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Serial Immunoprecipitation of 3xFLAG/V5-tagged Yeast Proteins to Identify Specific Interactions with Chaperone Proteins
连续免疫沉淀3xFLAG/V5标记的酵母蛋白以鉴定特异性互作伴侣蛋白   

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Abstract

This method was generated to isolate high affinity protein complexes from yeast lysate by performing serial affinity purification of doubly tagged 3xFLAG/V5 proteins. First, the bait protein of interest is immunoprecipitated by anti-FLAG-conjugated magnetic beads and gently eluted by 3xFLAG antigen peptide. Next, the bait protein is recaptured from the first eluate by anti-V5-conjugated magnetic beads and eluted with ionic detergent. In this manner, the majority of abundant, nonspecific proteins remain either bound to the first beads or in the first eluate, allowing pure, high affinity complexes to be obtained. This approach can be used to show specific interactions with notoriously ‘sticky’ chaperone proteins.

Keywords: Immunoprecipitation(免疫沉淀), Yeast(酵母), FLAG tag(FLAG标签), V5 tag(V5标签), Protein complexes(蛋白复合物)

Background

Immunoprecipitation followed by mass spectrometry (IP/MS) is an unbiased method to identify protein-protein interactions with a specific bait protein of interest. While this approach has been fruitfully applied to identify protein interaction networks, it is plagued by false positives–proteins that appear to interact but are actually non-specifically bound to the beads or antibodies used in the affinity purification. In particular, highly abundant proteins such as ribosomal proteins, metabolic enzymes and chaperone proteins are common contaminants. However, sometimes these common contaminants may be bona fide interaction partners, yet it is challenging to demonstrate specificity. To overcome this obstacle, we developed a serial affinity purification approach to isolate specific, high affinity complexes between bait proteins of interest tagged with two affinity epitopes–the 3xFLAG and V5 tags (Figure 1). We have generated a plasmid containing the 3xFLAG-V5 epitopes and a HIS3 selectable marker that can be amplified and used to C-terminally tag any yeast protein of interest in a single yeast transformation (Zheng et al., 2016). We originally applied our method to demonstrate a specific interaction between the heat shock transcription factor (Hsf1) and the major Hsp70 chaperone proteins present in the yeast cytosol, Ssa1/2. However, this approach can be generally applied to identify high affinity complexes involving a protein of interest. While this technique removes the bulk of false positive interactions, a major caveat is that low affinity and transient interactions are likely to be lost.


Figure 1. Schematic overview of the protocol. A 3xFLAG/V5 dual-tagged bait protein is serially purified with anti-FLAG beads followed by anti-V5 beads.

Materials and Reagents

  1. Pipette tips
  2. Glass culture tubes (20 x 150 mm) (Sigma-Aldrich, catalog number: C1048 )
  3. 50 ml Falcon tubes
  4. 1.5 ml microcentrifuge tubes
  5. Saccharomyces cerevisiae (W303 background) with 3xFLAG-V5 tagged bait protein of interest (Hsf1-3xFLAG/V5 in this protocol–Pincus lab strain DPY118: MATa ADE2 leu2-3,112 can1-100 ura3-1 his3-11,15 hsf1∆::KAN HSF1-3xFLAG/V5::TRP1)
  6. 3xFLAG peptide (Sigma-Aldrich, catalog number: F4799 )
  7. Liquid nitrogen
  8. Dry ice pellets
  9. Anti-FLAG magnetic beads (Sigma-Aldrich, catalog number: M8823 )
  10. Anti-V5 magnetic beads (MBL International, catalog number: M167-11 )
  11. 2% SDS
  12. Yeast extract (RPI, catalog number: Y20020 )
  13. Peptone (RPI, catalog number: P20240 )
  14. Glucose (Sigma-Aldrich, catalog number: G8270 )
  15. Dextrose (Sigma-Aldrich, catalog number: D9434 )
  16. HEPES (Sigma-Aldrich, catalog number: H3375 )
  17. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  18. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  19. Deoxycholate (Sigma-Aldrich, catalog number: D6750 )
  20. Ethylenediaminetetraacetate acid (EDTA) (Sigma-Aldrich, catalog number: EDS )
  21. cOmplete Mini EDTA-free protease inhibitor (Roche Molecular Systems, catalog number: 11836170001 )
  22. YPD media (see Recipes)
  23. IP lysis buffer (see Recipes)

