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Enzymatic Activity Assays in Yeast Cell Extracts
酵母细胞提取物的酶活性分析   

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Abstract

Saccharomyces cerevisiae (S. cerevisiae) (commonly known as baker’s yeast) is a model organism that has a similar upstream base excision repair (BER) pathway for the repair of methylated bases as that in mammalian cells, and it is very easy to maintain in the laboratory environment. Here, we described a method to prepare cell extracts from yeast to investigate their enzymatic activities. This protocol is a quick and efficient way to make yeast cell extracts without using commercial kits.

Keywords: DNA damage(DNA损伤), DNA polymerase(DNA聚合酶), DNA repair(DNA修复), Base excision repair(碱基切除修复), Ligation failure(结扎失败)

Materials and Reagents

  1. EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics, catalog number: 11836170001 )
  2. Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
  3. Dextrose (Sigma-Aldrich, catalog number: D9434 )
  4. Bacto-peptone (Sigma-Aldrich, catalog number: P5905 )
  5. Bacto-yeast extract (Sigma-Aldrich, catalog number: Y1625 )
  6. Adenine sulfate (Sigma-Aldrich, catalog number: A3159 )
  7. Trizma base (Sigma-Aldrich, catalog number: T1503 )
  8. EDTA (Sigma-Aldrich, catalog number: 93283 )
  9. NaCl (Sigma-Aldrich, catalog number: S7653 )
  10. β-mercaptoethanol (Sigma-Aldrich, catalog number: B0126 )
  11. Formamide (Sigma-Aldrich, catalog number: F9037 )
  12. Bromophenol blue (Sigma-Aldrich, catalog number: B0126)
  13. Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
  14. Dithiothreitol-DTT (Sigma-Aldrich, catalog number: D0632 )
  15. Sodium borohydride-NaBH4 (Sigma-Aldrich, catalog number: 247677 )
  16. Sterile water
  17. Yeast peptone dextrose adenine (YPDA) medium (see Recipes)
  18. Potassium phosphate buffer (see Recipes)
  19. Lysis buffer (see Recipes)
  20. 10x reaction buffer (see Recipes)
  21. Gel loading buffer (see Recipes)

Equipments

  1. 0.5 mm diameter glass beads (Bio Spec Products, catalog number: 11079105 )
  2. Eppendorf tubes
  3. Table-top centrifuge
  4. Table-top heat block

Procedure

  1. Preparation of yeast cell extracts
    1. The cell extracts from yeast Saccharomyces cerevisiae were prepared as described (Wang et al., 1995; Jazwinski, 1990) with modifications as below.
    2. Grow yeast culture in yeast peptone dextrose adenine (YPDA) medium at 30 °C overnight (O/N) with shaking.
    3. Prepare a fresh cell culture by diluting 10-fold from O/N culture in 50 ml YPDA medium.
    4. Grow the cells to stationary phase at 30 °C with vigorous shaking.
    5. When the culture reached an absorbance of 0.5-1.0 at 600 nm, centrifuge cells at 2,000 rpm (300 rcf) for 5 min at 4 °C.
    6. Wash the cell pellet with water and then with potassium phosphate buffer.
    7. Centrifuge cells again at 2,000 rpm (300 rcf) for 5 min at 4 °C.
    8. Resuspend the cell pellet in 500 μl of ice-cold lysis buffer.
    9. Transfer the suspension to an Eppendorf tube prefilled with pre-chilled beads (1:1 ratio of beads to lysate), and keep on ice.
    10. Disrupt the cells at 4 °C by vortexing the bead/lysate mixture: 10 cycles of 2 min vortex at maximum speed alternating with cycles of 1 min cooling on ice.
    11. Centrifuge the bead/lysate mixture at full speed at 4 °C for 15 min to remove cell debris.
    12. Transfer the supernatant fraction to a fresh Eppendorf tube. We recommend not collecting the entire supernatant fraction, instead leaving part of it to prevent touching pellet.
    13. Determine the protein concentration of the extract prepared by Bradford assay using dye reagent.

  2. Enzymatic activity assays in yeast cell extracts
    1. Prepare 10 μl of reaction mixture including 1x reaction buffer and 140 nM DNA substrate. DNA substrate used includes an adenylated uracil base at the 5′ end of the 3′-FAM–labeled oligonucleotide (Caglayan et al., 2014).
    2. Start the reaction by addition of yeast cell extract prepared as above (to a final amount of 50 μg) to the reaction mixture.
    3. Incubate the reaction mixture at 30 °C for 5, 15, and 30 min.
    4. Stabilize the reaction products by addition of 1 M freshly prepared and ice-cold NaBH4 to the final concentration of 100 mM.
    5. Incubate the reaction samples on ice for 30 min.
    6. Mix the reaction products with 10 μl of gel loading buffer.
    7. The reaction products in the yeast cell extracts were separated on a 15% polyacrylamide gel, the gel was scanned and the data were analyzed as reported (Caglayan et al., 2014). The result image for dRP lyase, FEN1 excision and DNA deadenylation enzymatic activities in yeast extracts has been reported (Caglayan et al., 2014).

