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Probing Yeast Protein Microarrays for Protein-protein Interactions Using V5-epitope Tagged Fusion Protein Probes
通过V5-抗原表位标签的融合蛋白标记酵母蛋白芯片探究蛋白之间的相互作用   

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Abstract

Protein microarray is probably the only technique currently available for systematic investigation of protein-protein interactions. This protocol describes an optimized method to probe yeast protein microarrays for protein-protein interactions using purified V5-epitope tagged fusion protein. It should also apply to any other proteins with appropriate modifications.

Keywords: Kinase(激酶), Protein-protein interaction(蛋白质-蛋白质相互作用), Phosphorylation(磷酸化), Protein microarray(蛋白芯片), MAP Kinase(MAP激酶)

Materials and Reagents

  1. Zirconia beads (Biospec Products, catalog number: 11079110zx 1.0mm dia)
  2. His-tag affinity resin (His Spintrap, GE Life sciences, catalog number:
    28-4013-53 )
  3. Protein microarray (Life Technologies, catalog number: PAH0525101 )
  4. V5-AlexaFluor 647 antibody (Life Technologies, catalog number: 451098 )
  5. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  6. Potassium chloride
  7. Sodium chloride
  8. Triton X-100
  9. Dithiothreitol (DTT) (Promega, catalog number: P1170 )
  10. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626-250MG )
  11. Imidazole (Sigma-Aldrich, catalog number: I5513-5G )
  12. ATP (for kinases)
  13. BSA
  14. Tween-20
  15. Yeast extract
  16. Peptone
  17. Galactose
  18. Yeast nitrogen base
  19. (NH4)2SO4
  20. Raffinose (Sigma-Aldrich, catalog number: R0514-100G )
  21. Agar
  22. Lysis buffer (see Recipes)
  23. Wash buffer 1 (see Recipes)
  24. Wash buffer 2 (see Recipes)
  25. Wash buffer 3 (see Recipes)
  26. Elution buffer (see Recipes)
  27. Probe buffer (see Recipes)
  28. Blocking buffer (see Recipes)
  29. 3x YEP-GAL (see Recipes)
  30. Synthetic complete minus uracil media (Sc-ura) (see Recipes)

Equipment

  1. FastPrep Cell Lyser (MP Bio, catalog number: 116004500 - 1 each )
  2. High speed centrifuges
  3. JA-10 (or comparable) rotor
  4. Axon 4200 AL microarray reader
  5. 50 ml conical tube
  6. Wheel
  7. Humidified chamber

Procedure

  1. Probe protein purification
    1. Construct a V5-His6-protein fusion construct (i.e. pYES-DEST52, Life Technologies, Invitrogen™) of the yeast protein you wish to profile by using the methods outlined by Fasolo and Snyder (2009).
    2. Yeast (Y258 strain optimized for protein purification (Zhu et al., 2001)) that have been transformed with a pYES-DEST52 fusion construct under control of the GAL1 inducible promoter are grown in Sc-Ura/Dex overnight in 5 ml starter cultures.
    3. The next day OD600 are determined for the starter cultures and used to inoculate a 400 ml. Sc-Ura/Raffinose culture to an OD600 = 0.1. The culture is grown at 30 °C shaking to an OD600 = 0.55-0.6 and induced with 200 ml 3XYEP-GAL for 6 h at 30 °C shaking.
    4. Cell pellet is harvested by centrifugation using a JA-10 (or comparable) rotor by spinning 400 ml volumes of cell suspension at 4,000 rpm, 4 °C, 5 min.
    5. Wash cell pellet in ice cold PBS buffer by transferring the cell pellet to 50 ml conical tube and centrifuging in a tabletop centrifuge at 3,000 rpm, 4 °C, 5 min.
    6. Re-suspend cell pellet in ice cold PBS once more and aliquot evenly into 4 screw cap 2.0 ml. FASTPREP compatible tubes (pellet weight is ~0.5 g/tube).
    7. Spin cell suspension at 14,000 rpm, 4 °C, 1 min in a tabletop microfuge and aspirate supernatant. Snap freeze cell pellet immediately in liquid nitrogen and store at -80 °C or lyse immediately (you can stop here and proceed on the following day if not prepared for following steps).
    8. Add 0.5 mm zirconia beads, and lysis buffer at a 1:1:1 ratio to cell pellet and store on ice.
    9. Place tubes on FastPrep machine for 6 cycles of lysis at maximum setting for 1 min each, with 1 min on ice in between each interval.
    10. Spin suspension down in tabletop microcentrifuge at 14,000 rpm, 4 °C, 10 min, 10 min and collect supernatant.
    11. Repeat steps 9 and 10 then discard extracted pellet.
    12. Combine supernatant from both extractions into a single tube and incubate with Ni2+, or Co2+ affinity resin for 3 h/4 °C/rotating on a wheel.
    13. Centrifuge resin and save small aliquot of cleared supernatant for quality control.
    14. Wash resin 2x 10 min in wash buffer 1 /4 °C/rotating on a wheel.
    15. Wash resin 2x 10 min in wash buffer 2 /4 °C/rotating on a wheel.
    16. Wash resin 1x 10 min in wash buffer 3 /4 °C/rotating on a wheel.
    17. Collect washed resin in G25 or comparable column and elute with elution buffer and store at -80 °C until ready to use in probing assay (stop here is not ready to proceed).

