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Purification of Rubisco from Chlamydomonas reinhardtii
莱茵衣藻中二磷酸核酮糖羧化酶的纯化   

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参见作者原研究论文

本实验方案简略版
Archives of Biochemistry and Biophysics
Feb 2015

Abstract

Chlamydomonas reinhardtii is a model organism for chloroplast studies. Besides other convenient features, the feasibility of chloroplast genome transformation distinguishes this unicellular alga as ideal for the manipulation of chloroplastic gene expression aiming biotechnological goals, such as improved biofuel and biomass production. Ribulose 1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) is the photosynthetic carbon-fixing enzyme which is considered crucial for biomass accumulation in algal cultures. Purification of wild type and site-directed mutants of Rubisco in C. reinhardtii is usually performed to study its catalytic properties and assess the carbon-fixing potential of the strains. In this protocol Rubisco is extracted through sonication of cell pellets, and purified by ammonium sulfate precipitation, sucrose gradient centrifugation (Goldwaithe and Bogorad, 1975) and anion exchange chromatography.

Keywords: Ribulose 1,5-bisphosphate carboxylase/oxygenase (二磷酸核酮糖的羧化酶/加氧酶), Protein purification (蛋白质纯化), Chlamydomonas reinhardtii (莱茵衣藻), Rubisco (Rubisco), Density gradient centrifugation (密度梯度离心法)

Materials and Reagents

  1. Vacuum driven disposable filtration system (Stericup 0.22 μm) (Merck Millipore Corporation, catalog number: P25628X1 )
  2. Graduated cylinder (50 ml)
  3. Sephadex G-25 columns (PD-10) (GE Healthcare, catalog number: 17-0851-01 )
  4. Glass rod
  5. Thick wall polycarbonate plastic tubes for ultracentrifugation (Beckman Coulter, catalog number: 355631 )
  6. Glass disposable micro pipet (Corning Inc., catalog number: 7099S-20 )
  7. Resource Q anion exchange column 6 ml (GE Healthcare, catalog number: 17-1179-01 )
  8. Chlamydomonas reinhardtii cells (about 8 g of wet weight collected from 2-3 liters of a 1.2 x 107 cells/ml photo-heterotrophic culture)
  9. Tris (2-amino, 2-hydroxymethyl, 1, 3-propanediol) (Trizma base) (Sigma-Aldrich, catalog number: T-1503 )
  10. Magnesium sulfate heptahydrate (MgSO4.7H2O) (vidraFOC, Panreac, catalog number: 131404-1210 )
  11. Magnesium chloride hexahydrate (MgCl2.6H2O) (Merck Millipore Corporation, catalog number: 1.05833 )
  12. Sodium chloride (NaCl) (AppliChem GmbH, Panreac, catalog number: 121659.1211 )
  13. Sodium bicarbonate (NaHCO3) (Merck Millipore Corporation, catalog number: 1.06329 )
  14. 2-mercaptoethanol (Merck Millipore Corporation, catalog number: 8.05740 )
  15. Sulfuric acid (H2SO4) (Sigma-Aldrich, Fluka, catalog number: 84718 )
  16. Hydrochloric acid (HCl) (VWR International, J.T.Baker®, catalog number: 6081 )
  17. Sucrose (Sigma-Aldrich, Fluka, catalog number: 84100 )
  18. cOmplete (Protease inhibitors tablets) (Roche Diagnostics, catalog number: 11697 498001 )
  19. Poly(vinylpolypyrrolidone) (Sigma-Aldrich, catalog number: P-6755 )
  20. Ammonium sulfate [(NH4)2SO4] (AppliChem GmbH, Panreac, catalog number: 141140 )
  21. dH2O (deionized water) (processed by the Milli-Q system from Merck Millipore Corporation)
  22. Coomassie Blue-stained SDS-PAGE (optional)
  23. Extraction buffer (EB) (see Recipes)
  24. Sucrose gradient buffer (SGB) (see Recipes)
  25. Sucrose density gradient (see Recipes)
  26. Chromatography buffer A (CBA) (see Recipes)
  27. Chromatography buffer B (CBB) (see Recipes)
  28. Activation buffer (AB) (see Recipes)