Representative data

The result image that shows dRP lyase, FEN1 excision and DNA deadenylation enzymatic activities in yeast extracts was published as Supplementary Figure 5 in Caglayan et al. (2014).

Notes

Enzymatic activity in yeast cell extracts may be lost after continuous vortexing if samples warm up. It might be necessary to extend the alternating cooling steps depending on the sample.

Recipes

  1. Yeast peptone dextrose adenine (YPDA) medium (1 L)
    20 g dextrose
    20 g Bacto-peptone
    10 g Bacto-yeast extract
    2 ml 0.5% adenine sulfate
  2. Potassium phosphate buffer
    25 mM KPO4 (pH 8.0)
  3. Lysis buffer
    25 mM Tris-HCl (pH 7.5)
    1 mM EDTA
    100 mM NaCl
    10 mM β-mercaptoethanol
    A protease inhibitor tablet
  4. 10x reaction buffer
    500 mM HEPES (pH 7.5)
    100 mM MgCl2
    200 mM KCl
    5 mM EDTA
    20 mM DTT
  5. Gel loading buffer
    95% formamide
    20 mM EDTA
    0.02% bromophenol blue
    0.02% xylene cyanol

Acknowledgments

This work was supported by the Intramural Research Program of the US National Institutes of Health, National Institute of Environmental Health Sciences (grants Z01 ES050158 and ES050159).

References

  1. Caglayan, M., Batra, V. K., Sassa, A., Prasad, R. and Wilson, S. H. (2014). Role of polymerase beta in complementing aprataxin deficiency during abasic-site base excision repair. Nat Struct Mol Biol 21(5): 497-499.
  2. Jazwinski, S. M. (1990). Preparation of extracts from yeast. Methods Enzymol 182: 154-174.
  3. Wang, Z., Wu, X. and Friedberg, E. C. (1995). The detection and measurement of base and nucleotide excision repair in cell-free extracts of the yeast Saccharomyces cerevisiae. Methods 7(2): 177-186.

简介

酿酒酵母(酿酒酵母)(通常称为面包酵母)是具有类似上游碱基切除修复(BER)途径以修复甲基化碱基的模式生物体 如在哺乳动物细胞中,并且在实验室环境中非常容易维持。 在这里,我们描述了一种从酵母中制备细胞提取物以研究其酶活性的方法。 此协议是一种快速和有效的方式来制作酵母细胞提取物,而不使用商业试剂盒。

关键字:DNA损伤, DNA聚合酶, DNA修复, 碱基切除修复, 结扎失败

材料和试剂

  1. 无EDTA蛋白酶抑制剂混合物片剂(Roche Diagnostics,目录号:11836170001)
  2. Bio-Rad蛋白测定染料试剂浓缩物(Bio-Rad Laboratories,目录号:500-0006)
  3. 葡萄糖(Sigma-Aldrich,目录号:D9434)
  4. 细菌用蛋白胨(Sigma-Aldrich,目录号:P5905)
  5. 细菌酵母提取物(Sigma-Aldrich,目录号:Y1625)
  6. 硫酸腺嘌呤(Sigma-Aldrich,目录号:A3159)
  7. Trizma碱(Sigma-Aldrich,目录号:T1503)
  8. EDTA(Sigma-Aldrich,目录号:93283)
  9. NaCl(Sigma-Aldrich,目录号:S7653)
  10. β-巯基乙醇(Sigma-Aldrich,目录号:B0126)
  11. 甲酰胺(Sigma-Aldrich,目录号:F9037)
  12. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  13. 二甲苯Cyanol(Sigma-Aldrich,目录号:X4126)
  14. 二硫苏糖醇-DTT(Sigma-Aldrich,目录号:D0632)
  15. 硼氢化钠 - NaBH 4(Sigma-Aldrich,目录号:247677)
  16. 无菌水
  17. 酵母蛋白胨葡萄糖腺苷(YPDA)培养基(参见配方)
  18. 磷酸钾缓冲液(见配方)
  19. 裂解缓冲液(见配方)
  20. 10x反应缓冲液(参见配方)
  21. 凝胶加载缓冲液(参见配方)

设备

  1. 0.5mm直径的玻璃珠(Bio Spec Products,目录号:11079105)
  2. Eppendorf管
  3. 台式离心机
  4. 桌面加热块

