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[Bio101] GST-tagged Yeast Protein Purification
[Bio101] 酵母中GST标签融合蛋白的纯化   

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Abstract

Glutation S-transferase (GST) tagging is the most commonly used purification strategy for recombinant protein. It was developed with the goal of preserving the enzymatic activity by utilizing gentle elution condition of the target protein from purification matrix (Poon and Hunt., 1994). The method described here can be applied from single protein to proteome scale purification of recombinant protein from yeast (Zhu et al., 2000; Zhu et al., 2001).

Materials and Reagents

  1. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E0396 )
  2. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  3. HEPES (Sigma-Aldrich, catalog number: 54457 )
  4. Roche protease inhibitor tablets (containing EDTA) (Roche Diagnostics, catalog number: 11697498001 )
  5. Phosphatase inhibitor (Roche Diagnostics, catalog number: 4906845001 )
  6. Glutathione (Axxora, catalog number: 157-002-G005 )
  7. Galactose (Mp Biomedicals, catalog number: 0210174701 )
  8. Zirconia/Silica beads (Biospec Products, catalog number: 11079105z )
  9. Glutathione beads (Thermo Fisher Scientific, catalog number: 16100 )
  10. Tris
  11. NaCl
  12. TritonX-100
  13. Glycerol
  14. Beta-mercaptoethanol (BME)
  15. Sc-ura liquid media with raffinose
  16. Lysis buffer (see Recipes)
  17. Wash buffer I (see Recipes)
  18. Wash buffer II (see Recipes)
  19. Elution buffer (see Recipes)

Equipment

  1. Table top centrifuge
  2. Bead beater
  3. 50 ml Falcon tubes

Procedure

  1. Strains are grown on sc-ura plates.
    1. Start a 5 ml starter culture of sc-ura or sc-ura/raffinose (I prefer to just do everything in autoclaved sc-ura/raffinose) and grow O/N or 16 h at 30 °C.
    2. Measure OD600 of starter culture and dilute cultures into 50 ml of fresh sc-ura/raffinose to an OD that allows at least 2 cell divisions. I typically inoculate to an OD of 0.1 and allow to grow to an OD 0.6-0.8 before inducing (typically requires roughly 3 h per doubling time for these strains).
    3. Induce with 20% galactose when cells at OD 0.6-0.8 to a final galactose concentration of 2%.
    4. Induce for 4-5 h.
    5. Spin down cells in 50 ml Falcon tubes at 3,000 rpm, wash pellet with water (keep cells on ice), transfer to 2 ml eppendorfs, wash with cold lysis buffer, spin and remove buffer. Freeze cell pellets at -80 °C.

  2. Protein pufication (everything on ice and done in cold room):
    1. Add ~ 400 μl of 0.5 mm Zirconia/Silica beads and 450 μl of lysis buffer containing protease and phosphatase inhibitors to cell pellet.
    2. Vortex the cells for 45 sec intervals with 2 min on ice for a total of 5 times.
    3. Spin tubes at 5,000 rpm for 4 min.
    4. Transfer lysate to fresh tubes on ice.
    5. Add another 450 μl lysis buffer containing protease and phosphatase inhibitors to cell pellet.
    6. Vortex again for another 5 times as above.
    7. Spin down the lysate and transfer to the previous lysate.
    8. Spin the combined lysate at 14,000 rpm for 10 min to clear lysate.
    9. Transfer lysate to fresh tubes being careful not to disturb cell junk at bottom.
      Note: At this point you can either do the binding on the glutathione beads in eppendorfs or in 15 ml Falcon tubes. Some claim that doing the binding step in a larger volume (by adding fresh lysis buffer to the lysate) helps the binding process by diluting inhibitors in the lysate that inhibit the binding process.
    10. Add 35 μl of glutathione beads (beads washed 4 times with lysis buffer and then diluted in lysis buffer to aid in transfer of beads uniformly to samples).
    11. Rock for 1 h in cold room.
    12. Wash beads 3 times with 400 μl of wash buffer I.
    13. Wash beads 3 times with 400 μl of wash buffer II.
    14. Elute proteins with 50 μl of elution buffer (rock for 30 min). Repeat for total elution of 100 μl.
    15. Aliquot elutions and freeze -80 °C.

