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Aug 2020
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In vitro Reconstitution Assays of Arabidopsis 20S Proteasome
拟南芥20S蛋白酶体的体外重组分析   

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Abstract

The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be controlled by the 20S proteasome. Relatively little is known about the specific functions of the 20S proteasome and the regulatory mechanisms of IDP degradation in plants compared to other species because there is a lack of systematic protocols for in vitro assembly of this complex to perform in vitro degradation assays. Here, we present a detailed protocol of in vitro reconstitution assay of the 20S proteasome in Arabidopsis by modifying previously reported methods. The main strategy to obtain the 20S core proteasome here is to strip away the 19S regulatory subunits from the 26S proteasome. The protocol has two major parts: 1) Affinity purification of 20S proteasomes from stable transgenic lines expressing epitope-tagged PAG1, an essential component of the 20S proteasome (Procedures A-D) and 2) an in vitro 20S proteasome degradation assay (Procedure E). We anticipate that these protocols will provide simple and effective approaches to study in vitro degradation by the 20S proteasome and advance the study of protein metabolism in plants.

Keywords: 26S proteasome (26S蛋白酶体), 20S proteasome (20S蛋白酶体), Ubiquitin-independent (游离泛素蛋白), Intrinsically disordered proteins (IDPs) (固有无序蛋白质(IDPs)), Protein degradation (蛋白质降解)

Background

In eukaryotes, protein degradation is carried out by the proteasome. The integrative 26S proteasome is comprised of two sub particles: one or two terminal 19S regulatory particle(s) (RP), which serve as a proteasome activator; and the 20S core proteasome (CP), which degrades proteins. Most eukaryotic proteins are polyubiquitinated and channeled into 26S proteasome for degradation. In contrast, proteins that contain intrinsically disordered regions have been found to be directly destroyed by the ubiquitin-independent 20S proteasome (Ben-Nissan et al., 2014). Methods to purify and assembly the 20S proteasome in vitro are well established for mammalian cells and yeast. This has led to an understanding and appreciation of the numerous ways by which IDPs interact with the 20S proteasome (Leggett et al., 2005). However, to date a detailed and efficient protocol has not been reported for the purification of the 20S proteasome from plants. Book et al. (2010) developed an affinity-based strategy to effectively isolate the 26S proteasome from Arabidopsis. In their approach, PAG1, one of the 14 core proteasome polypeptides, is epitope-tagged and immunoprecipitated with epitope-specific antibodies such that the 26S proteasome is recovered. From purification studies of the proteasome complex, there are two important components to monitor during the purification scheme: ATP amount and salt concentration (Verma et al., 2000; Leggett et al., 2002). It is known that the integrity of the RP-CP complex relies on ATP, and the abundance of RP subunits is substantially reduced if all purification steps do not include ATP. Similarly, RP subunit abundance is reduced when immunoprecipitates (IPs) are washed with a high salt buffer (800 mM NaCl) before elution (Book et al., 2010). Based on previous methods, we designed a simple approach to specifically isolate the 20S proteasome by immunoprecipitating PAG1 complexes from total protein extracts of stable transgenic lines expressing gPAG1-Flag-4Myc (gPAG1-FM) under its native promoter. In our protocol, the protein extracts are not supplied with ATP, and IPs are washed with a buffer containing 800 mM NaCl, as this stringent condition has been reported to strip the 19S regulatory subunits away from the 20S core proteasome. For in vitro protein degradation assays, we modified a protocol from yeast work with the 20S proteasome (Hsieh et al., 2015). Taken together, this protocol is easy to follow and can provide an effective strategy to study degradation of IDPs in plants. We hope that this protocol will advance research in protein metabolism and regulation in plants.

Materials and Reagents

  1. 15 ml conical tube

  2. 2 ml Eppendorf tube

  3. 1.5 ml Eppendorf tube

  4. Pipette tips

  5. 96-well Plate (Thermo Scientific, catalog number: 249935 )

  6. 10-day-old PPAG1-gPAG1-FM transgenic seedlings

  7. Liquid nitrogen

  8. Tris Base (Fisher Scientific, catalog number: BP152-10 )

  9. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )

  10. NaCl (Fisher Scientific, catalog number: BP358-10 )

  11. MgCl2 (Sigma-Aldrich, catalog number: M9272 )

  12. EDTA (Fisher Scientific, catalog number: BP120-1 )

  13. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43817 )

