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Expression and Purification of the GRAS Domain of Os-SCL7 from Rice for Structural Studies
表达、纯化水稻Os-SCL7的GRAS结构域用于结构研究   

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The Plant Cell
May 2016

Abstract

GRAS proteins, named after the first three members GAI, RGA and SRC, has been found in 294 embryophyta species and is represented by 1,035 sequences. They belong to a plant-specific protein family and play essential roles in plant growth and development. Proteins in this family are defined as minimally containing a conserved GRAS domain, which is about 350-450 resides and can be subdivided into five distinct motifs with their name derived from the most prominent amino acids: LRI (leucine-rich region I), VHIID, LRII (leucine-rich region II), PFYRE and SAW and mainly function in the interaction between GRAS proteins and their partners (Sun et al., 2012).By phylogenetic analysis, the GRAS family can be divided into more than ten subfamilies, of which SCL4/7 is one important subgroup and functions in response to environmental stresses. Here we describe a detailed protocol for the expression and purification of the GRAS domain of Os-SCL7, a SCL4/7 member in rice, which enables us to crystallize it and determine its structure.

Keywords: Expression (表达), Purification (纯化), GRAS (GRAS), Os-SCL7 (Os-SCL7), Rice (水稻), Structural studies (结构研究)

Background

The GRAS proteins are a large family that plays vital roles in plant development and signaling transduction. Findings indicate that some family members such as DELLAs function as a repressor of GA responsive plant growth and are key regulatory targets in the GA signaling pathway (Murase et al., 2008), NSP1 and NSP2 play important roles in regulating nodulation development and signaling (Kaló et al., 2005), the proteins SCR and SHR together play an important role in the control of radial patterning for both the root and shoot (Helariutta et al., 2000), AtLAS is a key regulator in the developmental processes of the axillary meristem (Greb et al., 2003), HAM functions in shoot meristem maintenance (Stuurman et al., 2002).

Based on sequence analyses, GRAS proteins include a variable N-terminal domain and a widely and highly conserved C-terminal domain known as the GRAS domain. The N-terminal domains constitute a plant-specific unfoldome and may act as molecular bait by initiating the key molecular recognition events (Uversky et al., 2010). And the C-terminal GRAS domain is highly conserved in the whole GRAS family, suggesting that these proteins share a similar function and/or a common mode-of-action (Sun et al., 2012).

Though many members of GRAS proteins have been studied, the functional mechanism of GRAS proteins is still unclear. Structural descriptions of GRAS proteins may deeply clarify the functional mechanism of this family. However as yet little structural analyses have been reported, mainly due to the difficulties in obtaining sufficient quality and quantity of GRAS proteins. Soto et al. (2014) reported the expression and purification of the GRAS domain of rice SLR1. They constructed a GST-SLR1 fusion protein and expressed it in E. coli. But the expression levels were low (0.5 mg [TB medium] or 0.2 mg [M9 medium] of purified protein from 1 L flask culture). With some modifications, they obtained 1-3 mg of stable isotope labeled purified protein at 87% purity from 1 L of fermenter culture. However, the expression levels and purity of SLR1 are both not enough for crystallization. Moreover, reducing the protein synthesis rate by low culture temperature and speed, co-expressing with chaperonins, expressing as a fusion protein with soluble tags such as glutathione S-transferase, thioredoxin and maltose-binding protein or mutating some hydrophobic or disulfide forming amino acids are usually used to improve the expression and solubility of target proteins. While using more purification methods and steps will help to improve the purity of target proteins. In order to get a high purity and quantity of GRAS protein, we established a protocol for easy expression and purification of the GRAS domain of Os-SCL7 from rice.

