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In vitro Detection of S-acylation on Recombinant Proteins via the Biotin-Switch Technique
通过生物素转换技术对重组蛋白中的S酰化作用进行体外检测   

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Abstract

Protein palmitoylation is the post-translational modification of proteins via the attachment of palmitate through acyl linkages. The nucleophile sulfhydryl group of cysteines is the common palmitoylation site. Covalent attachment of palmitate occurs on numerous proteins and is usually associated with directing protein localization to the endomembrane system. Detection of protein palmitoylation by in vivo labeling with tritium-labeled palmitic acid typically requires an autoradiographic exposure time of several months, and, thus is not suitable for rapid analyses. Here, we described an easy protocol for quick in vitro detection of protein S-acylation using the Arabidopsis protein kinase, PBS1, as an example. To determine whether PBS1 is modified through thioester linkage to acyl groups, we employed a “biotin switch” assay (Hemsley et al., 2008). This work was first published in Qi et al. (2014), but we expand on the method here. PBS1 functions within the basal immune system of plants, and is a target of the bacterial cysteine protease, AvrPphB (Shao et al., 2002; Zhang et al., 2010). It contains a predicted N-terminal S-acylation motif (MGCFSCFDS), with both Cys-3 and Cys-6 residues predicted to be palmitoylated by CSS-Palm 3.0 (http://csspalm.biocuckoo.org/; Ren et al., 2008). Our method utilizes hydroxylamine-induced cleavage of thioester bonds, which results in free sulfhydryl groups that can then be conjugated to a biotin derivative, 1-biotinamido-4-[4′-(maleimidomethyl) cyclohexanecarboxamido]-butane (Biotin-BMCC). The conjugates are detectable by Western blot with streptavidin-horseradish peroxidase. The whole process of in vitro labelling and detection took less than 3 days, allowing the fast detection of protein modifications via thioester bonds such as palmitoylation.

Materials and Reagents

  1. Nicotiana benthamiana (N. benthamiana) plants
  2. Agrobacterium tumefaciens strain GV3101 (pMP90)
  3. Bacto yeast extract (BD Biosciences, catalog number: 288620 )
  4. Bacto tryptone (BD Biosciences, catalog number: 211699 )
  5. Magnesium chloride hexahydrate (EMD Millipore, catalog number: 442611 )
  6. Acetosyringone (Sigma-Aldrich, catalog number: D134406-5G )
  7. Dexamethasone (Sigma-Aldrich, D4902-1G )
  8. Trizma Tris base (Sigma-Aldrich, catalog number: 93362 )
  9. Sodium chloride (EMD Millipore, catalog number: 567442 )
  10. Nonidet P-40(Sigma-Aldrich, catalog number: 21-3277 )
  11. Plant proteinase inhibitor cocktail (Sigma-Aldrich, catalog number: P9599-5M )
  12. SDS (Sigma-Aldrich, catalog number: L3771-100G )
  13. Glycerol (EMD Millipore, catalog number: 356350 )
  14. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148-25ML )
  15. EDTA (Sigma-Aldrich, catalog number: E6758-100G )
  16. BSA (Sigma-Aldrich, catalog number: A4503-100G )
  17. Bromphenol blue (EMD Millipore, catalog number: BX1410 )
  18. Anti-HA monoclonal antibody matrix (Roche Diagnostics, catalog number: 11867423001 )
  19. Tris-HEPES-SDS polyacrylamide gels (Thermo Fisher Scientific, catalog number: 25204 )
  20. Anti-HA peroxidase (Sigma-Aldrich, catalog number: H6533-1VL )
  21. High Sensitivity Streptavidin-HRP Conjugate (Thermo Fisher Scientific, catalog number: 21130 )
  22. ImmunoStar HRP Substrate Kit (Bio-Rad Laboratories, catalog number: 170-5070 )
  23. N-ethylmaleimide (Sigma-Aldrich, catalog number: E3876-5G )
  24. Hydroxylamine (Sigma-Aldrich, catalog number: 467804-10ML )
  25. 1-biotinamido-4-[4′(maleimidomethyl)-cyclohexane-carboxamido]-butane (EZ-link BMCC-Biotin) (Thermo Fisher Scientific, catalog number: 21900 )
  26. Nitrocellulose Membrane (Thermo Fisher Scientific, catalog number: WP4HY00010 )
  27. Metro-Mix 360 (Sun Gro Horticulture Canada)
  28. LB liquid medium (see Recipes)
  29. Lysis buffer (see Recipes)
  30. 4x SDS loading buffer (see Recipes)
  31. 5x nonreducing protein sample buffer (see Recipes)