  14. Glycerol (Sigma-Aldrich, catalog number: V900122 )

  15. PMSF (Sigma-Aldrich, catalog number: 78830 )

  16. Miracloth (Calbiochem, catalog number: 475855 )

  17. Anti-FLAG® M2 magnetic beads (Sigma-Aldrich, catalog number: M8823 )

  18. 3× FLAG peptide (DYKDDDDK) (Sigma-Aldrich, catalog number: F4799 )

  19. Anti-Flag (Sigma-Aldrich, catalog number: F1804 )

  20. Anti-Myc (Sigma-Aldrich, catalog number: C3956 )

  21. Anti-SE (Agrisera, catalog number: AS09 532A)

  22. DMSO (Sigma-Aldrich, catalog number: D4540 )

  23. MG132 (Calbiochem, catalog number: 474787 )

  24. Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC) (Sigma-Aldrich, catalog number: S6510 )

  25. SilverQuestTM Staining Kit (Invitrogen, catalog number: LC6070 )

  26. Bradford Reagent (Sigma-Aldrich, catalog number: B6916 )

  27. 2× SDS-PAGE loading buffer (see Recipes)

  28. Extraction buffer (see Recipes)

  29. Washing buffer (see Recipes)

  30. Reaction Buffer (see Recipes)

Equipment

  1. -80 °C freezer

  2. Pipettes

  3. Centrifuge

  4. DynaMagTM-2 (Invitrogen, model: 12321D )

  5. PolyATtract® System 1000 Magnetic Separation Stand (Promega Corporation, model: Z5410 )

  6. Rugged Rotator (Glas-Col, model: 099A RD4512 )

  7. Thermomixer R (Eppendorf, model: 022679810)

  8. Microplate spectrophotometer (PerkinElmer, model: VICTORTM X3)

  9. Gel imaging system (Bio-Rad, model: Universal Hood II )

  10. Vortex mixer (VWR, model: 945300 )

Procedure

Note: Please see Figure 1 for a schematic diagram of the steps described below.



Figure 1. A schematic diagram of procedure


  1. Preparation of 10-day-old PPAG1-gPAG1-FM stable transgenic plants (Li et al., 2020)

    1. Transform a binary vector pBA002a-PPAG1-gPAG1-FM into Col-0 ecotype of Arabidposis thaliana by the floral-dip transformation method (Zhang et al., 2006) to generate PPAG1-gPAG1-FM stable transgenic plants.

    2. Sterilize and place the seeds from the stable transgenic line expressing PPAG1-gPAG1-FM on MS medium (Zhang et al., 2006) and stratify seeds by keeping them in the dark at 4 °C for 3 days.

    3. Germinate seeds and grow the seedlings under a 12 h light-12 h dark cycle at 22 °C for 10 days.

    4. Collect 5 g of 10-day-old seedlings, ground to fine powder in liquid nitrogen, and stored at -80 °C.


  2. Affinity purification of 20S proteasomes (Book et al., 2010)

    1. Re-suspend the 5 g powder sample in 8 ml of extraction buffer.

    2. Keep the sample on ice for min. Keeping the sample cold, homogenize it well with a vortex mixer 2-3 times.

    3. Centrifuge the fully dissolved protein extract at 4 °C for 15 min at 21,000 × g and then filter the protein extract through one layer of pre-wet Miracloth.

    4. After filtration, centrifuge the cleared protein extract again at 4 °C for 15 min at 21,000 × g.

    5. Collect the supernatant from Step B4 in a pre-cooled 15 ml conical tube and keep it on ice.

    6. Prepare Anti-FLAG beads for immunoprecipitation during the centrifugation steps. Add 200 μl bed volume Anti-FLAG® M2 magnetic beads (for 5 g plant tissue) into a new 2 ml Eppendorf tube on ice.

    7. Wash the magnetic FLAG-beads with 600 μl of 0.1 M glycine HCl (pH 3.0) to remove un-conjugated antibody.

    8. Invert the tube gently and leave the mixture in the tube for 1.5 min.

    9. Immediately re-equilibrate the Anti-FLAG® M2 magnetic beads with 1 ml buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0).

    10. Remove the equilibration buffer, followed by washing the beads with 1 ml extraction buffer for three times using DynaMag10. Remove the equilibration buffer, followed by washing the beads with 1 ml extraction buffer three times using DynaMagTM-2.

    11. Completely remove the extraction buffer and add 200 μl new extraction buffer into the tube to re-suspend the beads.

    12. Add 200 μl equilibrated magnetic beads into sample.

    13. Rotate the 15 ml tube at 4 °C for 30 min using a rugged rotator.

    14. At the end of Step B13, prepare 3× FLAG peptide solution for elution of Flag-4Myc-tagged PAG1 from the Anti-FLAG® M2 magnetic beads.