Equipment

  1. Pipettes
  2. Incubator shaker (e.g., Eppendorf, New BrunswickTM, model: Innova® 44 , catalog number: M1282-0004)
  3. 500 ml flask
  4. Spectrophotometer (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: OrionTM AquaMate 7000 , catalog number: AQ7000)
  5. Centrifuge for 50 ml Falcon tubes (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM LegendTM XT , catalog number: 75004505)
  6. Cryogenic tissue grinder (Bio Spec Products, catalog number: CTG111 )
  7. 200 ml glass beaker
  8. Intelli-mixer rotating mixer (Labscoop, catalog number: EL-RM2L )
  9. Magnetic separation rack (New England Biolabs, catalog number: S1506S )
  10. Thermomixer (Eppendorf, model: ThermoMixer® C , catalog number: 5382000023)
  11. Vortex mixer (VWR, catalog number: 97043-562 )

Procedure

  1. Inoculate the yeast strain harboring Hsf1-FLAG-V5 in a culture tube containing 3 ml YPD liquid media (see Recipes) and grow overnight at 30 °C in a shaker set at 200 rpm.
  2. Dilute 2 ml overnight grown culture into the 500 ml flask containing 100 ml YPD media, and grow to OD600 = 0.5-0.8 OD at 30 °C in a shaker set at 200 rpm (~4 h).
  3. Pour the growth media into two 50 ml Falcon tubes, and centrifuge at 4,000 x g for 4 min. Discard the media and submerge the Falcon tubes containing the yeast pellets in liquid nitrogen. The pellets can be stored at -80 °C for up to 3 months.
  4. Add dry ice pellets into a cryogenic tissue grinder pre-chilled at 4 °C (a simple coffee grinder will work as well) and grind it into powder that covers the blades. The dry ice allows the cells to be lysed while remaining frozen. Dry ice should be handled with care and never confined in an airtight compartment.
  5. Add the yeast pellets to the crushed dry ice and grind the yeast pellet with the dry ice for 30 sec.
  6. Repeat the grinding 5 more times for a total of 6 grindings. Add more dry ice pellets as needed to keep the blades fully covered with dry ice.
  7. Transfer the pulverized cells/dry ice powder into beaker, and sublimate/evaporate dry ice at room temperature.
  8. Add 1 ml lysis buffer and incubate the lysate at 4 °C for 5 min with intermittent swirling to resuspend.
  9. Transfer the lysate into a 1.5 ml centrifuge tube, and centrifuge at 20,000 x g for 10 min. Reserve the supernatant (cleared lysate). Take a 10 µl sample of the cleared lysate for analysis by Western blot (input sample).
  10. Add 25 µl anti-FLAG magnetic beads to a 1.5 ml tube. Place the tube on a magnetic separation rack and remove the buffer. Wash with 200 µl lysis buffer (see Recipes) on the magnetic rack and discard the wash.
  11. Pipet the cleared lysate into the 1.5 ml tube containing the anti-FLAG beads and incubate at 4 °C for 2 h on an inversion rotating mixer.
  12. Place the tube on the magnetic separation rack. When the solution clears (~2 min), take a 10 µl sample for analysis by Western blot (unbound sample).
  13. Discard the unbound fraction and wash the beads 3 times with 500 µl lysis buffer. Gently vortex the beads with each wash step and incubate on ice for 5 min. Return to the magnetic separation rack and discard washes.
  14. Elute the Hsf1-FLAG-V5 complex with 500 µl lysis buffer containing 100 µg/ml 3xFLAG peptide. Incubate on ice for 30 min. Repeat for a total of 2 elutions, and pool the eluate fractions.
  15. Add 25 µl anti-V5 magnetic beads to a 1.5 ml tube. Place the tube on a magnetic separation rack and remove the buffer. Wash with 200 µl lysis buffer on the magnetic rack and discard the wash.
  16. Pipet the pooled 3xFLAG eluate into the tube with the V5 beads and incubate at 4 °C for 2 h on an inversion rotating mixer.
  17. Place the tube on the magnetic separation rack. Discard the unbound fraction and wash the beads 3 times with 500 µl lysis buffer. Vortex the beads at half-maximal setting for 5 sec with each wash step and incubate on ice for 5 min. Return to the magnetic separation rack and discard washes.
  18. Add 100 µl lysis buffer + 2% SDS. Incubate for 15 min at 95 °C. Place tube on the magnetic separation rack and reserve solution.
  19. Use 10 µl for analysis by Western blot (final eluate: this is 10x concentrated compared to input and unbound fraction).
  20. Submit remaining 90 µl samples to a mass spectrometry core facility (e.g., Whitehead Institute Proteomics Facility) for analysis as described (Zheng et al., 2016). Mass spectrometry is extremely sensitive to contamination by proteases, so wearing gloves, working quickly and keeping samples on ice prior to analysis is of paramount importance.