Equipment

  1. Confocal microscope system (Leica Microsystem, model: TCS SP5 )
  2. Mini-Protean Electrophoresis and Blotting system (Bio-Rad Laboratories)
  3. Benchtop Centrifuge 5424 (Eppendorf)
  4. Lab tube rotator (Thermo Fisher Scientific)
  5. Gene Pulser (Bio-Rad Laboratories, catalog number: 1652076 )
  6. 1 ml syringe (BD Biosciences, catalog number: 305945 )

Procedure

  1. Transient expression assays in N. benthamiana
    Transient expression assays were performed as described previously (DeYoung et al., 2012). Briefly:
    1. N. benthamiana plants were grown in small plastic pots filled with Metro-Mix 360 under an 8/16 h photoperiod at 150 μmol/m/s and 23 °C.
    2. Dexamethasone-inducible constructs of N20sYFP:HA and n20sYFP:HA were mobilized into Agrobacterium tumefaciens strain GV3101 (pMP90) by electroporation.
    3. After overnight culture in Luria-Bertani liquid medium, cells (7 ml culture) were pelleted and resuspended in 10 mM MgCl2 solution with 100 μm acetosyringone and suspensions were diluted to OD600 = 0.3 before infiltration.
    4. The cells were incubated for 2 h at room temperature and infiltrated with a needleless 1 ml syringe into leaves of 4-week-old (best age for transient expression) N. benthamiana plants. A minimum of six fully expanded leaves should be infiltrated for one experiment.
    5. Transgenes were induced by spraying leaves with 50 µM dexamethasone 40 h after inoculation. Samples were harvested for protein extraction 4 to 6 h after dexamethasone application.
    6. Laser scanning confocal microscopy was performed at 5 h after dexamethasone application to confirm the expression of sYFP fusions. A 1 cm x 1 cm tissue was clipped from the infiltrated leaf area and placed in a drop of ddH2O in the center of a slide. After a cover slip was placed, the slide was examined using the Leica SP5 system.

  2. Immunoprecipitation and Immunoblotting
    1. For total protein extraction, six infiltrated leaves were ground in lysis buffer. Tissues can be flash-frozen in liquid nitrogen and stored at -80 °C till needed.
    2. Homogenates were centrifuged twice at 13,000 rpm at 4 °C for 10 min, and supernatants were transferred to new tubes.
    3. 100 μl an anti-HA monoclonal antibody matrix was added into each homogenate. The mixture was then incubated at 4 °C overnight and rotated end-to-end.
    4. The resin was pelleted by centrifuging at 16,000 x g for 10 sec and washed three times with 1 ml lysis buffer.
    5. For immuno-blot, the immuno-precipitates were mixed with 4x SDS loading buffer at a ratio of 3:1 and boiled for 10 min before resolving on 4% to 20% (w/v) gradient Tris-HEPES-SDS PAGE.
    6. Protein samples were then transferred to a nitrocellulose membrane for probing with anti-HA peroxidase.
    7. The ImmunoStar HRP Substrate Kit was used for detecting antibody complexes.

  3. Detection of S-Acylation
    The in vitro detection of PBS1 S-acylation was performed as described by Berzat et al. (2005). Briefly:
    1. The immuno-precipitates of N20-sYFP:HA or n20-sYFP:HA were incubated in 1 ml lysis buffer with 50 mM N-ethylmaleimide on an end-to-end rotator for 48 h at 4 °C to block free sulfhydryl groups.
    2. The immuno-precipitates were then washed, incubated in 500 μl 1 M hydroxylamine solution on an end-to-end rotator for 1 h at room temperature to hydrolyze Cys-palmitate thioester bonds.
    3. The immuno-precipitates were washed and incubated in 500 μl 50 mM Tris (pH 7.0) solution containing 1 μm EZ-Link Biotin-BMCC on an end-to-end rotator for 2 h at room temperature to label cleaved thioester bonds.
    4. The immuno-precipitates were washed, resuspended in nonreducing protein sample buffer, resolved on 4% to 20% (w/v) gradient Tris-HEPES-SDS PAGE.
    5. The protein samples were transferred to a nitrocellulose membrane.
    6. The membrane was blocked in 5% biotin-free BSA TBST solution for 1 h and then probed with streptavidin-horseradish peroxidase.
    7. The ImmunoStar HRP Substrate Kit was used for detecting biotin-streptavidin complexes.