Materials and Reagents

  1. Amicon Ultra centrifugal filters, 30 K (Merck Millipore, catalog number: UFC903096 )
  2. Amicon Ultra centrifugal filters 30,000 MWCO (EMD Millipore, catalog number: UFC803024 )
  3. Economical biotech dialysis membrane, 14 KD (Sangon Biotech, catalog number: TX0111 )
  4. Escherichia coli (E. coli) (BL21) (Thermo Fisher Scientific, InvitrogenTM, catalog number: C6000-03 )
  5. pET32aM (Novagen, modified from pET32a by inserting a TEV protease cut site inside the NcoI cleavage site) (Figure 1)
  6. Rice (NM_001057650, residues 201-578)
  7. Ampicillin (Sangon Biotech, catalog number: A100741 )
  8. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sangon Biotech, catalog number: A100487 )
  9. Chelating Sepharose Fast FLow (GE Healthcare, catalog number: 17057502 )
  10. Nickel(II) sulfate hexahydrate (NiSO4·6H2O) (Sangon Biotech, catalog number: A600658 )
  11. Imidazole (Sangon Biotech, catalog number: A500529 )
  12. TEV (tobacco etch virus) protease (produced in-house) (Fang et al., 2007)
  13. Tryptone (Oxoid, catalog number: LP0042 )
  14. Yeast extract (Oxoid, catalog number: LP0021 )
  15. Sodium chloride (NaCl) (Sangon Biotech, catalog number: A501218 )
  16. Agar (Oxoid, catalog number: LP0011 )
  17. Tris-base (Sangon Biotech, catalog number: A100826 )
  18. Hydrochloric acid (HCl) (Sinopharm Chemical Reagent, catalog number: 7647-01-0 )
  19. Glycerol (Sangon Biotech, catalog number: A100854 )
  20. Ethylenediaminetetraacetic acid disodium salt (EDTA) (Sangon Biotech, catalog number: A100105 )
  21. β-mercaptoethanol (AMRESCO, catalog number: 60-24-2 )
  22. Tween-20 (Sangon Biotech, catalog number: A100777 )
  23. Ethanol anhydrous (Sangon Biotech, catalog number: A500737 )
  24. Phosphoric acid ortho (85%) (Sangon Biotech, catalog number: A502803 )
  25. Acetic acid (Sangon Biotech, catalog number: A501931 )
  26. Coomassie Brilliant Blue G-250 (Sangon Biotech, catalog number: A100615 )
  27. Coomassie Brilliant Blue R-250 (Sangon Biotech, catalog number: A100472 )
  28. SDS (Sangon Biotech, catalog number: A100227 )
  29. Isopropanol (Sangon Biotech, catalog number: A507048 )
  30. LB medium (see Recipes)
  31. LB-agar plates (see Recipes)
  32. Lysis buffer (see Recipes)
  33. Chromatography buffer (see Recipes)
  34. Coomassie Brilliant Blue G-250 buffer (see Recipes)
  35. SDS-PAGE staining solution (see Recipes)
  36. SDS-PAGE destaining solution (see Recipes)


    Figure 1. The map of pET32aM plasmid

Equipment

  1. 2 L flask
  2. Spectrophotometer device (Kebo instrument, model: UV-1100 )
  3. Sonicator (Scientz, model: IID )
  4. Refrigerated centrifuge (Eppendorf, model: 5810 R )
  5. Chromatography system (GE Healthcare, model: INV-907 )
    Note: This product has been discontinued.
  6. HiLoad 16/600 Superdex 200(GE Healthcare, catalog number: 28-9893-35 )
  7. Electrophoresis system (Liuyi, model: DYY-6D )