程序

  1. 酵母细胞提取物的制备
    1. 来自酵母酿酒酵母的细胞提取物被制备为 描述(Wang等人,1995; Jazwinski,1990),修改为
    2. 在酵母蛋白胨葡萄糖腺苷(YPDA)培养基中在30℃下振荡培养酵母培养物过夜(O/N)。
    3. 准备新鲜的细胞培养物通过稀释10倍从O/N文化在50ml YPDA培养基。
    4. 在30℃下剧烈摇动使细胞生长至稳定期。
    5. 当培养物在600nm处达到0.5-1.0的吸光度时,在4℃下以2,000rpm(300rcf)离心细胞5分钟。
    6. 用水洗涤细胞沉淀,然后用磷酸钾缓冲液洗涤。
    7. 在4℃下以2,000rpm(300rcf)再次离心细胞5分钟
    8. 重悬细胞沉淀在500μl冰冷的裂解缓冲液。
    9. 将悬浮液转移到预填充的Eppendorf管中 预先冷却的珠子(珠与裂解物的比例为1:1),并保持在冰上
    10. 通过涡旋珠/裂解物混合物在4℃下破碎细胞:10 2分钟涡流的最大速度的循环与1分钟的循环交替   在冰上冷却。
    11. 将珠/裂解物混合物在4℃下以全速离心15分钟以除去细胞碎片。
    12. 将上清液部分转移到新鲜的Eppendorf管中。 我们 建议不要收集整个上清液部分 留下一部分以防止接触丸粒
    13. 确定使用染料试剂通过Bradford测定法制备的提取物的蛋白质浓度。

  2. 酵母细胞提取物中的酶活性测定
    1. 准备10微升反应混合物,包括1×反应缓冲液和140 nM DNA底物。 所用的DNA底物包括腺苷酸化的尿嘧啶碱基 在3'-FAM标记的寡核苷酸的5'末端(Caglayan等人, 2014)。
    2. 通过向反应混合物中加入如上制备的酵母细胞提取物(至最终量50μg)开始反应
    3. 将反应混合物在30℃下孵育5,15和30分钟
    4. 通过加入1M新鲜制备的和冰冷的NaBH 4 4至100mM的终浓度来稳定反应产物。
    5. 在冰上孵育反应样品30分钟
    6. 将反应产物与10μl凝胶上样缓冲液混合
    7. 将酵母细胞提取物中的反应产物在   15%聚丙烯酰胺凝胶,扫描凝胶并分析数据 如报道(Caglayan等人,2014)。 dRP裂解酶的结果图像, FEN1切除和酵母中的DNA去腺苷酸化酶活性 提取物已被报道(Caglayan等人,2014)。

代表数据

在Caglayan等人中公开了在酵母提取物中显示dRP裂解酶,FEN1切除和DNA去腺苷酸化酶活性的结果图像作为补充图5。 (2014)。

笔记

如果样品变热,在连续涡旋后酵母细胞提取物中的酶活性可能丧失。 根据样品,可能需要延长交替冷却步骤。

食谱

  1. 酵母蛋白胨葡萄糖腺嘌呤(YPDA)培养基(1L)
    20g葡萄糖
    20克细菌蛋白胨 10g细菌 - 酵母提取物
    2ml 0.5%腺嘌呤硫酸盐
  2. 磷酸钾缓冲液
    25mM KPO 4(pH 8.0)
  3. 裂解缓冲液
    25mM Tris-HCl(pH7.5) 1mM EDTA
    100 mM NaCl
    10mMβ-巯基乙醇 蛋白酶抑制剂片剂
  4. 10x反应缓冲液
    500mM HEPES(pH7.5) 100mM MgCl 2/v/v 200 mM KCl
    5 mM EDTA
    20 mM DTT
  5. 凝胶加载缓冲液
    95%甲酰胺 20 mM EDTA
    0.02%溴酚蓝
    0.02%二甲苯蓝

致谢

这项工作是由美国国立卫生研究院,国家环境健康科学研究院(授予Z01 ES050158和ES050159)的校内研究计划支持。

参考文献

  1. Caglayan,M.,Batra,V.K.,Sassa,A.,Prasad,R.and Wilson,S.H。(2014)。 在脱碱基位点碱基切除修复期间聚合酶β在补充aprataxin缺乏中的作用 < em> Nat Struct Mol Biol 21(5):497-499
  2. Jazwinski,S.M。(1990)。 从酵母中提取提取物的准备方法Enzymol 182: 154-174。
  3. Wang,Z.,Wu,X。和Friedberg,E.C。(1995)。 检测和测量酵母的无细胞提取物中的碱基和核苷酸切除修复酿酒酵母。 方法 7(2):177-186。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Çağlayan, M. and Wilson, S. H. (2014). Enzymatic Activity Assays in Yeast Cell Extracts. Bio-protocol 4(23): e1312. DOI: 10.21769/BioProtoc.1312.
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