Recipes

  1. Lysis buffer
    50 mM
    Tris (pH 7.5)
    100 mM
    NaCl
    1 mM
    EGTA
    0.1%
    TritonX-100
    0.1%
    BME
    0.5 mM
    PMSF
    Roche protease inhibitor tablets (containing EDTA)
    BME, PMSF, and inhibitor tablets are added freshly.
  2. Wash buffer I
    Exactly the same as lysis buffer except 500 mM NaCl
  3. Wash buffer II or your preferred kinase buffer
    50 mM
    HEPES (pH 7.5)
    100 mM
    NaCl
    10%
    glycerol
  4. Elution Buffer or your preferred kinase buffer with glycerol and glutathione:
    50 mM
    HEPES (pH 7.5)
    100 mM
    NaCl
    20%
    glycerol
    20 mM
    glutathione (reduced form)
    Make sure the pH is around 7.5.

References

  1. Poon, R. Y. and Hunt, T. (1994). Reversible immunoprecipitation using histidine- or glutathione S-transferase-tagged staphylococcal protein A. Anal Biochem 218(1): 26-33.
  2. Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean, R. A., Gerstein, M. and Snyder, M. (2001). Global analysis of protein activities using proteome chips. Science 293(5537): 2101-2105.
  3. Zhu, H., Klemic, J. F., Chang, S., Bertone, P., Casamayor, A., Klemic, K. G., Smith, D., Gerstein, M., Reed, M. A. and Snyder, M. (2000). Analysis of yeast protein kinases using protein chips. Nat Genet 26(3): 283-289.

简介

Gligation S转移酶(GST)标签是重组蛋白最常用的纯化策略。 它的开发目的是通过利用来自纯化基质的目标蛋白的温和洗脱条件来保持酶活性(Poon和Hunt,1994)。 本文所述的方法可以从单一蛋白质应用于来自酵母的重组蛋白质的蛋白质组规模纯化(Zhu et al。,2000; Zhu et al。,2001)。

材料和试剂

  1. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E0396)
  2. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  3. HEPES(Sigma-Aldrich,目录号:54457)
  4. Roche蛋白酶抑制剂片剂(含有EDTA)(Roche Diagnostics,目录号:11697498001)
  5. 磷酸酶抑制剂(Roche Diagnostics,目录号:4906845001)
  6. 谷胱甘肽(Axxora,目录号:157-002-G005)
  7. 半乳糖(Mp Biomedicals,目录号:0210174701)
  8. 氧化锆/硅石珠(Biospec Products,目录号:11079105z)
  9. 谷胱甘肽珠(Thermo Fisher Scientific,目录号:16100)
  10. Tris
  11. NaCl
  12. TritonX-100
  13. 甘油
  14. β-巯基乙醇(BME)
  15. Sc-ura液体培养基用棉子糖
  16. 裂解缓冲液(见配方)
  17. 洗涤缓冲液I(见配方)
  18. 洗涤缓冲液II(参见配方)
  19. 洗脱缓冲液(见配方)

设备

  1. 台式离心机
  2. 珠磨机
  3. 50ml Falcon管

程序

  1. 菌株在sc-ura平板上生长
    1. 开始一个5毫升sc-ura或sc-ura /棉子糖的发酵剂培养物(我喜欢 只是做高压灭菌的sc-ura /棉子糖)的一切,并生长O/N或16小时 在30℃
    2. 测量起始培养物和稀释培养物的OD 600 加入到50ml新鲜的sc-ura /棉子糖中至允许至少2的OD 细胞分裂。 我通常接种到OD为0.1并允许生长 到诱导前的OD 0.6-0.8(通常需要大约3小时/ 这些菌株的倍增时间)。
    3. 当细胞OD为0.6-0.8,最终半乳糖浓度为2%时,用20%半乳糖诱导
    4. 诱导4-5小时。
    5. 旋转细胞在50ml Falcon管在3,000 rpm,洗涤沉淀   水(保持细胞在冰上),转移到2毫升eppendorfs,用冷洗 裂解缓冲液,旋转和去除缓冲液。 在-80℃下冷冻细胞沉淀。