Procedure

  1. Clone the gene encoding the GRAS domain Os-SCL7 from rice (NM_001057650, residues 201-578) into the modified pET32a vector (pET32aM) (Figure 1).
  2. Transform the recombinant plasmid (OsSCL7201-578-pET32aM), which was verified by DNA sequencing, into Escherichia coli strain BL21 (DE3) by heat shock method (Hanahan, 1983) and plate on LB-agar plates containing 100 mg/L ampicillin for 12 h at 37 °C.
  3. Pick a single colony and culture the E. coli in 10 ml LB medium containing 100 mg/L ampicillin at 37 °C for 5 h. Then transfer 10 ml culture to 1 L fresh LB medium containing 100 mg/L ampicillin (in a 2 L flask) and culture at 37 °C until OD600 nm reaches 0.4-0.6.
  4. Cool the cultures to about 16 °C and then induce protein expression with 0.3 mM IPTG for 12-14 h at 16 °C, 180 rpm.
  5. Harvest the cells by centrifugation at 8,800 x g for 5 min and discard the supernatant, then suspend the cells in 50 ml lysis buffer by vortexing.
  6. Sonicate the resuspended cells on ice with the power of 380 W for 30 min (ultrasonication is set to 2 sec ‘on’ and 6 sec ‘off’, and repeat this cycle).
    Note: The proteins should always be kept on ice and avoided of high energy produced during the ultrasonicaton process. Having an interval time should be better in this process (here the ultrasonication process is set as three times with 10 min for each time and 2 min interval between every two times).
  7. After sonication, centrifuge the sample at 18,000 x g for 30 min.
  8. Transfer the supernatant to a new tube, add imidazole into the supernatant to a final concentration of 20 mM and mix it thoroughly. Keep the pellet (containing the cell debris and protein aggregates) on ice before being subjected to SDS-PAGE (step 11).
    Note: Adding a final concentration of 20 mM imidazole into the supernatant can effectively reduce non-specific proteins binding.
  9. Load the sample onto a nickel-sepharose affinity resin (Chelating sepharose Fast FLow) which was pre-equilibrated with lysis buffer.
  10. Elute the bound protein with lysis buffers containing a gradient of imidazole (10 mM, 20 mM, 50 mM and 500 mM).
  11. Subject the eluted protein mixed with loading buffer to SDS-PAGE (Figure 2a). The protein of interest should be between 45 kDa and 66.2 kDa, according to the molecular weight 59 kDa of OsSCL7 GRAS domain fused with TRX and 6His-tag.
    Note: In order to get high purity proteins, it’s better to use a series concentration of imidazole to wash the nonspecific binding proteins and make sure each concentration of imidazole could not nearly elute any protein (see Notes). The target proteins are in the 500 mM imidazole eluent.
  12. Add TEV protease to the eluted proteins of interest (mTEV:mprotein = 1:10) to remove the TRX and 6His-tag from OsSCL7 GRAS domain. Then transfer the mixture to a dialysis membrane and dialyze it in lysis buffer on a rotator overnight at 4 °C.
    Note: In this step, the lysis buffer is used to dialyze imidazole to a low concentration. Generally it is lower than 5 mM.
  13. Load the cleaved protein onto the nickel-sepharose affinity resin again. The target proteins are in the flow through and subsequent 20 mM imidazole eluent. The band of the target protein is between 35 and 45 kDa, according to the molecular weight 42 kDa of the OsSCL7 GRAS domain (Figure 2b).
  14. Then load the sample into a HiLoad 16/600 Superdex 200 column pre-equilibrated with 1.2 column volume of chromatography buffer (Figure 3a). The samples of OsSCL7 monomer and dimer were assessed by SDS-PAGE (Figure 3b).
  15. Pool the fractions containing target protein and concentrate the sample to 5 to 8 mg/ml, measured by Bradford method on spectrophotometer device (Bradford, 1976), with Amicon Ultra centrifugal filters for subsequent crystallization experiment.

Data analysis



Figure 2. 15% SDS-PAGE analysis of purified OsSCL7 GRAS domain by nickel-sepharose affinity resin stained with Coomassie Brilliant Blue. a. Lane 1: The aggregates (produced in step 8); Lane 2: The flow (step 8); Lane 3: Eluted with 20 mM imidazole; Lane 4: Eluted with 500 mM imidazole; M: Marker. The arrow indicates the similar MW with the predicted MW (59 kDa) of OsCL7 GRAS domain (41.5 kDa) + Trx-tag (17.5 kDa); b. Lane 1: The flow of the second nickel-sepharose affinity resin purification (produced in step 11, The TRX and 6His-tag were cut off from the OsSCL7 GRAS domain), according to the MW of OsSCL7 GRAS domain, around 42 kDa; Lane 2: Elution with 20 mM imidazole, which also contains the OsSCL7 GRAS domain; M: Marker.