Representative data

Representative data are shown in Figure 1. This experiment was repeated three times using this protocol with similar results.


Figure 1. S-Acylation mediates PBS1 localization to the PM. A. The predicted N-terminal S-acylation motif in PBS1 is required for plasma membrane localization. N20:sYFP indicates fusion of the first 20 amino acids of PBS1, which contains the predicted palmitoylation motif, to the super yellow fluorescent protein. n20 is the fragment with the G2A/C3AC/6A triple mutations. Both fusion proteins were transiently expressed in Nicotiana benthamiana. Confocal microscopy was performed at 5 h post dexamethasone induction. All images are three-dimensional projections from a Z-stack. The N20:sYFP localized to the plasma membrane and mobile vesicle-like structures were observed, which was reported when acylated proteins were overexpressed (Vilas et al., 2006; Joensuu et al., 2010). However, the n20:sYFP fusion was detected exclusively as cytoplasmic strands. The YFP signal was showed in the false color of green. B. PBS1 is S-acylated. Cell extracts of N. benthamiana tissue expressing N20:sYFP-HA or n20:sYFP-HA were subjected to immunoprecipitation using anti-HA matrix (IP). Immunoprecipitates were subjected to immuno-blot with anti-HA antibodies (IB) as the loading control. At the same time, the immunoprecipitates were treated with 50 mM N-ethylmaleimide to block free sulfhydryl groups, incubated with 1 M hydroxylamine to hydrolyze any Cys-palmitate thioester bonds, and then treated with 1 μm EZ-Link Biotin-BMCC in 50 mM Tris (pH 7.0) to label the newly exposed free sulfhydryl groups resulting from cleaved thioester bonds. The modified immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blot (WB) with streptavidin horseradish peroxidase. Reproduced from Qi et al. (2014) (Copyright American Society of Plant Biologists).

Notes

  1. No strong reductants, such as DTT or β-mercaptoethanol, can be used in any step of the sample preparation.
  2. Confocal microscopy was employed merely for quick confirmation of protein expression. It is not necessary for the following immuno-precipitation and in vitro labelling.
  3. This technique can be extended to recombinant proteins expressed in other eukaryotic heterologous expression systems such as Pichia pastoris, Spodoptera frugiperda, and fibroblast, etc.

Recipes

  1. LB liquid medium (per liter)
    10 g tryptone
    5 g yeast extract
    10 g NaCl
  2. Lysis buffer
    50 mM Tris (pH 7.5)
    150 mM NaCl
    0.1% (v/v) Nonidet P-40
    1% (v/v) plant proteinase inhibitor cocktail
  3. 4x SDS loading buffer
    50 mM Tris-HCl (pH 6.8)
    2% (w/v) SDS
    10% (v/v) glycerol
    1% (v/v) β-mercaptoethanol
    12.5 mM EDTA
    0.02% (w/v) bromphenol blue
  4. 5x nonreducing protein sample buffer
    10% (w/v) SDS
    1 M Tris-HCl (pH 6.8)
    25% (w/v) sucrose
    0.01% (w/v) bromphenol blue

Acknowledgments

This work was supported by the National Institute of General Medical Sciences at the National Institutes of Health (grant no. R01 GM046451 to R.W.I). This protocol was adapted from a previous work by Qi et al. (2014).