  2. 蛋白质纯化(冰上的一切,在寒冷的室内完成):
    1. 加入〜400μl的0.5mm氧化锆/二氧化硅珠和450μl裂解缓冲液 含有蛋白酶和磷酸酶抑制剂到细胞沉淀
    2. 涡旋细胞45秒间隔在冰上2分钟共5次。
    3. 旋转管以5000rpm离心4分钟
    4. 将溶胞产物转移到冰上的新鲜管中
    5. 向细胞沉淀中加入另外450μl含蛋白酶和磷酸酶抑制剂的裂解缓冲液
    6. 再次涡旋5次,如上所述。
    7. 旋转裂解物并转移到先前的裂解物
    8. 将混合的裂解物以14,000rpm旋转10分钟以澄清裂解物
    9. 将裂解液转移到新鲜管中,小心不要弄乱底部的细胞沉淀 注意:此时您可以在谷胱甘肽上进行结合 珠在eppendorfs或在15ml Falcon管中。 有人声称做 结合步骤在更大的体积(通过加入新鲜的裂解缓冲液 裂解物)通过稀释裂解物中的抑制剂来帮助结合过程 抑制绑定过程。
    10. 加入35微升谷胱甘肽珠   (珠用裂解缓冲液洗涤4次,然后在裂解中稀释 缓冲剂以帮助珠均匀地转移到样品)
    11. 在寒冷的房间里摇曳1小时。
    12. 用400μl洗涤缓冲液I洗涤珠子3次。
    13. 用400μl洗涤缓冲液II洗珠子3次
    14. 用50μl洗脱缓冲液(摇动30分钟)洗脱蛋白质。 重复,总洗脱100μl。
    15. 等分试样洗脱并冷冻-80℃。

食谱

  1. 裂解缓冲液
    50 mM
    Tris(pH 7.5)
    100 mM
    NaCl
    1 mM
    EGTA
    0.1%
    TritonX-100
    0.1%
    BME
    0.5 mM
    PMSF
    罗氏蛋白酶抑制剂片剂(含EDTA)
    新鲜加入BME,PMSF和抑制剂片剂
  2. 洗涤缓冲液I
    与除500mM NaCl之外的裂解缓冲液完全相同
  3. 洗涤缓冲液II或您首选的激酶缓冲液
    50 mM
    HEPES(pH7.5)
    100 mM
    NaCl
    10%
    甘油
  4. 洗脱缓冲液或您喜欢的甘油和谷胱甘肽激酶缓冲液:
    50 mM
    HEPES(pH7.5)
    100 mM
    NaCl
    20%
    甘油
    20 mM
    谷胱甘肽(还原形式)
    确保pH值为7.5左右。

参考文献

  1. Poon,R.Y。和Hunt,T。(1994)。 使用组氨酸或谷胱甘肽S-转移酶标记的葡萄球菌蛋白A的可逆免疫沉淀。 em> Anal Biochem 218(1):26-33
  2. Zhu,H.,Bilgin,M.,Bangham,R.,Hall,D.,Casamayor,A.,Bertone,P.,Lan,N.,Jansen,R.,Bidlingmaier,S.,Houfek, Mitchell,T.,Miller,P.,Dean,RA,Gerstein,M。和Snyder,M。(2001)。 使用蛋白质组芯片进行蛋白质活动的全球分析 科学 293(5537):2101-2105
  3. Zhu,H.,Klemic,J.F.,Chang,S.,Bertone,P.,Casamayor,A.,Klemic,K.G.,Smith,D.,Gerstein,M.,Reed,M.A.and Snyder,M。(2000)。 使用蛋白质芯片分析酵母蛋白激酶 Nat Genet 26(3):283-289。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Im, H. (2011). GST-tagged Yeast Protein Purification. Bio-protocol Bio101: e141. DOI: 10.21769/BioProtoc.141;
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May I ask you the recipe for preparing sc-ura/raffinose? Thanks
7/13/2012 7:10:24 AM Reply
I have a question. What exactly is a bead beater? And where in the procedure would I use this? And if I don't have one is there something that does relatively the same thing? Would a vortex work?
1/31/2012 3:07:50 AM Reply
Hogune Im
Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical Schoo, USA

It's a machine that holds the tube and shakes; almost like a vortexes. There are various types which can handle one or many tubes at once. Vortexer also has head accessory parts that does the similar job. However, in our hands bead beater was more effective for cell lysis.

1/31/2012 11:20:42 AM