Figure 3. Purification of OsSCL7 GRAS domain by size-exclusion chromatography. a. Size-exclusion chromatography (HiLoad 16/600 Superdex 200 column), the dimer and monomer of OsSCL7 GRAS domain appear at 72.26 and 79.68 ml respectively, their corresponding molecular weights are 84 kDa and 42 kDa. b. 15% SDS-PAGE analysis of purified OsSCL7 GRAS domain from fractions of the observed peak in size-exclusion chromatography, showing high purity.

Notes

  1. The TEV protease prepared here must be pure enough (more than 90%) and the second nickel-sepharose affinity resin step is important for OsSCL7-GRAS domain purification.
  2. A pre-experiment was performed to determine the concentration of imidazole would elute the target protein well.
  3. A series of different concentrations of imidazole is used for washing the nonspecific binding proteins (the highest concentration of imidazole used in this step must be lower than which is for elution of the target protein). And Coomassie Brilliant Blue G-250 buffer (see Recipes) is used to detect no proteins can be washed down any longer by each concentration of imidazole.

Recipes

  1. LB medium
    10 g/L tryptone
    5 g/L yeast extract
    10 g/L NaCl
  2. LB-agar plates
    10 g/L tryptone
    5 g/L yeast extract
    10 g/L NaCl
    1.5% agar
  3. Lysis buffer
    500 mM NaCl
    50 mM Tris-HCl buffer (pH 8.0)
    5% glycerol
    0.2 mM EDTA
    2 mM β-mercaptoethanol
    0.1% Tween-20
  4. Chromatography buffer
    100 mM NaCl
    25 mM Tris-HCl buffer (pH 8.0)
    5% glycerol
  5. Coomassie Brilliant Blue G-250 buffer
    47.5 ml ethanol anhydrous
    100 ml phosphoric acid ortho (85%)
    0.1 g G-250
    Add ddH2O to 1 L
  6. SDS-PAGE staining solution
    300 ml ethanol anhydrous
    100 ml acetic acid
    1 g Brilliant Blue R-250
    Add ddH2O to 1 L
  7. SDS-PAGE destaining solution
    300 ml ethanol anhydrous
    100 ml acetic acid
    Add ddH2O to 1 L

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31500218).

References

  1. 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.
  2. Fang, L., Jia, K. Z., Tang, Y. L., Ma, D. Y., Yu, M. and Hua, Z. C. (2007). An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization. Protein Expr Purif 51(1): 102-109.
  3. Greb, T., Clarenz, O., Schafer, E., Muller, D., Herrero, R., Schmitz, G. and Theres, K. (2003). Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation. Genes Dev 17(9): 1175-1187.
  4. Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166(4): 557-580.
  5. Helariutta, Y., Fukaki, H., Wysocka-Diller, J., Nakajima, K., Jung, J., Sena, G., Hauser, M.T. and Benfey, P. N. (2000). The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell 101(5): 555-567.
  6. Kaló, P., Gleason, C., Edwards, A., Marsh, J., Mitra, R. M., Hirsch, S., Jakab, J., Sims, S., Long, S. R., Rogers, J., Kiss, G. B., Downie, J. A. and Oldroyd, G. E. (2005). Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science 308(5729): 1786-1789.
  7. Murase, K., Hirano, Y., Sun, T. P. and Hakoshima, T. (2008). Gibberellin-induced DELLA recognition by the gibberellin receptor GID1. Nature 456(7221): 459-463.
  8. Sato, T., Miyanoiri, Y., Takeda, M., Naoe, Y., Mitani, R., Hirano, K., Takehara, S., Kainosho, M., Matsuoka, M., Ueguchi-Tanaka, M. and Kato, H. (2014). Expression and purification of a GRAS domain of SLR1, the rice DELLA protein. Protein Expr Purif 95: 248-258.
  9. Stuurman, J., Jaggi, F. and Kuhlemeier, C. (2002). Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells. Genes Dev 16(17): 2213-2218.
  10. Sun, X., Jones, W. T. and Rikkerink, E. H. (2012). GRAS proteins: the versatile roles of intrinsically disordered proteins in plant signalling. Biochem J 442(1): 1-12.
  11. Uversky, V. N. (2010). The mysterious unfoldome: structureless, underappreciated, yet vital part of any given proteome. J Biomed Biotechnol 2010: 568068.