Data analysis

Mass spectrometry data should be analyzed by first ensuring that you observed your bait protein with reasonable coverage (> 25%). Putative interacting proteins should be validated by repeated experiments and alternative detection methods, such as Western blotting (Figure 2). For examples of peptide counts of a bait protein, binding partners and nonspecific contaminants, see Figure 1–source data 1 in Zheng et al., 2016.


Figure 2. Western blot of immuno-precipitated Hsf1-3xFLAG/V5. 3xFLAG/V5-tagged Hsf1 was serially purified with anti-FLAG and anti-V5 beads from cells under control (-) and heat shock conditions (+). Hsf1 is low abundance and cannot be easily detected in the input, but is enriched following immunoprecipitation. Hsf1 migrates slower in the gel under heat shock conditions due to phosphorylation (HS = heat shock).

Notes

Despite the stringency of the serial affinity protocol, background contaminants will still be observed in the mass spectrometry data. In general, the repertoire of highly abundant ribosomal proteins and metabolic enzymes that are nonspecific contaminants is not very reproducible, though a subset is always present. Thus, with enough replicates, most of these contaminants can be discarded.

Recipes

  1. YPD media
    1% yeast extract
    2% peptone
    2% glucose
    Autoclave before adding glucose or filter sterilize
  2. IP lysis buffer
    50 mM HEPES pH 8.0
    150 mM NaCl
    1% Triton X-100
    0.1% deoxycholate
    5 mM EDTA
    1x cOmplete Mini EDTA-free protease inhibitor

Acknowledgments

This protocol was adapted from our previous work (Zheng et al., 2016). This work was supported by a grant from the Office of the Director of the National Institutes of Health to D.P. (DP5 OD017941-01).

References

  1. Zheng, X., Krakowiak, J., Patel, N., Beyzavi, A., Ezike, J., Khalil, A. S. and Pincus, D. (2016). Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation. Elife 5.

简介

产生这种方法通过双重标记的3xFLAG / V5蛋白的连续亲和纯化来分离来自酵母裂解物的高亲和力蛋白质复合物。 首先,感兴趣的诱饵蛋白通过抗FLAG缀合的磁珠免疫沉淀,并用3xFLAG抗原肽轻轻洗脱。 接下来,通过抗V5共轭磁珠从第一洗脱液中回收诱饵蛋白质并用离子型洗涤剂洗脱。 以这种方式,大多数丰富的非特异性蛋白质仍然与第一珠粒或第一洗脱液结合,从而获得纯的高亲和性复合物。 这种方法可用于显示与臭名昭着的“粘滞”伴侣蛋白的特定相互作用。
【背景】通过质谱(IP / MS)进行免疫沉淀是鉴定与特定目标诱饵蛋白质的蛋白质 - 蛋白质相互作用的无偏见方法。虽然这种方法被有效地应用于鉴定蛋白质相互作用网络,但是它被假阳性困扰 - 似乎相互作用但实际上非特异性结合到亲和纯化中使用的珠粒或抗体的蛋白质。特别地,高度丰富的蛋白质如核糖体蛋白质,代谢酶和伴侣蛋白质是常见的污染物。然而,有时这些常见的污染物可能是真正的交互作用伙伴,但是展现特异性却具有挑战性。为了克服这个障碍,我们开发了一种串联亲和纯化方法,以分离两个亲和表位(3xFLAG和V5标签)标记的感兴趣的诱饵蛋白之间的特异性,高亲和力复合物(图1)。我们已经产生了含有3xFLAG-V5表位的质粒和HIS3选择性标记,其可以在单个酵母转化中被扩增并用于C末端标记目的酵母蛋白质(Zheng等,2016)。我们最初应用我们的方法来证明热休克转录因子(Hsf1)和存在于酵母胞质溶胶Ssa1 / 2中的主要Hsp70伴侣蛋白之间的特异性相互作用。然而,该方法通常可用于鉴定涉及感兴趣的蛋白质的高亲和力复合物。虽然这种技术消除了大量的假阳性相互作用,但主要的警告是低亲和力和瞬时相互作用可能会丢失。