    15. Add 35 μl 3× FLAG elution buffer stock (4 mg/ml) into 245 μl extraction buffer to make a final concentration 500 ng/μl of 3× FLAG peptide. Mix well and put it on ice.

    16. After Step B13 is done, load the 15 ml tube on the PolyATtract® System 1000 Magnetic Separation Stand for a few seconds, then slowly pour out the supernatant. Supernatant can be discarded.

    17. Re-suspend the beads with 6 ml washing buffer and transfer all the beads to a new, clean precooled 15 ml tube.

    18. Wash the beads with 6 ml washing buffer three times at 4 °C, each time for 5 min using the rugged rotator.

    19. After washing three times, add 2 ml of extraction buffer to a 15 ml tube to re-suspend the beads.

    20. Transfer all the beads carefully to a clean 2 ml Eppendorf tube.

    21. Load the 2 ml tube into DynaMagTM-2, and remove the extraction buffer.

    22. Add 250 μl 3× FLAG elution buffer into the 2 ml tube, incubate the tube for 30 min at 4°C with 1,200 rpm shaking using Thermomixer R.

    23. Load the 2 ml tube into DynaMagTM-2. Transfer the eluates to the 1.5 ml Eppendorf tube and store at -80 °C.


  3. Silver staining of purified proteasome

    1. Add 20 μl 2× SDS-PAGE loading buffer into 20 μl of the eluted sample and boil at 95 °C for 8 min.

    2. Load 20 μl eluted sample onto two 10% SDS-PAGE gels and subject to electrophoresis for 2.5 h at 80 V.

    3. Stain one of the gels with SilverQuestTM Staining Kit (Figure 2).



      Figure 2. A representative silver-staining image of immunoprecipitated PAG1-FM-containing 20S resolved by SDS-PAGE. The immunoprecipitation was performed using the anti-FLAG® M2 magnetic beads with the protein extracts prepared from PPAG1-gPAG1-FM transgenic seedlings (gPAG1-FM) or from Col-0 (control), respectively. The bracket indicates subunits of the 20S core proteasome (CP).


    4. Do Western blot analysis for the other gel using anti-Myc or anti-Flag antibodies.

    5. Take images of silver staining gel using a gel imaging documentation system.


  4. Proteasome activity assay (Yang et al., 2004; Han et al., 2019)

    1. Prepare reaction buffer containing 50 μM Suc-LLVY-AMC substrate, which is widely used as a fluorogenic substrate for measuring the chymotrypsin – like activity of the 20S proteasome (Reidlinger et al., 1997).

    2. Add 10 μl eluted sample into 90 μl reaction Buffer, mix well and add into 96-well Plate.

    3. Add 10 μl extraction buffer into 90 μl reaction Buffer as a blank control and repeat each reaction three times.

    4. Incubate the reaction mixtures at 37 °C for 20, 40, 60, 80, 100, 120 min.

    5. Monitor the fluorescence reading of the released AMC at the indicated times using a Microplate spectrophotometer by fluorescence using 380 nm excitation and 440 nm emission wavelengths.

    6. Plot proteasome activity in relative fluorescence units per 1 μl of reaction mixtures, using free AMC as a standard.

      Note: You can also use other representations to display your data, such as relative fluorescence units per 10 μl of eluted sample.


  5. In vitro 20S proteasome degradation assay (Hsieh et al., 2015)

    1. Prepare purified proteins for testing.

    2. Estimate the concentration of the purified proteasome and test proteins by the Bradford method (Bradford, 1976). (Method: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/b6916bul.pdf).

    3. Prepare in vitro 20S proteasome-decay reaction mixtures on ice as follows (Table 1):


      Table 1. In vitro 20S proteasome degradation reaction system

      One reaction Reaction mixtures for five indicated times
      150 nM Purified test protein According to the concentration
      10 nM Purified 20S proteasome According to the concentration
      50 mM Tris-HCl (pH 7.5) 10 μl [1 M Tris-HCl (pH 7.5) ]
      2% DMSO or 50 μM MG132 4 μl (2.5 mM MG132, dissolved in DMSO)
      Add H2O to final volume 40 μl Add H2O to final volume 200 μl
      Note: The amount of the purified 20S proteasome and test protein in the reaction mixture is only a reference, the reaction condition needs to be optimized for different proteins.