  2. Protein microarray probing
    1. Dilute the purified protein probe over a concentration range of 5-500 μg/ml in the probe buffer (concentration of probe is empirically determined for each protein-protein interaction assay).
    2. Block the arrays in blocking buffer for 1 h by shaking at 50 rpm on a stage at 4 °C.
    3. After blocking, transfer the arrays to a humidified chamber, and add 90 μl of diluted probe directly to the array surface. Overlay the arrays with a raised lifter slip and incubate static (no shaking) in the humidor for 1.5 h.
    4. Wash the arrays 3 times for 1 min each in probe buffer in three 50-ml conical tubes.
    5. To detect interactions, dilute the V5-AlexaFluor 647 antibody to 260 ng/ml in probe buffer and mix thoroughly by shaking on a wheel for 30 min at 4 °C.
    6. After washing the arrays several times as indicated, add antibody solution directly to array and overlay with a raised lifter slip as before. Incubate the arrays for 30 min/static/4 °C.
    7. Finally, wash the arrays for 1 min (3x) in probe buffer, and spin in a 50-ml conical tube at 800 x g in a tabletop centrifuge for 5 min at RT. Air-dry the arrays in a slide holder in the dark for 30 min prior to scanning the array at 647 nm on an Axon 4200 AL microarray reader (or comparable).

Notes

  1. Protein microarrays used in this protocol are produced using full-length GST-fusion fusion proteins that were purified using glutathione sepharose beads. Protein arrays produced using proteins purified with antibody-conjugated matrices may result in high background when probed with anti-V5 antibody for detection due to the presence of residual IgG contamination. It is therefore important to test the background of each array with a negative control consisting of the detection antibody alone.
  2. Protein probe concentration must be high enough to dilute to working concentration in probe buffer without effecting solute concentration of probe buffer. This can be accomplished by scaling up culture to >10 L if necessary. Downstream applications like molecular weight cut-off columns may be used to exchange solutions and remove excess imidazole from final probe solution.
  3. Imidazole gradient in wash steps may be altered to optimize protein purification which can vary from protein to protein; protein probes must be at ~80-90% pure in order to which can be assessed by mass spectrometry and SDS-PAGE.