关键字:免疫沉淀, 酵母, FLAG标签, V5标签, 蛋白复合物

材料和试剂

  1. 移液器提示
  2. 玻璃培养管(20×150mm)(Sigma-Aldrich,目录号:C1048)
  3. 50ml Falcon管
  4. 1.5 ml微量离心管
  5. 具有3xFLAG-V5标记的感兴趣的饵料蛋白(W303背景)(本方案中的Hsf1-3xFLAG/V5) - Pincus实验室菌株DPY118:MATa ADE2 leu2-3,112 can1-100 ura3- 1 his3-11,15hsf1Δ:: KAN HSF1-3xFLAG/V5 :: TRP1 )
  6. 3xFLAG肽(Sigma-Aldrich,目录号:F4799)
  7. 液氮
  8. 干冰球粒
  9. 抗FLAG磁珠(Sigma-Aldrich,目录号:M8823)
  10. 抗V5磁珠(MBL International,目录号:M167-11)
  11. 2%SDS
  12. 酵母提取物(RPI,目录号:Y20020)
  13. 胨(RPI,目录号:P20240)
  14. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  15. 葡萄糖(Sigma-Aldrich,目录号:D9434)
  16. HEPES(Sigma-Aldrich,目录号:H3375)
  17. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  18. Triton X-100(Sigma-Aldrich,目录号:X100)
  19. 脱氧胆酸盐(Sigma-Aldrich,目录号:D6750)
  20. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:EDS)
  21. cOmplete迷你EDTA无蛋白酶抑制剂(Roche Molecular Systems,目录号:11836170001)
  22. YPD媒体(见配方)
  23. IP裂解缓冲液(参见食谱)

设备

  1. 移液器
  2. 孵化器振动筛(例如,Eppendorf,New Brunswick TM ,型号:Innova 44,目录号:M1282-0004)
  3. 500毫升烧瓶
  4. 分光光度计(例如Thermo Fisher Scientific,Thermo Scientific TM,型号:Orion TM AquaMate 7000,目录号:AQ7000)
  5. 离心机用于50ml Falcon管(例如,Thermo Fisher Scientific,Thermo Scientific TM ,型号为:Sorvall TM Legend sup> XT,目录号:75004505)
  6. 低温组织研磨机(Bio Spec Products,目录号:CTG111)
  7. 200毫升玻璃烧杯
  8. 智能搅拌机旋转搅拌机(Labscoop,目录号:EL-RM2L)
  9. 磁分离架(New England Biolabs,目录号:S1506S)
  10. Thermomixer(Eppendorf,型号:ThermoMixer ®,目录号:5382000023)
  11. 涡街搅拌机(VWR,目录号:97043-562)

程序

  1. 将含有Hsf1-FLAG-V5的酵母菌株接种在含有3ml YPD液体培养基的培养管中(参见食谱),并在设定为200rpm的振荡器中在30℃下生长过夜。
  2. 将2ml过夜培养的培养物稀释到含有100ml YPD培养基的500ml烧瓶中,并在以200rpm(〜4h)设定的摇床中在30℃下生长至OD 600> 0.5-0.8OD, 。
  3. 将生长培养基倒入两个50ml的Falcon管中,并以4,000 x g离心4分钟。丢弃介质并将含有酵母颗粒的Falcon管浸入液氮中。颗粒可以在-80℃下储存长达3个月。
  4. 将干冰颗粒加入到在4℃预冷却的低温组织研磨机中(一个简单的咖啡研磨机也可以工作),并将其研磨成覆盖叶片的粉末。干冰允许细胞裂解,同时保持冷冻。干冰应小心处理,不要局限在气密舱内。
  5. 将酵母颗粒加入破碎的干冰中,用干冰研磨酵母沉淀30秒
  6. 重复研磨5次,共6次研磨。根据需要添加更多的干冰球,以保持刀片完全被干冰覆盖。
  7. 将粉碎的细胞/干冰粉末转移到烧杯中,并在室温下升华/蒸发干冰
  8. 加入1ml裂解缓冲液,并在4℃孵育裂解物5分钟,间歇旋转重新悬浮。
  9. 将裂解物转移到1.5ml离心管中,并以20,000×g离心10分钟。保留上清液(澄清裂解物)。取出10μl样品的清除裂解物,进行蛋白质印迹分析(输入样品)
  10. 将25μl抗FLAG磁珠加入到1.5ml管中。将管放在磁性分离架上并取出缓冲液。用磁力架上的200μl溶解缓冲液(参见食谱)洗涤,弃去洗涤物。
  11. 将清除的裂解物吸入含有抗FLAG珠粒的1.5ml管中,并在反转旋转混合器上在4℃下孵育2小时。
  12. 将管放在磁性分离架上。当溶液清除(〜2分钟)时,取10μl样品进行Western印迹分析(未结合的样品)。
  13. 弃去未结合的部分,用500μl裂解缓冲液洗涤珠珠3次。每个洗涤步骤轻轻涡旋珠子,并在冰上孵育5分钟。返回到磁性分离架并丢弃洗涤。
  14. 用含有100μg/ml 3xFLAG肽的500μl裂解缓冲液洗脱Hsf1-FLAG-V5复合物。在冰上孵育30分钟。重复2次洗脱,并汇集洗脱液。
  15. 将25μl抗V5磁珠加入到1.5ml管中。将管放在磁性分离架上并取出缓冲液。用磁力架上的200μl裂解缓冲液洗涤并弃去洗涤物。
  16. 将合并的3xFLAG洗脱液用V5珠子吸入管中,并在反转旋转搅拌器上在4℃下孵育2小时。
  17. 将管放在磁性分离架上。弃去未结合的部分,用500μl裂解缓冲液洗涤珠珠3次。在每个洗涤步骤中将珠子以半最大设置旋转5秒,并在冰上孵育5分钟。返回到磁性分离架并丢弃洗涤。
  18. 加入100μl裂解缓冲液+ 2%SDS。在95℃孵育15分钟。将管放在磁分离架上并保留溶液。
  19. 使用10μl进行Western印迹分析(最终洗脱液:与输入和未结合部分相比,浓度为10倍)。
  20. 将剩余的90μl样品提交到质谱核心设施(例如,Whitehead Institute Proteomics Facility)进行分析(Zheng等人,2016)。质谱对蛋白酶的污染非常敏感,所以佩戴手套,快速工作,并在分析前将样品保存在冰上是至关重要的。