简介

以前3个成员GAI,RGA和SRC命名的GRAS蛋白质已被发现在294个胚胎种,并由1,035个序列表示。它们属于植物特异性蛋白质家族,在植物生长和发育中起重要作用。该家族中的蛋白质被定义为最低限度地含有保守的GRAS结构域,其约为350-450个,并且可以细分为五个不同的基序,其名称源自最突出的氨基酸:LRI(富含亮氨酸的区域I),VHIID ,LRII(富含亮氨酸的区域II),PFYRE和SAW,并且主要在GRAS蛋白质与其配偶体之间的相互作用中起作用(Sun等人,2012)。通过系统发育分析,GRAS家族可以分为十多个亚科,其中SCL4 / 7是一个重要的亚组,对应对环境压力有作用。这里我们描述了Os-SCL7(水稻SCL4 / 7成员)的GRAS结构域的表达和纯化的详细方案,使我们能够使其结晶并确定其结构。

背景 GRAS蛋白是一个大家族,在植物发育和信号转导中起重要作用。结果表明,一些家庭成员如DELLAs起到GA响应植物生长的抑制作用,是GA信号通路(Murase等人,2008)中的关键调控目标,NSP1和NSP2起重要作用在调节结瘤发育和信号传导(Kaló等人,2005)中,蛋白质SCR和SHR一起在控制根和芽的径向图案中起重要作用(Helariutta et al。等等,2000),AtLAS是腋生分生组织(Greb等人,2003)的发育过程中的关键调节因子,射枝分生组织维持中的HAM功能(Stuurman et al。,2002)。
 基于序列分析,GRAS蛋白包括可变的N末端结构域和广泛和高度保守的被称为GRAS结构域的C末端结构域。 N末端结构域构成植物特异性的解折叠体,并且可以通过引发关键的分子识别事件作为分子诱饵(Uversky等人,2010)。而在整个GRAS家族中,C末端GRAS结构域是高度保守的,这表明这些蛋白质具有相似的功能和/或共同的作用模式(Sun等人,2012)。
 尽管研究了GRAS蛋白的许多成员,但GRAS蛋白的功能机制尚不清楚。 GRAS蛋白的结构描述可以深入阐明该家族的功能机制。然而,迄今为止报道的结构分析尚不多,主要是由于难以获得足够的质量和数量的GRAS蛋白质。 Soto等人(2014)报道了水稻SLR1的GRAS结构域的表达和纯化。他们构建了一种GST-SLR1融合蛋白,并在E中表达。大肠杆菌。但是表达水平较低(0.5毫克[TB培养基]或0.2毫克[M9培养基] 1升烧瓶培养物中的纯化蛋白)。通过一些修改,他们从1升发酵罐培养物中获得了1-3毫克的稳定同位素标记的纯化蛋白质,纯度为87%。然而,SLR1的表达水平和纯度都不足以进行结晶。此外,通过低培养温度和速度降低蛋白质合成速率,与伴侣蛋白共表达,用可溶性标签如谷胱甘肽S-转移酶,硫氧还蛋白和麦芽糖结合蛋白表达融合蛋白或突变一些疏水或二硫键形成的氨基酸通常用于改善靶蛋白的表达和溶解度。使用更多的纯化方法和步骤有助于提高靶蛋白的纯度。为了获得高纯度和高含量的GRAS蛋白,我们建立了一个方便表达和纯化水稻Os-SCL7的GRAS结构域的方法。