Recipes

  1. Lysis buffer
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    150 mM NaCl
    10% glycerol
    0.1% Triton X-100
    0.5 mM DTT
    1 mM PMSF
    1x complete protease inhibitor tablet (F. Hoffmann-La Roche)
  2. Wash buffer 1
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    150 mM NaCl
    50 mM Imidazole
    10% glycerol
    0.1% Triton X-100
  3. Wash buffer 2
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    150 mM NaCl
    100 mM Imidazole
    10% glycerol
    0.1% Triton X-100
  4. Wash buffer 3
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    150 mM NaCl
    150 mM Imidazole
    10% glycerol
    0.1% Triton X-100
  5. Elution buffer
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    0.1% Triton X-100
    500 mM NaCl
    0.5 mM DTT
    500 mM imidazole
    2 mM MgCl2
    25% glycerol (if freezing at -80 °C)
  6. Probe buffer
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    1% glycerol
    2 mM MgCl2
    0.5 mM DTT
    0.05% Triton X-100
    50 mM NaCl
    500 μM ATP (for kinases)
    1% BSA
  7. Blocking buffer
    PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride) (pH 7.4)
    1% BSA
    0.1% Tween-20
  8. 3x YEP-GAL (yeast extract–peptone–galactose)
    30 g yeast extract
    60 g peptone
    Make up to 700 ml with dH2O
    Add 300 ml of sterile filtered 20% galactose to media after autoclaving.
  9. Synthetic complete minus uracil media (Sc-ura)
    1.5 g yeast nitrogen base
    5 g (NH4)2SO4
    2 g Sc-ura drop out mix (commercially available)
    20 g raffinose (or dextrose for starter culture media and SD-ura plates)
    20 g agar (for plates)

Acknowledgments

This protocol was developed in the Snyder Lab, Department of Genetics, Stanford University, Stanford, CA, USA. It was adapted from Fasolo et al. (2011), and Fasolo and Snyder, (2009). This work was supported by the NIH.

References

  1. Fasolo, J., Sboner, A., Sun, M. G., Yu, H., Chen, R., Sharon, D., Kim, P. M., Gerstein, M. and Snyder, M. (2011). Diverse protein kinase interactions identified by protein microarrays reveal novel connections between cellular processes. Genes Dev 25(7): 767-778.
  2. Fasolo, J. and Snyder, M. (2009). Protein microarrays. Methods Mol Biol 548: 209-222.

简介

蛋白质微阵列可能是目前唯一可用于系统调查蛋白质 - 蛋白质相互作用的技术。 该协议描述了使用纯化的V5-表位标记的融合蛋白探测蛋白质 - 蛋白质相互作用的酵母蛋白质微阵列的优化方法。 它也应适用于具有适当修饰的任何其他蛋白质。

关键字:激酶, 蛋白质-蛋白质相互作用, 磷酸化, 蛋白芯片, MAP激酶

材料和试剂

  1. 氧化锆珠(Biospec Products,目录号:11079110zx 1.0mm直径)
  2. His标签亲和树脂(His Spintrap,GE Life sciences,目录号:
    28-4013-53)
  3. 蛋白质微阵列(Life Technologies,目录号:PAH0525101)
  4. V5-AlexaFluor 647抗体(Life Technologies,目录号:451098)
  5. 甘油(Sigma-Aldrich,目录号:G5516)
  6. 氯化钾
  7. 氯化钠
  8. Triton X-100
  9. 二硫苏糖醇(DTT)(Promega,目录号:P1170)
  10. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626-250MG)
  11. 咪唑(Sigma-Aldrich,目录号:I5513-5G)
  12. ATP(激酶)
  13. BSA
  14. 吐温-20
  15. 酵母提取物
  16. 蛋白胨
  17. 半乳糖
  18. 酵母氮基
  19. (NH 4) 2 4
  20. 棉子糖(Sigma-Aldrich,目录号:R0514-100G)
  21. Agar
  22. 裂解缓冲液(见配方)
  23. 洗涤缓冲液1(见配方)
  24. 洗涤缓冲液2(见配方)
  25. 洗涤缓冲液3(见配方)
  26. 洗脱缓冲液(见配方)
  27. 探针缓冲区(参见配方)
  28. 阻止缓冲区(参见配方)
  29. 3x YEP-GAL(请参阅配方)
  30. 合成完全减去尿嘧啶培养基(Sc-ura)(参见配方)

