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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).

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

生物化学 > 蛋白质 > 分离和纯化
作者: Hogune Im
10/5/2011, 7984 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.141

[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).

[Abstract] 谷胱甘肽S 转移酶(GST)标记是最常用的重组蛋白的纯化方法。通过利用温和的洗脱条件以保护酶的活性,使靶蛋白高纯化(1)。这里介绍的方法可以适用于从单一的蛋白质,以及蛋白质组大规模纯化重组蛋白酵母(2,3)。

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.

材料与试剂

 

1.        Tris

2.        氯化钠

3.        EGTASigma公司E0396

4.        TritonX - 100

5.        β-巯基乙醇(BME

6.        PMSFSigma - Aldrich公司P7626

7.        罗氏公司的蛋白酶抑制剂片(含EDTA)(罗氏诊断11697498001

8.        HEPESSigma - Aldrich公司54457

9.        甘油

10.    谷胱甘肽(Axxora Llc 157 - 002 - G005

11.    Sc-ura  棉白糖的液体培养基

12.    半乳糖(MP生物医学公司0210174701

13.    氧化锆/硅珠(Biospec产品11079105z

14.    磷酸酶抑制剂(罗氏诊断4906845001

15.    谷胱甘肽珠(Thermo Scientific 16100

 

设备

 

1.        水平离心机

2.        搅拌器

 

程序

 

1.        菌株是生长在sc-ura板上。

1)      起初是用5 mlsc-ura 或者 sc-ura/raffinose培养(我喜欢使用高压灭菌的sc-ura/raffinose)起初培养生长在O / N3016个小时。

2)      测量起初培养OD600值,并用新鲜sc-ura/raffinose稀释成50ml,至少2盘细胞分裂培养。我通常接种到0.1 OD,并允许将增长到0.6- 0.8 OD 诱导前(这些菌株倍增时间,通常需要大约3小时)。

3)      20%半乳糖诱导细胞至0.6 - 0.8 OD,调节半乳糖终浓度2%。

4)      诱导为4-5小时。

5)      离心细胞在3000 RPM,用清水洗净沉淀(保持细胞在冰上),转移到2ml eppendorfs管,冷裂解液洗,旋转和去除缓冲液。-80oC冻存。

2.        蛋白质纯化(一切在冰上操作,并保持低温):

1)      新增400μl0.5mm的锆石/二氧化硅珠(#11079105z Biospec产品)和450μl含有蛋白酶和磷酸酶抑制剂的裂解缓冲液质细胞??沉淀中。

2)      将细胞在冰上震荡2分钟,每次间隔45秒,共5次。

3)      将管用5000转离心4分钟。

4)      在冰上将裂解液转到新鲜管中。

5)      另外添加450μl含有蛋白酶和磷酸酶抑制剂的裂解液至细胞沉淀。

6)      如上描述再次振荡5次。

7)      离心裂解产物并转移到上述的裂解产物中。

8)      14,000 10分钟的转速旋转相结合的裂解液,以清除裂解。

9)      小心转移裂解产物到新管中,注意不要搅乱管底部的细胞碎片。

注:在这一点上,你可以将谷胱甘肽珠绑定在eppendorfs或在15ml Falcon管中。有人认为在绑定步骤用一个体积较大的容器中(将新鲜的裂解缓冲液中加入裂解产物),有助于稀释结合过程中的裂解物抑制剂,抑制绑定过程。

10)  添加35μl谷胱甘肽珠(珠裂解液洗4次,然后在裂解缓冲液稀释,以帮助珠均匀转移到样品)

11)  在低温室内摇动1小时。

12)  400μl缓冲液洗I洗珠3

13)  400μl缓冲液洗II洗珠3

14)  50μl洗脱蛋白洗脱缓冲液(摇动30分钟)。重复共洗脱100μl

15)  分装并- 80℃保存。

 

配制溶液

 

1.        裂解缓冲液:

50 mM       Tris pH7.5

100 mM      氯化钠

1 mM        EGTA

0.1        TritonX - 100

0.1        β-巯基乙醇(BME

0.5 mM      PMSF

罗氏公司的蛋白酶抑制剂药片(含EDTA

加入新鲜的BMEPMSF和抑制剂药片。

2.        洗涤缓冲液I

完全与裂解缓冲液相同,除了添加500 mM NaCl

3.        洗涤缓冲液II或您的首选激酶缓冲液:

50 mM     HEPES pH7.5

100 mM    氯化钠

10%的甘油

4.        洗脱缓冲液,或您与甘油和谷胱甘肽的首选激酶缓冲液:

50 mM   HEPES pH7.5

100 mM  氯化钠

20     甘油

20 mM    谷胱甘肽(简化形式)

确保pH值约7.5

 

参考文献

 

1.         Poon R.Y., Hunt T. (1994). Reversible immunoprecipitation using histidine- or glutathione S-transferase-tagged staphylococcal protein A. Analytical Biochemistry 218(1): 26-33. 

2.         Zhu H., Klemic J.F., Chang S., Bertone P., Casamayor A., Klemic K.G., Smith D., Gerstein M., Reed M.A., Snyder M. (2000). Analysis of yeast protein kinases using protein chips. Nature Genetics 26(3): 283-9. 

3.         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., Snyder M. (2001). Global analysis of protein activities using proteome chips. Science 293(5537): 2101-5.

 

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How to cite this protocol: Im, H. (2011). GST-tagged Yeast Protein Purification. Bio-protocol Bio101: e141. DOI: 10.21769/BioProtoc.141; Full Text



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7/13/2012 7:10:24 AM  

May I ask you the recipe for preparing sc-ura/raffinose? Thanks

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1/31/2012 3:07:50 AM  

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 11:20:42 AM  

Hogune Im (Author)
Stanford University

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.

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