关键字:表达, 纯化, GRAS, Os-SCL7, 水稻, 结构研究

材料和试剂

  1. Amicon超离心过滤器,30K(Merck Millipore,目录号:UFC903096)
  2. Amicon超离心过滤器30,000 MWCO(EMD Millipore,目录号:UFC803024)
  3. 经济型生物技术透析膜,14KD(Sangon Biotech,目录号:TX0111)
  4. 大肠杆菌(大肠杆菌)(BL21)(Thermo Fisher Scientific,Invitrogen TM,目录号:C6000-03)
  5. pET32aM(Novagen,通过在NcoI切割位点插入TEV proteasecut位点从pET32a修饰)(图1)
  6. 水稻(NM_001057650,残留201-578)
  7. 氨苄青霉素(Sangon Biotech,目录号:A100741)
  8. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Sangon Biotech,目录号:A100487)
  9. 螯合Sepharose Fast FLow(GE Healthcare,目录号:17057502)
  10. 硫酸镍(II)六水合物(NiSO 4·6H 2 O)(Sangon Biotech,目录号:A600658)
  11. 咪唑(Sangon Biotech,目录号:A500529)
  12. TEV(tabacco蚀刻病毒)蛋白酶(室内生产)(Fang等人,2007)
  13. 胰蛋白胨(Oxoid,目录号:LP0042)
  14. 酵母提取物(Oxoid,目录号:LP0021)
  15. 氯化钠(NaCl)(Sangon Biotech,目录号:A501218)
  16. 琼脂(Oxoid,目录号:LP0011)
  17. 三碱基(Sangon Biotech,目录号:A100826)
  18. 盐酸(HCl)(国药化学试剂,目录号:7647-01-0)
  19. 甘油(Sangon Biotech,目录号:A100854)
  20. 乙二胺四乙酸二钠盐(EDTA)(Sangon Biotech,目录号:A100105)
  21. β-巯基乙醇(AMRESCO,目录号:60-24-2)
  22. 吐温-20(Sangon Biotech,目录号:A100777)
  23. 无水乙醇(Sangon Biotech,目录号:A500737)
  24. 磷酸邻(85%)(Sangon Biotech,目录号:A502803)
  25. 乙酸(Sangon Biotech,目录号:A501931)
  26. 考马斯亮蓝G-250(Sangon Biotech,目录号:A100615)
  27. 考马斯亮蓝R-250(Sangon Biotech,目录号:A100472)
  28. SDS(Sangon Biotech,目录号:A100227)
  29. 异丙醇(Sangon Biotech,目录号:A507048)
  30. LB培养基(参见食谱)
  31. LB-琼脂平板(参见食谱)
  32. 裂解缓冲液(见配方)
  33. 色谱缓冲液(参见食谱)
  34. 考马斯亮蓝G-250缓冲液(参见食谱)
  35. SDS-PAGE染色溶液(参见食谱)
  36. SDS-PAGE脱色溶液(参见食谱)


    图1. pET32aM质粒的图谱

设备

  1. 2升烧瓶
  2. 分光光度计装置(Kebo仪器,型号:UV-1100)
  3. 超声波仪(Scientz,型号:IID)
  4. 冷冻离心机(Eppendorf,型号:5810 R)
  5. 色谱系统(GE Healthcare,型号:INV-907)
    注意:本产品已停产。
  6. HiLoad 16/600 Superdex 200(GE Healthcare,目录号:28-9893-35)
  7. 电泳系统(Liuyi,型号:DYY-6D)