设备

  1. FastPrep Cell Lyser(MP Bio,目录号:116004500-1各自)
  2. 高速离心机
  3. JA-10(或类似的)转子
  4. Axon 4200 AL微阵列阅读器
  5. 50ml锥形管

  6. 加湿室

程序

  1. 探针蛋白纯化
    1. 使用Fasolo和Snyder(2009)概述的方法构建希望分布的酵母蛋白的V5-His6-蛋白融合构建体(即


      pYES-DEST52,Life Technologies, br />
    2. 用在GAL1诱导型启动子控制下的pYES-DEST52融合构建体转化的酵母(用于蛋白质纯化优化的Y258菌株(Zhu et al。,2001))在Sc-Ura/Dex在5ml起始培养物中过夜
    3. 第二天测定起始培养物的OD 600,并用于接种400ml。 Sc-Ura /棉子糖培养至OD 600 = 0.1。将培养物在30℃摇动下生长至OD 600 = 0.55-0.6,并在30℃摇动下用200ml 3XYEP-GAL诱导6小时。
    4. 使用JA-10(或相当的)转子通过在4,000rpm,4℃,5分钟下旋转400ml体积的细胞悬浮液来离心收获细胞沉淀。
    5. 通过将细胞沉淀转移到50ml锥形管中,并在台式离心机中以3,000rpm,4℃,5分钟离心,在冰冷的PBS缓冲液中洗涤细胞沉淀。
    6. 再次将冰冻PBS中的细胞沉淀再悬浮一次,并均匀地分装到2个螺旋盖2.0ml。 FASTPREP相容管(颗粒重量为〜0.5g /管)
    7. 旋转细胞悬浮在14,000 rpm,4°C,1分钟在桌面微量离心机和吸出上清液。快速冷冻细胞丸立即在液氮中,存储在-80°C或立即裂解(你可以 停在这里,如果没有准备好接下来的步骤,在第二天继续。)
    8. 将0.5mm氧化锆珠和裂解缓冲液以1:1:1的比例加入细胞沉淀中并储存在冰上。
    9. 将管置于FastPrep机器上,在最大设置下每次1分钟裂解6个循环,在每个间隔之间在冰上1分钟。
    10. 在桌面微量离心机中以14,000rpm,4℃,10分钟,10分钟旋转悬浮液并收集上清液。
    11. 重复步骤9和10,然后丢弃提取的颗粒
    12. 将来自两次提取的上清液合并到单个管中并与Ni 2+或Co 2+亲和树脂孵育3小时/4℃/在轮上旋转。 br />
    13. 离心树脂并保存少量等分的澄清上清液以进行质量控制
    14. 在洗涤缓冲液1/4℃/在轮上旋转,洗涤树脂2x 10分钟
    15. 在洗涤缓冲液2/4℃/在轮上旋转,洗涤树脂2x 10分钟
    16. 在洗涤缓冲液3/4℃/在轮上旋转,洗涤树脂1x 10分钟
    17. 收集洗涤的树脂在G25或类似的列和洗脱缓冲液洗脱,存储在-80°C,直到准备用于探测测定(停止这里没有准备进行)。

  2. 蛋白质微阵列探测
    1. 将纯化的蛋白质探针在探针缓冲液中浓度范围为5-500μg/ml(通过每种蛋白质 - 蛋白质相互作用测定经验确定探针的浓度)稀释。
    2. 在封闭缓冲液封闭阵列1小时,通过在4℃下在舞台上以50rpm摇动
    3. 封闭后,将阵列转移到加湿室,并将90μl稀释的探针直接添加到阵列表面。用升高的提升滑块覆盖阵列,并在加湿器中静止(无振荡)1.5小时
    4. 在三个50-ml锥形管中的探针缓冲液中洗涤阵列3次,每次1分钟
    5. 为了检测相互作用,将V5-AlexaFluor 647抗体在探针缓冲液中稀释至260ng/ml,并通过在4℃下在轮上摇动30分钟充分混合。
    6. 在如所指示的将阵列洗涤几次后,将抗体溶液直接加入阵列,并如前所述用升高的升降机漏片覆盖。孵育阵列30分钟/静态/4℃
    7. 最后,在探针缓冲液中洗涤阵列1分钟(3x),并在台式离心机中在800xg的锥形管中在室温下旋转5分钟。在黑暗中将阵列在玻片保持器中空气干燥30分钟,然后在Axon 4200AL微阵列阅读器(或可比较的)上以647nm扫描阵列。