References

  1. Ade, J., DeYoung, B. J., Golstein, C. and Innes, R. W. (2007). Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial protease. Proc Natl Acad Sci U S A 104(7): 2531-2536.
  2. Berzat, A. C., Buss, J. E., Chenette, E. J., Weinbaum, C. A., Shutes, A., Der, C. J., Minden, A. and Cox, A. D. (2005). Transforming activity of the Rho family GTPase, Wrch-1, a Wnt-regulated Cdc42 homolog, is dependent on a novel carboxyl-terminal palmitoylation motif. J Biol Chem 280(38): 33055-33065.
  3. DeYoung, B. J., Qi, D., Kim, S. H., Burke, T. P. and Innes, R. W. (2012). Activation of a plant nucleotide binding-leucine rich repeat disease resistance protein by a modified self protein. Cell Microbiol 14(7): 1071-1084.
  4. Hemsley, P. A., Taylor, L. and Grierson, C. S. (2008). Assaying protein palmitoylation in plants. Plant Methods 4: 2.
  5. Joensuu, J. J., Conley, A. J., Lienemann, M., Brandle, J. E., Linder, M. B. and Menassa, R. (2010). Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana. Plant Physiol 152(2): 622-633.
  6. Qi, D., Dubiella, U., Kim, S. H., Sloss, D. I., Dowen, R. H., Dixon, J. E. and Innes, R. W. (2014). Recognition of the protein kinase AVRPPHB SUSCEPTIBLE1 by the disease resistance protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 is dependent on S-acylation and an exposed loop in AVRPPHB SUSCEPTIBLE1. Plant Physiol 164(1): 340-351.
  7. Ren, J., Wen, L., Gao, X., Jin, C., Xue, Y. and Yao, X. (2008). CSS-Palm 2.0: an updated software for palmitoylation sites prediction. Protein Eng Des Sel 21(11): 639-644.
  8. Shao, F., Merritt, P. M., Bao, Z., Innes, R. W. and Dixon, J. E. (2002). A Yersinia effector and a Pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis. Cell 109(5): 575-588.
  9. Vilas, G. L., Corvi, M. M., Plummer, G. J., Seime, A. M., Lambkin, G. R. and Berthiaume, L. G. (2006). Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events. Proc Natl Acad Sci U S A 103(17): 6542-6547.
  10. Zhang, J., Li, W., Xiang, T., Liu, Z., Laluk, K., Ding, X., Zou, Y., Gao, M., Zhang, X., Chen, S., Mengiste, T., Zhang, Y. and Zhou, J. M. (2010). Receptor-like cytoplasmic kinases integrate signaling from multiple plant immune receptors and are targeted by a Pseudomonas syringae effector. Cell Host Microbe 7(4): 290-301.

简介

蛋白质棕榈酰化是通过棕榈酸酯通过酰基键连接的蛋白质的翻译后修饰。半胱氨酸的亲核巯基是常见的棕榈酰化位点。棕榈酸酯的共价附着发生在许多蛋白质上,并且通常与将蛋白质定位到内膜系统相关。通过体内标记氚标记的棕榈酸来检测蛋白质棕榈酰化通常需要几个月的放射自显影曝光时间,因此不适合快速分析。在这里,我们描述了使用拟南芥蛋白激酶(PBS1)作为实例的快速体外检测蛋白S-酰化的简单方案。为了确定PBS1是否通过硫酯键连接到酰基修饰,我们采用"生物素开关"测定法(Hemsley等人,2008)。这项工作首次发表在Qi。et al。(2014),但我们在这里扩展方法。 PBS1在植物的基础免疫系统内起作用,并且是细菌半胱氨酸蛋白酶AvrPphB的靶(Shao等人,2002; Zhang等人,2010) 。它含有预测的N-末端S - 酰基化基序(MGCFSCFDS),其中Cys-3和Cys-6残基预测为被CSS-Palm 3.0棕榈酰化(http://csspalm.biocuckoo。 org /; Ren等人,2008)。我们的方法利用羟胺诱导的硫酯键裂解,这导致游离的巯基,然后可以与生物素衍生物1-生物素酰氨基-4- [4' - (马来酰亚胺甲基)环己烷甲酰胺基] - 丁烷(生物素-BMCC)缀合。通过蛋白质印迹用链霉亲和素 - 辣根过氧化物酶可检测缀合物。体外标记和检测的整个过程花费不到3天,允许通过硫酯键(例如棕榈酰化)快速检测蛋白修饰。