程序

  1. 将编码GRAS结构域Os-SCL7的基因(NM_001057650,残基201-578)克隆到修饰的pET32a载体(pET32aM)中(图1)。
  2. 将通过DNA测序验证的重组质粒(OsSCL7 201-578 -pET32aM)通过热休克法(Hanahan,1983)转化到大肠杆菌菌株BL21(DE3) ),并在含有100mg/L氨苄青霉素的LB-琼脂平板上在37℃下培养12小时
  3. 选择一个殖民地并培养E。大肠杆菌在含有100mg/L氨苄青霉素的10ml LB培养基中在37℃下培养5小时。然后将10ml培养物转移到含有100mg/L氨苄青霉素(2L烧瓶)的1L新鲜LB培养基中,并在37℃下培养直至OD 600nm达到0.4-0.6。
  4. 将培养物冷却至约16℃,然后在16℃,180rpm下用0.3mM IPTG诱导蛋白质表达12-14小时。
  5. 通过以8,800×g离心收获细胞5分钟并丢弃上清液,然后通过涡旋将细胞悬浮在50ml裂解缓冲液中。
  6. 用380W的功率将重悬细胞在冰上超声处理30分钟(超声处理设置为2秒'开6秒',并重复该循环)。
    注意:蛋白质应始终保持在冰上,避免超声波过程中产生的高能量。在这个过程中,间隔时间应该更好(这里超声波处理设置为三次,每次10分钟,每两次间隔2分钟)。
  7. 超声处理后,将样品以18,000 x g离心30分钟。
  8. 将上清液转移到新管中,向上清液中加入咪唑至最终浓度为20 mM,并彻底混合。保持沉淀(含有细胞碎片和蛋白质聚集体)在冰上进行SDS-PAGE(步骤11)。
    注意:将最终浓度为20 mM的咪唑加入上清液可以有效降低非特异性蛋白质的结合。
  9. 将样品加载到用裂解缓冲液预平衡的镍 - 琼脂糖亲和树脂(Chelating sepharose Fast FLow)上。
  10. 用含有咪唑(10mM,20mM,50mM和500mM)梯度的裂解缓冲液洗脱结合的蛋白质。
  11. 将洗脱的蛋白质与加载缓冲液混合以进行SDS-PAGE(图2a)。根据与TRX和6His标签融合的OsSCL7 GRAS结构域的分子量59kDa,感兴趣的蛋白质应在45kDa至66.2kDa之间。
    注意:为了获得高纯度蛋白质,最好使用一系列浓度的咪唑洗涤非特异性结合蛋白,并确保咪唑浓度几乎不能洗脱任何蛋白质(见注释)。目标蛋白质在500mM咪唑洗脱液中。
  12. 将TEV蛋白酶加入目标洗脱的蛋白质(mTEV:mprotein = 1:10),以从OsSCL7 GRAS结构域中除去TRX和6His标签。然后将混合物转移到透析膜上,并在旋转器上的裂解缓冲液中在4℃下透析过夜。
    注意:在该步骤中,裂解缓冲液用于将咪唑透析至低浓度,通常低于5mM。
  13. 将裂解的蛋白质再次装入镍 - 琼脂糖亲和树脂上。目标蛋白质流经和随后的20mM咪唑洗脱液。根据OsSCL7 GRAS结构域的分子量42kDa,靶蛋白的条带在35和45kDa之间(图2b)。
  14. 然后将样品加载到预先用1.2柱体积的色谱缓冲液预平衡的HiLoad 16/600超级200柱(图3a)中。通过SDS-PAGE评估OsSCL7单体和二聚体的样品(图3b)。
  15. 收集含有目标蛋白的级分,并将样品浓缩至5至8mg/ml,通过Bradford方法在分光光度计装置(Bradford,1976)上测量,使用Amicon Ultra离心过滤器进行随后的结晶实验。

数据分析



图2.用考马斯亮蓝染色的镍琼脂糖亲和树脂纯化的OsSCL7 GRAS结构域的15%SDS-PAGE分析。 a。泳道1:聚集体(在步骤8中生产);泳道2:流(步骤8);泳道3:用20mM咪唑洗脱;泳道4:用500mM咪唑洗脱; M:标记箭头表示与预期的MW(59kDa)的OsCL7GRAS结构域(41.5KDa)+ Trx-标签(17.5KDa)相似的MW。 b。泳道1:根据OsSCL7 GRAS结构域的MW,约42kDa,第二次镍琼脂糖亲和力树脂纯化(在步骤11中产生,TRX和6His标签从OsSCL7 GRAS结构域切断)的流动;泳道2:用20mM咪唑洗脱,其还含有OsSCL7 GRAS结构域; M:标记。


图3.通过大小排阻色谱法纯化OsSCL7 GRAS域。 a。尺寸排阻色谱法(HiLoad 16/600 superdex 200柱),OsSCL7 GRAS结构域的二聚体和单体分别为72.26和79.68 ml,分子量分别为84 kDa和42 kDa。 b。 15%SDS-PAGE分析纯化的OsSCL7 GRAS结构域,从大小排阻层析中观察到的峰的部分,显示出高纯度。

