol Microbiol 872):430-445

2.     LacksS.and HotchkissR.D。(1960)。 对确定肺炎球菌中的酶的遗传物质的研究 a> Biochim Biophys Acta 39508-518

 

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How to cite this protocol: Frias, M. J., Melo-Cristino, J. and Ramirez, M. (2013). Preparation of Pneumococcal Proteins for Western Blot Analysis. Bio-protocol 3(13): e807. DOI: 10.21769/BioProtoc.807; Full Text



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