材料和试剂

  1. 本塞姆氏烟草( N。benthamiana )植物
  2. 根癌土壤杆菌菌株GV3101(pMP90)
  3. 细菌酵母提取物(BD Biosciences,目录号:288620)
  4. Bacto胰蛋白胨(BD Biosciences,目录号:211699)
  5. 氯化镁六水合物(EMD Millipore,目录号:442611)
  6. 乙酰丁香酮(Sigma-Aldrich,目录号:D134406-5G)
  7. 地塞米松(Sigma-Aldrich,D4902-1G)
  8. Trizma Tris碱(Sigma-Aldrich,目录号:93362)
  9. 氯化钠(EMD Millipore,目录号:567442)
  10. Nonidet P-40(Sigma-Aldrich,目录号:21-3277)
  11. 植物蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P9599-5M)
  12. SDS(Sigma-Aldrich,目录号:L3771-100G)
  13. 甘油(EMD Millipore,目录号:356350)
  14. β-巯基乙醇(Sigma-Aldrich,目录号:M3148-25ML)
  15. EDTA(Sigma-Aldrich,目录号:E6758-100G)
  16. BSA(Sigma-Aldrich,目录号:A4503-100G)
  17. 溴酚蓝(EMD Millipore,目录号:BX1410)
  18. 抗HA单克隆抗体基质(Roche Diagnostics,目录号:11867423001)
  19. Tris-HEPES-SDS聚丙烯酰胺凝胶(Thermo Fisher Scientific,目录号:25204)
  20. 抗HA过氧化物酶(Sigma-Aldrich,目录号:H6533-1VL)
  21. 高灵敏度链霉亲和素-HRP偶联物(Thermo Fisher Scientific,目录号:21130)
  22. ImmunoStar HRP底物试剂盒(Bio-Rad Laboratories,目录号:170-5070)
  23. N,N-乙基马来酰亚胺(Sigma-Aldrich,目录号:E3876-5G)
  24. 羟胺(Sigma-Aldrich,目录号:467804-10ML)
  25. 1 - 生物素酰氨基-4- [4'(马来酰亚胺甲基) - 环己烷 - 甲酰氨基] - 丁烷(EZ-link BMCC-生物素)(Thermo Fisher Scientific,目录号:21900)
  26. 硝基纤维素膜(Thermo Fisher Scientific,目录号:WP4HY00010)
  27. Metro-Mix 360(加拿大Sun Gro园艺)
  28. LB液体介质(见配方)
  29. 裂解缓冲液(见配方)
  30. 4x SDS加样缓冲液(见配方)
  31. 5x非还原蛋白样品缓冲液(见配方)

设备

  1. 共聚焦显微镜系统(Leica Microsystem,型号:TCS SP5)
  2. 微型蛋白质电泳和印迹系统(Bio-Rad Laboratories)
  3. 台式离心机5424(Eppendorf)
  4. 实验室管旋转器(Thermo Fisher Scientific)
  5. Gene Pulser(Bio-Rad Laboratories,目录号:1652076)
  6. 1ml注射器(BD Biosciences,目录号:305945)

程序

  1. 本生烟草中的瞬时表达测定
    如先前所述进行瞬时表达测定(DeYoung等人,2012)。 简要地说:
    1. N。 本生烟草植物生长在装满的小塑料盆中 Metro-Mix 360在8/16小时光周期下以150μmol/m/s和23℃进行
    2. N20sYFP:HA和n20sYFP:HA的地塞米松诱导型构建体   动员到根癌土壤杆菌菌株GV3101(pMP90)中 电穿孔。
    3. 在Luria-Bertani液体中过夜培养   培养基,细胞(7ml培养物)沉淀并重悬于10mM MgCl 2溶液和100μm的乙酰丁香酮,并稀释悬浮液 到渗透前OD 600 = 0.3。
    4. 将细胞孵育   在室温下孵育2小时,并用无针1ml注射器浸润 到4周龄(用于瞬时表达的最佳年龄)的叶中。 本生植物。 至少应有六个完全展开的叶 渗入一次实验。
    5. 通过诱导转基因 在接种后40小时用50μM地塞米松喷洒叶子。 样品   收获地塞米松4至6 h后进行蛋白提取 应用。
    6. 在进行激光扫描共聚焦显微镜检查   施用地塞米松5 h后确认sYFP的表达 融合。 从浸润的叶面积剪下1cm×1cm的组织   并放置在载玻片中心的ddH 2 O 2滴中。 封面后 放置载玻片,使用Leica SP5系统检查载玻片。

  2. 免疫沉淀和免疫印迹
    1. 对于总蛋白提取,研磨六个浸润的叶 裂解缓冲液。 组织可以在液氮中快速冷冻并储存   在-80℃直至需要
    2. 将匀浆在4℃下以13,000rpm离心两次,每次10分钟,将上清液转移至新管中。
    3. 每个中加入100μl抗HA单克隆抗体基质 匀浆。 然后将混合物在4℃下孵育过夜并旋转   端到端。
    4. 通过在16,000×g离心10秒钟使树脂沉淀,并用1ml裂解缓冲液洗涤三次。
    5. 对于免疫印迹,将免疫沉淀物与4×SDS混合 加样缓冲液以3:1的比例混合,煮沸10分钟,然后分离 在4%至20%(w/v)梯度Tris-HEPES-SDS PAGE上。
    6. 然后将蛋白质样品转移到硝酸纤维素膜上,用抗HA过氧化物酶探测
    7. ImmunoStar HRP底物试剂盒用于检测抗体复合物