    4. Then distribute the mixtures evenly into five PCR or 1.5 ml tubes and incubate the tubes at 22 °C.

    5. Stop the reaction by adding 40 μl 2× SDS-PAGE loading buffer at the indicated times (0, 5, 10, 20, 30 min) followed by Western blot analysis (Figure 3).



      Figure 3. A representative image of in vitro 20S proteasome-mediated protein degradation assay. Recombinant 6xHis-SUMO-SE protein was incubated with the PAG1-FM immunoprecipitate from PPAG1-gPAG1-FM transgenic plants or control IP from Col-0, respectively. The reaction mixture was stopped at the indicated time intervals. Western blot assay of 6xHis-SE was probed with an anti-SE antibody.

Data analysis

For additional reference images of silver staining and in vitro 20S proteasome degradation assay, see Figures 4c and 4d from Li et al. (2020), respectively. For additional reference images of the proteasome activity assay, see the Extended Data Figure 7b from Li et al. (2020).

Notes

  1. In step B, all the buffers and tubes required for purification should be pre-cooled to 4 °C before use.

  2. In step B, Miracloth is pre-wet with extraction buffer.

  3. In step B, divide the final elution samples into several tubes to avoid freeze-thaw samples in future use, which will effect proteasome activity.

  4. In step E, optimize the in vitro degradation conditions according to test proteins, including pH, degradation time, degradation temperature and the concentration of the 20S proteasome and test proteins.

  5. In step E, prepare and evenly distribute the reaction mixtures quickly and keep on ice. Make sure all time points start the reaction at the same time.

Recipes

  1. 2× SDS-PAGE loading buffer

    0.125 mM Tris-HCl, pH 6.8

    20% glycerol

    4% SDS

    0.2 M DTT

    0.02% bromophenol blue

  2. Extraction buffer

    50 mM Tris-HCl, pH 7.5

    25 mM NaCl

    2 mM MgCl2

    1 mM EDTA

    5% glycerol

    2 mM PMSF (add just before use)

  3. Washing buffer

    50 mM Tris-HCl, pH 7.5

    800 mM NaCl

    2 mM MgCl2

    1 mM EDTA

    5% glycerol

    2 mM PMSF (add just before use)

  4. Reaction Buffer

    50 mM Tris-HCl, pH 7.5

    25 mM NaCl

    2 mM MgCl2

    1 mM EDTA

    2 mM dithiothreitol (DTT)

    5% glycerol

    50 μM Suc-LLVY-AMC substrate

Acknowledgments

The work was supported by grants from the NIH (grant GM127742) to X.Z. The protocol presented here was developed from several previous publications (Yang et al., 2004; Zhang et al., 2006; Book et al., 2010; Hsieh et al., 2015; Han et al., 2019) and optimized in our recent work (Li et al., 2020).

Competing interests

The authors declare no competing interests.

References

  1. Ben-Nissan, G. and Sharon, M. (2014). Regulating the 20S proteasome ubiquitin-independent degradation pathway. Biomolecules 4(3): 862-884.
  2. Book, A. J., Gladman, N. P., Lee, S. S., Scalf, M., Smith, L. M. and Vierstra, R. D. (2010). Affinity purification of the Arabidopsis 26S proteasome reveals a diverse array of plant proteolytic complexes. J Biol Chem 285: 25554-25569.
  3. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  4. Han, J. J., Yang, X., Wang, Q., Tang, L., Yu, F., Huang, X., et al. (2019). The β5 subunit is essential for intact 26S proteasome assembly to specifically promote plant autotrophic growth under salt stress. New Phytol 221(3): 1359-1368.
  5. Hsieh, L. S., Su, W. M., Han, G. S. and Carman, G. M. (2015). Phosphorylation regulates the ubiquitin-independent degradation of yeast Pah1 phosphatidate phosphatase by the 20S proteasome. J Biol Chem 290(18): 11467-11478.
  6. Leggett, D. S., Glickman, M. H. and Finley, D. (2005). Purification of proteasomes, proteasome subcomplexes, and proteasome-associated proteins from budding yeast. In: Ubiquitin-Proteasome Protocols. Springer pp: 57-70.
  7. Leggett, D. S., Hanna, J., Borodovsky, A., Crosas, B., Schmidt, M., Baker, R. T., et al. (2002). Multiple associated proteins regulate proteasome structure and function. Molecular cell 10(3): 495-507.
  8. Li, Y., Sun, D., Ma, Z., Yamaguchi, K., Wang, L., Zhong, S., et al. (2020). Degradation of SERRATE via ubiquitin-independent 20S proteasome to survey RNA metabolism. Nature Plants 6: 970-982.
  9. Reidlinger, J., Pike, A. M., Savory, P. J., Murray, R. Z. and Rivett, A. J. (1997). Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities. J Biol Chem 272(40): 24899-24905.
  10. Verma, R., Chen, S., Feldman, R., Schieltz, D., Yates, J., Dohmen, J., et al. (2000). Proteasomal proteomics: identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol Biol Cell 11(10): 3425-3439.
  11. Yang, P., Fu, H., Walker, J., Papa, C. M., Smalle, J., Ju, Y. M., et al. (2004). Purification of the Arabidopsis 26 S proteasome biochemical and molecular analyses revealed the presence of multiple isoforms. J Biol Chem 279(8): 6401-6413.
  12. Zhang, X. R., Henriques, R., Lin, S. S., Niu, Q. W. and Chua, N. H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1(2): 641-646. 