数据分析

应首先分析质谱数据,确保您以合理的覆盖率(> 25%)观察您的诱饵蛋白质。推定的相互作用蛋白应通过重复实验和替代检测方法(如Western blotting)进行验证(图2)。关于诱饵蛋白质,结合配偶体和非特异性污染物的肽计数的实例,请参见Zheng等人,2016年的图1-源数据。


图2.免疫沉淀的Hsf1-3xFLAG/V5的Western印迹将3xFLAG/V5标记的Hsf1用来自控制( - )和热休克的细胞的抗FLAG和抗V5珠粒连续纯化条件(+)。 Hsf1是低丰度的,不能在输入中容易地检测到,但是在免疫沉淀后富集。由于磷酸化(HS =热休克),Hsf1在热休克条件下在凝胶中迁移较慢。

笔记

尽管连续亲和力方案的严格性,在质谱数据中仍然会观察到背景污染物。一般来说,高度丰富的核糖体蛋白质和非特异性污染物的代谢酶的谱系不是很可重复,尽管子集总是存在。因此,通过足够的重复,这些污染物中的大多数可以被丢弃。

食谱

  1. YPD媒体
    1%酵母提取物
    2%蛋白胨
    2%葡萄糖
    添加葡萄糖或过滤消毒之前高压灭菌
  2. IP裂解缓冲液
    50 mM HEPES pH 8.0
    150 mM NaCl
    1%Triton X-100
    0.1%脱氧胆酸盐
    5 mM EDTA
    1x完全迷你EDTA无蛋白酶抑制剂

致谢

这个协议是从我们以前的工作(Zheng等人,2016)改编而来的。这项工作得到了美国国家卫生研究院院长办公室给D.P.的资助。 (DP5 OD017941-01)。

参考

  1. Zheng,X.,Krakowiak,J.,Patel,N.,Beyzavi,A.,Ezike,J.,Khalil,AS和Pincus,D。(2016)。< a class ="ke-insertfile"href = "http://www.ncbi.nlm.nih.gov/pubmed/27831465"target ="_ blank">通过伴侣开关和磷酸化在热休克期间Hsf1的动态控制。 5。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Zheng and Pincus. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zheng, X. and Pincus, D. (2017). Serial Immunoprecipitation of 3xFLAG/V5-tagged Yeast Proteins to Identify Specific Interactions with Chaperone Proteins. Bio-protocol 7(12): e2348. DOI: 10.21769/BioProtoc.2348.
  2. Zheng, X., Krakowiak, J., Patel, N., Beyzavi, A., Ezike, J., Khalil, A. S. and Pincus, D. (2016). Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation. Elife 5.
提问与回复

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当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。