  3. 检测 -Acylation
    如Berzat等人(2005)所述进行PBS1 S - 酰化的体外检测。 简要地说:
    1. 将N20-sYFP:HA或n20-sYFP:HA的免疫沉淀物在1℃下孵育   ml裂解缓冲液与50mM N,N-乙基马来酰亚胺在端对端旋转器上 在4℃下封闭48小时以封闭游离的巯基。
    2. 的 然后洗涤免疫沉淀物,在500μl1M中温育 羟胺溶液在端对端旋转器上室温下1小时 温度水解Cys-棕榈酸酯硫酯键
    3. 的 洗涤免疫沉淀物并在500μl50mM Tris(pH 7.4)中孵育 7.0)溶液,其中含有1微米的EZ-Link生物素-MBC,端对端 旋转器在室温下标记切割的硫酯键2小时。
    4. 洗涤免疫沉淀物,重悬于非还原中 蛋白质样品缓冲液,在4%至20%(w/v)梯度上分离 Tris-HEPES-SDS PAGE
    5. 将蛋白质样品转移到硝酸纤维素膜上。
    6. 将膜在5%无生物素的BSA TBST溶液中封闭1小时   然后用链霉亲和素 - 辣根过氧化物酶探测
    7. ImmunoStar HRP底物试剂盒用于检测生物素 - 链霉亲和素复合物。

代表数据

代表性数据如图1所示。使用该方案重复该实验三次,得到类似的结果

图1.S-酰化介导PBS1定位到PM。 A。 PBS1中预测的N-末端S-酰化基序是质膜定位所需的。 N20:sYFP表示的前20个氨基酸的融合 PBS1,其包含预测的棕榈酰化基序,到超黄色荧光蛋白。 n20是具有G2A/C3AC/6A三重突变的片段。两种融合蛋白在本塞姆氏烟草中瞬时表达。在地塞米松诱导后5小时进行共聚焦显微镜检查。所有图像是来自Z堆叠的三维投影。观察到N20:sYFP定位于质膜,并且观察到移动的囊泡样结构,当酰化蛋白过表达时,报道了这种结构(2006年; Vilas等人,2006; Joensuu& em]等人,2010)。然而,n20:sYFP融合仅作为胞质链检测。 YFP信号显示为绿色的假色。 B.PBS1是S-酰化的。细胞提取物。本发明的表达N20:sYFP-HA或n20:sYFP-HA的组织使用抗HA基质(IP)进行免疫沉淀。将免疫沉淀物用抗HA抗体(IB)作为上样对照进行免疫印迹。同时,用50mM N-乙基马来酰亚胺处理免疫沉淀物以封闭游离巯基,与1M羟胺孵育以水解任何Cys-棕榈酸酯硫酯键,然后用在50mM中的1μmEZ-Link生物素-BMCC处理Tris(pH 7.0)以标记由裂解的硫酯键产生的新暴露的游离巯基。通过SDS-PAGE分辨修饰的免疫沉淀,并通过蛋白质印迹(WB)用链霉亲和素辣根过氧化物酶进行分析。摘自Qi等人(2014)(版权美国植物生物学家协会)。

笔记

  1. 在样品制备的任何步骤中都不能使用强还原剂,例如DTT或β-巯基乙醇。
  2. 共聚焦显微镜仅用于快速确认蛋白质表达。以下免疫沉淀和体外标记不是必需的。
  3. 这种技术可以扩展到在其他真核异源表达系统如毕赤酵母(Pichia pastoris),草地贪夜蛾(Spodoptera frugiperda)和成纤维细胞等表达的重组蛋白质。 br />

食谱

  1. LB液体培养基(每升)
    10g胰蛋白胨
    5g酵母提取物
    10克NaCl
  2. 裂解缓冲液
    50mM Tris(pH7.5) 150mM NaCl 0.1%(v/v)Nonidet P-40
    1%(v/v)植物蛋白酶抑制剂混合物
  3. 4x SDS上样缓冲液
    50mM Tris-HCl(pH6.8)
    2%(w/v)SDS
    10%(v/v)甘油 1%(v/v)β-巯基乙醇 12.5mM EDTA
    0.02%(w/v)溴酚蓝
  4. 5x非还原性蛋白质样品缓冲液
    10%(w/v)SDS
    1 M Tris-HCl(pH 6.8)
    25%(w/v)蔗糖 0.01%(w/v)溴酚蓝