简介

[摘要]大多数细胞蛋白的s的降解通过26S在真核生物蛋白酶。但是,内在无序的蛋白质(IDPs)包含大量的非结构化区域,并且内在地不稳定,因此很容易通过不依赖泛素的20S蛋白酶体降解。越来越多的证据最近显示ň植物境内流离失所者的平衡也可以通过20S蛋白酶控制。但是,由于缺乏用于体外分离20S蛋白酶体和降解测定的系统协议,因此我们对植物中IDP和20S蛋白酶体降解的功能和调控机制的研究和理解一直处于婴儿期与其他生物。在这里,我们通过采用和修改先前公开的方法,对拟南芥中20S蛋白酶体进行体外重组测定的详细方案。在此获得20S核心蛋白酶体的主要策略是从26S蛋白酶体中去除19S调节亚基。该协议包括两个主要部分:1)的来自表达表位标记的PAG1稳定的转基因品系20S蛋白酶体亲和纯化,的20S蛋白酶(程序AD)的基本组成部分; 2 )体外20S蛋白酶体降解测定法(方法E)。我们预计该协议将提供一种简单有效的方法来研究体外20S蛋白酶体降解,并促进植物中蛋白质代谢的研究。

[背景]蛋白质的降解通常是通过真核生物中的蛋白酶体来实现的。整合的26S蛋白酶体由两个亚颗粒组成:一个或两个末端的19S调节颗粒(RP),用作蛋白酶体激活剂;和20S核心蛋白酶体(CP),执行降解过程。大多数真核蛋白被多聚泛素化并导入26S蛋白酶体进行降解。然而,含有固有蛋白质无序已发现的区域直接通过破坏一个由20S蛋白酶的泛素依赖性降解(本日产等人,2014) 。20S蛋白酶体的体外纯化已在哺乳动物和酵母菌中得到了很好的确立,从而导致人们对许多IDP的功能和法规有了更多的了解和赞赏(Leggett等,2005 )。然而,迄今为止,尚未公开用于植物中20S蛋白酶体纯化的详细且友好的使用方案。先前,Book等人。(2010年)开发了一种基于亲和力的策略,可从拟南芥中有效分离26S蛋白酶体。在该方法中,用表位标记14种核心蛋白酶多肽之一的PAG1,并用特异性抗体免疫沉淀标记的PAG1 ,以便回收26S蛋白酶体。RP-CP复合物的完整性依赖于ATP,并且对高盐敏感(Verma等,2000 ;Leggett等,2002 )。也有报道说,如果不使用ATP进行所有纯化步骤,则RP亚基的丰度将大大降低,在洗脱前用高盐(800 mM NaCl )洗涤免疫沉淀物(IPs)的情况也是如此(Book)等人,2010)。基于以前的研究和方法,在这里我们提供了一种简单的方法,可以通过从天然启动子下表达g PAG1-Flag-4Myc (g PAG 1- FM )的稳定转基因品系的总蛋白提取物中免疫沉淀出PAG1复合物来特异性分离20S蛋白酶体。在我们的方案中,蛋白质提取物不使用ATP,而IPs用含有800 mM NaCl的缓冲液洗涤,因为这种严格条件会使19S调节亚基与20S核心蛋白酶体分离。对于体外降解测定,我们修改了酵母研究的方案(Hsieh等,2015 )。综上所述,该协议易于遵循,并且可以提供一种有效的策略来研究植物中的IDP。我们希望该协议能促进植物蛋白质代谢和调控的研究。