笔记

  1. 本方案中使用的蛋白质微阵列使用全长GST-融合融合蛋白产生,其使用谷胱甘肽琼脂糖珠纯化。使用用抗体缀合的基质纯化的蛋白质产生的蛋白质阵列可能由于存在残留的IgG污染而在用抗V5抗体检测时导致高背景。因此,重要的是用仅由检测抗体组成的阴性对照测试每个阵列的背景
  2. 蛋白质探针浓度必须足够高以在探针缓冲液中稀释至工作浓度,而不影响探针缓冲液的溶质浓度。如果需要,这可以通过将培养物放大至> 10L来实现。下游应用如分子量截留柱可用于交换溶液并从最终探针溶液中除去过量的咪唑
  3. 可改变洗涤步骤中的咪唑梯度以优化蛋白质纯化,其可随蛋白质而变化; 蛋白质探针必须为〜80-90%纯度,以便可以通过质谱法和SDS-PAGE进行评估

食谱

  1. 裂解缓冲液
    PBS(0.01M磷酸盐缓冲液,0.0027M氯化钾,0.137M氯化钠)(pH7.4) 150mM NaCl 10%甘油 0.1%Triton X-100 0.5 mM DTT
    1mM PMSF
    1x完全蛋白酶抑制剂片剂(F.Hoffmann-La Roche)
  2. 洗涤缓冲液1
    PBS(0.01M磷酸盐缓冲液,0.0027M氯化钾,0.137M氯化钠)(pH7.4) 150mM NaCl 50mM咪唑 10%甘油 0.1%Triton X-100
  3. 洗涤缓冲液2
    PBS(0.01M磷酸盐缓冲液,0.0027M氯化钾,0.137M氯化钠)(pH7.4) 150mM NaCl 100mM咪唑 10%甘油 0.1%Triton X-100
  4. 洗涤缓冲液3
    PBS(0.01M磷酸盐缓冲液,0.0027M氯化钾,0.137M氯化钠)(pH7.4) 150mM NaCl 150mM咪唑 10%甘油 0.1%Triton X-100
  5. 洗脱缓冲液
    PBS(0.01M磷酸盐缓冲液,0.0027M氯化钾,0.137M氯化钠)(pH7.4) 0.1%Triton X-100 500 mM NaCl
    0.5 mM DTT
    500mM咪唑 2mM MgCl 2/
    25%甘油(如果在-80℃冷冻)
  6. 探测缓冲区
    PBS(0.01M磷酸盐缓冲液,0.0027M氯化钾,0.137M氯化钠)(pH7.4) 1%甘油 2mM MgCl 2/
    0.5 mM DTT
    0.05%Triton X-100 50mM NaCl 500μMATP(激酶)
    1%BSA
  7. 阻塞缓冲区
    PBS(0.01M磷酸盐缓冲液,0.0027M氯化钾,0.137M氯化钠)(pH7.4) 1%BSA
    0.1%Tween-20
  8. 3x YEP-GAL(酵母提取物 - 蛋白胨 - 半乳糖)
    30克酵母提取物
    60 g蛋白胨
    用dH 2 O/
    补充至700ml 高压灭菌后,向培养基中加入300ml无菌过滤的20%半乳糖
  9. 合成完全减去尿嘧啶培养基(Sc-ura)
    1.5g酵母氮源
    5g(NH 4)2 SO 4 4
    2g Sc-ura drop out混合物(市售)
    20g棉子糖(或用于起始培养基和SD-ura平板的右旋糖) 20克琼脂(平板)

致谢

这个协议是在斯奈德大学斯坦福大学斯坦德实验室,美国加利福尼亚州的遗传学系开发的。 它从Fasolo等人(2011)和Fasolo和Snyder(2009)改编而来。 这项工作得到了NIH的支持。

参考文献

  1. Fasolo,J.,Sboner,A.,Sun,M.G.,Yu,H.,Chen,R.,Sharon,D.,Kim,P.M.,Gerstein,M.and Snyder, 由蛋白质微阵列鉴定的不同蛋白激酶相互作用揭示了细胞过程之间的新型连接。 Genes Dev 25(7):767-778。
  2. Fasolo,J。和Snyder,M。(2009)。 Protein microarrays。 Methods Mol Biol 548:209-
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fasolo, J. and Snyder, M. (2012). Probing Yeast Protein Microarrays for Protein-protein Interactions Using V5-epitope Tagged Fusion Protein Probes. Bio-protocol 2(5): e123. DOI: 10.21769/BioProtoc.123.
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