致谢

这项工作得到国立卫生研究院国家综合医学科学研究所(授予号R01 GM046451至R.W.I)的支持。该协议改编自Qi等人之前的工作。(2014)。

参考文献

  1. Ade,J.,DeYoung,B.J.,Golstein,C.and Innes,R.W。(2007)。 由细菌蛋白酶间接激活植物核苷酸结合位点 - 富含亮氨酸的重复蛋白。 Proc Natl Acad Sci USA 104(7):2531-2536。
  2. Berzat,A.C.,Buss,J.E.,Chenette,E.J.,Weinbaum,C.A.,Shutes,A.,Der,C.J.,Minden,A.and Cox,A.D。(2005)。 Rho家族GTPase,Wrch-1(Wnt调节的Cdc42同源物)的转化活性,依赖于新的羧基末端棕榈酰化基序。 J Biol Chem 280(38):33055-33065。
  3. DeYoung,B.J.,Qi,D.,Kim,S.H.,Burke,T.P.and Innes,R.W。(2012)。 激活植物核苷酸结合富亮氨酸重复疾病 抗性蛋白通过修饰的自身蛋白。细胞微生物 14(7):1071-1084。
  4. Hemsley,P.A.,Taylor,L.and Grierson,C.S。(2008)。 在植物中测定蛋白质棕榈酰化。植物方法 4: 2.
  5. Joensuu,J.J.,Conley,A.J.,Lienemann,M.,Brandle,J.E.,Linder,M.B.and Menassa,R。(2010)。 疏水蛋白融合蛋白,用于本氏烟草中的高水平瞬时蛋白表达和纯化。 。 Plant Physiol 152(2):622-633。
  6. Qi,D.,Dubiella,U.,Kim,S.H.,Sloss,D.I.,Dowen,R.H.,Dixon,J.E。和Innes,R.W。(2014)。 抗病蛋白抗体对蛋白激酶AVRPPHB SUSCEPTIBLE1的识别抗性对SYRINGAE5的依赖取决于S-酰化和在AVRPPHB SUSCEPTIBLE1中暴露的环。 植物生理学 164(1):340-351。
  7. Ren,J.,Wen,L.,Gao,X.,Jin,C.,Xue,Y。和Yao, CSS-Palm 2.0:更新的棕榈酰化位点预测软件 蛋白Eng Des Sel 21(11):639-644。
  8. Shao,F.,Merritt,P.M.,Bao,Z.,Innes,R.W.and Dixon,J.E。(2002)。 Yersinia效应物和假单胞菌无毒蛋白定义了一个家族 半胱氨酸蛋白酶在细菌发病机理中起作用。 Cell 109(5):575-588。
  9. Vilas,G.L.,Corvi,M.M.,Plummer,G.J.,Seime,A.M.,Lambkin,G.R。和Berthiaume,L.G。(2006)。 胱天蛋白酶活化的p21活化蛋白激酶2(PAK2)的翻译后豆蔻酰化增强晚期凋亡事件。/a> Proc Natl Acad Sci USA 103(17):6542-6547。
  10. Zhang,J.,Li,W.,Xiang,T.,Liu,Z.,Laluk,K.,Ding,X.,Zou,Y.,Gao,M.,Zhang,X.,Chen, Mengiste,T.,Zhang,Y.and Zhou,JM(2010)。 受体样细胞质激酶整合来自多种植物免疫受体的信号传导,并被假单胞菌 syringae 效应子。 Cell Host Microbe 7(4):290-301。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Qi, D. and Innes, R. W. (2014). In vitro Detection of S-acylation on Recombinant Proteins via the Biotin-Switch Technique. Bio-protocol 4(22): e1296. DOI: 10.21769/BioProtoc.1296.
  2. Qi, D., Dubiella, U., Kim, S. H., Sloss, D. I., Dowen, R. H., Dixon, J. E. and Innes, R. W. (2014). Recognition of the protein kinase AVRPPHB SUSCEPTIBLE1 by the disease resistance protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 is dependent on S-acylation and an exposed loop in AVRPPHB SUSCEPTIBLE1. Plant Physiol 164(1): 340-351.
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