关键字:26S蛋白酶体, 20S蛋白酶体, 游离泛素蛋白, 固有无序蛋白质(IDPs), 蛋白质降解



材料和试剂


1. 15毫升锥形管     
2. 2毫升Eppendorf管     
3. 1.5毫升Eppendorf管     
4. P ipette提示     
5. 96孔板(Thermo Scientific,目录号:249935)     
6. 10日龄P PAG1 -gPAG1-FM转基因幼苗     
7.液氮     
8. Tris Base(Fisher Scientific,目录号:BP152-10)     
9.十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)     
10. NaCl (Fisher Scientific,目录号:BP358-10) 
11. MgCl 2 (西格玛奥德里奇,目录号:M9272) 
12. EDTA(Fisher Scientific,目录号:BP120-1) 
13.二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43817) 
14.甘油(Sigma-Aldrich,目录号:V900122) 
15. PMSF(Sigma-Aldrich,目录号:78830) 
16. Miracloth (Calbiochem ,目录号:475855) 
17.抗FLAG ® M2磁珠(Sigma-Aldrich公司,目录号:M8823) 
18. 3 × FLAG肽(DYKDDDDK)(Sigma-Aldrich,目录号:F4799) 
19.防国旗(Sigma-Aldrich,目录号:F1804) 
20.抗Myc (Sigma-Aldrich,目录号:C3956) 
21. Anti-SE(Agrisera ,目录号:AS09 532A) 
22. DMSO(Sigma-Aldrich,目录编号:D4540) 
23. MG132 (Calbiochem ,目录号:474787) 
24.琥珀酰-Leu-Leu-Val-Tyr-7-酰胺基-4-甲基香豆素(Suc -LLVY-AMC)(Sigma-Aldrich,目录号:S6510) 
25. SilverQuest TM染色试剂盒(Invitrogen,目录号:LC6070) 
26. Bradford试剂(Sigma-Aldrich,目录号:B6916) 
27. 2 × SDS-PAGE加载缓冲区(请参见食谱) 
28.提取缓冲液(请参阅食谱) 
29.洗涤缓冲液(请参阅食谱) 
30.反应缓冲液(请参见食谱) 

设备


-80°C冷冻室
P ipettes
ç entrifuge
Dyna Mag TM -2(Invitrogen,代码:12321D)
PolyATtract ®系统1000磁性分离架(Promega公司制,型号:Z5410)
坚固耐用的转子(Glas -Col,型号:099A RD4512)
Thermomixer R(Eppendorf,型号:022679810)
微板分光光度计(PerkinElmer公司,米Odel等:VICTOR TM X3)
凝胶成像系统(Bio-Rad,型号:Universal Hood II)
涡旋混合器(VWR,型号945300)

程序


注意:请参见图1以获得以下步骤的示意图。



图1.过程示意图


制备10天大的P PAG1 -gPAG1-FM稳定转基因植物(Li等,2020)
变换二元载体pBA002a-P PAG1 -gPAG1-FM成的Col-0中生态型Arabidposis拟南芥通过浸花转化法(张等人,2006 ),以生成P PAG1 -gPAG1-FM稳定的转基因植物。
STER ilize并放置来自表达稳定的转基因系的种子P PAG1 -gPAG1-FM的MS培养基上(张等人。,2006),并通过在4℃下使它们保持在黑暗中3天种子分层。
发芽种子,并在22 °C下12 h光照12 h暗循环下使幼苗生长10天。
收集5 g 1个0天大的幼苗,在液氮中研磨成细粉,并在-80°C下保存。