笔记

  1. 这里制备的TEV蛋白质必须足够纯(超过90%),而第二种镍 - 琼脂糖亲和性树脂步骤对于OsSCL7-GRAS结构域纯化是重要的。
  2. 进行预实验以确定咪唑的浓度将很好地洗脱靶蛋白
  3. 咪唑的系列浓度用于洗涤非特异性结合蛋白(该步骤中使用的最高浓度的咪唑必须低于靶蛋白的洗脱浓度)。考马斯亮蓝G-250缓冲液(参见食谱)用于检测每个浓度的咪唑可以再次洗涤蛋白质。

食谱

  1. LB培养基
    10g/L胰蛋白胨
    5克/升酵母提取物
    10g/L NaCl
  2. LB-琼脂板
    10g/L胰蛋白胨
    5克/升酵母提取物
    10g/L NaCl
    1.5%琼脂
  3. 裂解缓冲液
    500 mM NaCl
    50mM Tris-HCl缓冲液(pH8.0)
    5%甘油
    0.2 mM EDTA
    2mMβ-巯基乙醇
    0.1%Tween-20
  4. 色谱缓冲液
    100 mM NaCl
    25mM Tris-HCl缓冲液(pH8.0)
    5%甘油
  5. 考马斯亮蓝G-250缓冲液
    无水乙醇47.5ml 100毫克磷酸邻(85%)
    0.1 g G-250
    将ddH 2 O添加到1 L
  6. SDS-PAGE染色溶液
    300毫升乙醇无水乙腈 100 ml乙酸
    1克亮蓝R-250
    将ddH 2 O添加到1 L
  7. SDS-PAGE去污解决方案
    300毫升乙醇无水乙腈 100 ml乙酸
    将ddH 2 O添加到1 L

致谢

这项工作得到了中国国家自然科学基金(31500218)的支持。

参考文献

  1. Bradford,MM(1976)。  一种快速敏感的方法用于利用蛋白质 - 染料结合原理定量微量的蛋白质。
    Anal Biochem 72:248-254。
  2. 方法,L.,Jia,KZ,Tang,YL,Ma,DY,Yu,M.和Hua,ZC(2007)。  用于大肠杆菌中高水平生产TEV蛋白酶的改进策略及其纯化和表征 蛋白质清除剂 51(1):102-109。
  3. Greb,T.,Clarenz,O.,Schafer,E.,Muller,D.,Herrero,R.,Schmitz,G。和Theres,K.(2003)。 >拟南芥揭示了腋生分生组织形成的保守控制机制。 Genes Dev 17(9):1175-1187。
  4. Hanahan,D。(1983)。  关于转化的研究具有质粒的大肠杆菌。 J Mol Biol 166(4):557-580。
  5. Helariutta,Y.,Fukaki,H.,Wysocka-Diller,J.,Nakajima,K.,Jung,J.,Sena,G.,Hauser,M.T。和Benfey,PN(2000)。  短基因基因通过径向信令控制拟南芥根的放射状图案。 101(5):555-567。 >
  6. Kal,P.,Gleason,C.,Edwards,A.,Marsh,J.,Mitra,RM,Hirsch,S.,Jakab,J.,Sims,S.,Long,SR,Rogers,J.,Kiss, GB,Downie,JA和Oldroyd,GE(2005)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15961668"target ="_ blank" >豆类中的结瘤信号需要NSP2,GRAS家族成员转录调节因子。 科学 308(5729):1786-1789。
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  9. Stuurman,J.,Jaggi,F.and Kuhlemeier,C。(2002)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/12208843"目标="_ blank">射击分生组织维护由区分细胞的GRAS 介导的信号控制。 Genes Dev 16(17):2213-2218 。
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引用:Li, S., Zhao, Y. and Wu, Y. (2017). Expression and Purification of the GRAS Domain of Os-SCL7 from Rice for Structural Studies. Bio-protocol 7(3): e2122. DOI: 10.21769/BioProtoc.2122.
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