20S蛋白酶体的亲和纯化(Book等,2010 )
将5 g粉末样品重悬于8 ml提取缓冲液中。
保持所述样品在冰上为8分钟,并同时均匀它以及与涡旋混合器2-3次。
离心在4℃下15分钟,完全溶解蛋白提取物在21 ,00 0 ×克和则f ILTER蛋白质提取物通过的预先润湿的一个层的Miracloth 。
过滤后,在4℃下再次离心清除蛋白提取物在15分钟21 ,00 0 ×克。
收集的上清液小号TEP B4到预-冷却的15毫升锥形管中并保持在冰上。
准备抗-FLAG珠,在离心步骤中进行免疫沉淀。加入200微升床体积抗FLAG ® M2磁珠(对于5克植物组织)到一个新的2毫升Eppendorf管中于冰上。
用600μl的0.1 M甘氨酸HCl (pH 3.0)洗涤磁性Flag-beads以去除未结合的抗体。
轻轻颠倒试管,将混合物留在试管中1.5分钟。
立即重新平衡的抗Flag ® M2的磁珠用1毫升缓冲液(50米的Tris -盐酸,150毫氯化钠,pH 8.0)中 。
除去平衡缓冲液,然后使用DynaMag TM -2用1 ml提取缓冲液洗涤磁珠3次。
完全除去提取缓冲液,然后向管中添加200μl新的提取缓冲液,以重新悬浮珠子。
将200μl平衡的磁珠添加到样品中。
然后使用坚固的旋转器将15 ml试管在4°C下旋转30分钟。
在的端小号TEP B13,制备3 × ˚F LAG用于从所述的Flag-4Myc标记的PAG1的洗脱肽溶液抗FLAG ® M2磁珠。
添加35微升3 × ˚F LAG洗脱缓冲液原液(4毫克/毫升)到245微升的提取缓冲液,使最终浓度为500ng /μ升3 × ˚F LAG pepti德。拌匀,放在冰上。
步骤B13完成后,加载上的15ml管中PolyATtract ®系统1000磁性分离架几秒钟,然后慢慢倒入上清至垃圾桶。
用6 ml洗涤缓冲液重悬珠子,并将所有珠子转移到新的干净的预冷1 5 ml管中。
在4°C下用6 ml洗涤缓冲液洗涤磁珠3次,每次3次,每次使用坚固的旋转器,持续5分钟。
洗涤3次后,将2 ml提取缓冲液添加到15 ml管中,以重新悬浮珠子。
小心地将所有珠子转移到干净的2 ml Eppendorf管中。
将2 ml试管装入DynaMag TM -2,然后除去提取缓冲液。
加入250微升3 × FLAG在4℃下的洗脱缓冲液进2毫升管,孵育管30分钟与1 ,使用Thermomixer中R. 200rpm振荡
将2 ml试管装入DynaMag TM -2。转移ELU一个TES到1.5毫升Eppendorf管并储存于- 80℃。

              纯化蛋白酶体的银染
加入20微升2 × SDS - PAGE上样缓冲液到20微升的洗脱样品中,并在95煮沸℃8分钟。
负载20微升洗脱的样品到两个10%SDS-PAGE凝胶上并受到电泳2.5小时,在80 V.
使用SilverQue st TM染色试剂盒对其中一种凝胶进行染色(图2)。

˚F igure的2.代表银染色图像免疫沉淀的PAG1 -含FM-20S在SDS解决- PAGE。使用进行了免疫沉淀的抗FLAG ® M2磁珠从制备的蛋白提取物P PAG1 - gPAG1 - FM转基因幼苗(gPAG1-FM分别从)或Col-0中(对照)。括号表示20S核心蛋白酶体(CP)的亚基。


做W¯¯西部时代印迹分析使用抗其他凝胶的Myc或抗Flag抗体。
使用gel成像文档系统拍摄银染凝胶的照片。

蛋白酶体活性测定(Yang等,2004 ; Han等,2019 )
制备含有50反应缓冲液μM的Suc -LLVY-AMC底物,其被广泛用作荧光底物用于测定胰凝乳蛋白酶 –类似于20S蛋白酶体的活性(Reidlinger等,1997)。
将10μl洗脱的样品添加到90μl反应缓冲液中,充分混合并添加到96孔板中。
将10μl提取缓冲液添加到90μl反应缓冲液中作为空白对照,并将每个反应重复3次。
孵育反应混合物在37℃下为20,4 0,60,80,100,120分钟。
使用Microplate分光光度计,通过380 nm激发波长和440 nm发射波长的荧光监测所指示时间释放的AMC的荧光读数。
使用游离AMC作为标准品,以每1μl反应混合物的相对荧光单位绘制蛋白酶体活性。
注意:您还可以使用其他表示形式来显示数据,例如每10μl洗脱样品的相对荧光单位。


体外20S蛋白酶体降解试验(Hsieh等,2015 )
准备用于测试的纯化蛋白。
通过Bradford方法估算了纯化的蛋白酶体和测试蛋白的浓度(Bradford,1976)。(Mothod :https : //www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/b6916bul.pdf)。
如下在冰上制备体外20S蛋白酶体-衰变反应混合物(表1):

Ť能够1.我Ñ体外20蛋白酶体降解反应系统


注意:Ť他相当于在反应混合物中的纯化的20S蛋白酶和测试蛋白质的只是一个参考,该反应条件需要小号为不同的蛋白质进行优化。


然后将混合物均匀分配到5个PCR或1.5 ml试管中,并在22 °C下孵育。
该反应将通过添加40停止微升2 × SDS-PAG在随后所指示的时间(0,5,10,20,30分钟)电子样缓冲液W¯¯西部时代印迹分析(图3)。


˚F igure的3.一种代表性图像体外20S蛋白酶体介导的蛋白质降解测定。重组的6xHis - SUMO - SE蛋白与PAG1孵育- FM免疫沉淀从P PAG1 - gPAG1 - FM的转基因植物或对照IP,分别从Col-0中。以指定的时间间隔停止反应混合物。用抗SE抗体探测6xHis-SE的Western印迹法。


数据分析


银染色和体外20S蛋白酶体降解试验的代表性图像也可以参考Li等人的图4c和4d 。(2020 )。蛋白酶体活性的代表性数据可以参考Li等人的扩展数据图7b 。(2020年)。


笔记


在STE p乙,全部为纯化所需的缓冲液和管应预先-在使用之前冷却到4℃。
在步骤B中,Miracloth被提取缓冲液预湿。
在步骤B中,将最终洗脱样品分成几支试管,以避免将来使用时冻融样品,因为冻融样品会影响蛋白酶体的活性。
在步骤E中,根据测试蛋白质优化体外降解条件,包括pH,降解时间,降解温度以及20S蛋白酶体和测试蛋白质的浓度。
在步骤E中,快速准备反应混合物并将其均匀分布并保持在冰上。确保所有时间点都开始同时做出反应。

菜谱


2 × SDS-PAGE加载缓冲区
0.125 mM的Tris-HCl ,pH 6.8


20%甘油


4%SDS


20万DTT


0.02%溴酚蓝


提取缓冲液
50 mM的Tris-HCl ,pH 7.5


25毫米氯化钠


2毫米氯化镁2


1毫米EDTA


5%甘油


2 mM PMSF(在使用前添加)


              洗涤缓冲液
50 mM的Tris-HCl ,pH 7.5


800毫米氯化钠


2毫米氯化镁2


1毫米EDTA


5%甘油


2 mM PMSF(在使用前添加)


反应缓冲液
50 mM的Tris-HCl ,pH 7.5


25毫米氯化钠


2毫米氯化镁2


1毫米EDTA


2 mM二硫苏糖醇(DTT)


5%甘油


50 μM的Suc -LLVY-AMC底物


致谢


NIH(授予GM127742)对XZ的资助支持了这项工作。此处介绍的协议是根据以前的出版物(Yang等,2004;Zhang等,2006 ;Book等,2010 ;Hsieh等)开发的。 。,2015年;汉等人,2019 ),并在我们最近的工作优化(李等人,2020 )。


利益争夺


作者宣称没有利益冲突。


参考


本尼桑,G 。和Sharon,M.(2014年)。 调节20S蛋白酶体泛素独立的降解途径。生物分子4 (3):862-884。
Book,AJ,Gladman ,NP,Lee,SS,Scalf ,M.,Smith,LM和Vierstra ,RD(2010)。拟南芥26S蛋白酶体的亲和纯化揭示了多种植物蛋白水解复合物。Ĵ生物学化学285:25554-25569。
MM,布拉德福德(1976)。利用蛋白质-染料结合原理,快速而灵敏的定量微克蛋白质的方法。肛门乙iochem 72:248-254。
Han,JJ,Yang,X.,Wang,Q.,Tang,L.,Yu,F.,Huang,X.,et al。(2019)。β5亚基对于完整的26S蛋白酶体组装对于在盐胁迫下特异性促进植物自养生长至关重要。新植物221 (3):1359-1368。
Hsieh,L.S. ,Su,W.M. ,Han,G.S .和Carman,GM(2015)。磷酸化调节20S蛋白酶体对酵母Pah1磷脂酰磷酸酶的泛素依赖性降解。Ĵ生物学化学290 (18):11467-11478。
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Leggett,DS,Hanna,J.,Borodovsky ,A.,Crosas ,B.,Schmidt,M.,Baker,RT等。(2002)。多种相关蛋白调节蛋白酶体的结构和功能。分子细胞10 (3):495-507。
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引用:Li, Y., Sun, D., Yan, X., Wang, Z. and Zhang, X. (2021). In vitro Reconstitution Assays of Arabidopsis 20S Proteasome. Bio-protocol 11(7): e3967. DOI: 10.21769/BioProtoc.3967.
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