Equipment

  1. Ultrasonic processor (Sonics Vibracell, model: VCX 500 ) equipped with a 19 mm high-gain probe
  2. Optical microscope (Microscope Central, Nikon, model: Alphaphot 2 YS2 )
  3. Magnetic stirrer (Bibby Scientific, Stuart, model: SB161-3 ) and magnetic bars
  4. Preparative centrifuge (GMI, Beckman Coulter, model: J2-HS ) equipped with a fixed-angle rotor (Beckman Coulter, model: JA-20 )
  5. Ultracentrifuge (GMI, Beckman Coulter, model: L-70 ) equipped with a fixed-angle rotor (Beckman Coulter, model: 55.2 Ti )
  6. Two-chamber (2 x 8 ml) gradient mixer (homemade)
  7. UV Monitor (GE Healthcare, Amersham-Pharmacia Biotech, model: UV-1 with Rec112 register )
  8. Fast Performance Liquid Chromatography equipment (FPLC) (GE Healthcare, Amersham-Pharmacia Biotech, model: Äkta Prime )
  9. Peristaltic pump (GE Healthcare, Pharmacia, model: P-1 )
  10. UV-V spectrophotometer (Shimadzu Scientific Instruments, model: UV-1603 )
  11. Water jet vacuum pump (aspirator) (KARTELL SPA VIA DELLE INDUSTRIE, model: 1395 )
  12. Ultrasonic water bath (Scientific Support, Branson, model: 2200 )

Procedure

  1. Extraction and ammonium sulfate precipitation of Rubisco
    1. Rubisco can be purified either from freshly harvested cultures (pelleted by centrifugation at 2,000 x g for 5 min) or from frozen pellets (which may be conserved indefinitely if immediately frozen in liquid nitrogen). In the latter case, the frozen cells should be thawed on ice in the presence of extraction buffer (EB) with protease inhibitors (see next step).
    2. Resuspend about 8 g (wet weight) of cell pellets in 32 ml of ice-cold EB (containing protease inhibitors).
    3. Sonicate the cell suspension in an ice bath with 6 pulses (of 30 sec each, separated by 60 sec standby intervals) of a 19 mm-high gain probe delivering 100 W. The extent of cell wall breakage may be checked by optical microscopy.
    4. Add 0.64 g of insoluble polyvinylpolypyrrolidone to the sonicated suspension and stir with a magnetic bar for 5 min in a cold room (4 °C).
    5. Centrifuge at 25,000 x g for 15 min at 4 °C, collect the supernatant (avoiding the dark green layer on top of the pellet) and measure its volume in a graduated cylinder.
    6. Add 0.194 g of solid ammonium sulfate per ml of supernatant (this will bring the solution to 35% salt saturation) while stirring with a magnetic bar. Keep stirring for 30 min in the cold room (4 °C).
    7. Centrifuge at 25,000 x g for 10 min at 4 °C, collect the clear supernatant and measure its volume in a graduated cylinder again.
    8. Add 0.151 g of solid ammonium sulfate per ml of supernatant (in order to bring the solution to 60% salt saturation) while stirring again for 30 min in the cold room (4 °C).
    9. Centrifuge at 25,000 x g for 10 min at 4 °C and discard the supernatant. Resuspend the precipitated protein in less than 3 ml of sucrose gradient buffer (SGB) by mixing carefully with a glass rod (do not vortex because this would develop perdurable foam).
    10. Bring the volume of solution to 5 ml and load it onto two Sephadex G-25 (PD-10) desalting columns (2.5 ml each) previously equilibrated with SGB at 4 °C. Elute each column with 3.5 ml of the same SGB, finally pooling the eluates.

  2. Sucrose gradient centrifugation
    1. By using a two-chamber gradient mixer, prepare four plastic thick-wall centrifuge tubes containing a 16 ml linear gradient of 0.2 to 0.8 M sucrose dissolved in SGB.
    2. Distribute the 7 ml of the desalted eluate on top of the four 16 ml sucrose gradients and balance the tubes for equal weight by pairs.
    3. Ultracentrifuge the tubes at 132,000 x g for 4 h in a fixed angle rotor at 4 °C. Select a slow initial acceleration to avoid perturbation of the gradient.
    4. After centrifugation, fractionate the content of each tube using a glass cannula (e.g., a disposable micro pipet) stuck to the bottom of the tube, connected to an aspiring peristaltic pump in line with a UV-monitor adjusted to 280 nm (Figure 1).


      Figure 1. Assembly for collecting fractions from the sucrose gradient after ultracentrifugation

    5. Collect separately the distinct Rubisco peak which appears within 2 and 5 ml from the bottom of the tube (see Figure 2). Combine the collected peaks from the four tubes and store frozen at -20 °C unless the ensuing chromatography is to be performed immediately. Gradient fractions may be stored at -20 °C indefinitely.


      Figure 2. Typical record of the optical density at 280 nm of the sucrose gradient collected from the bottom of the tube after ultracentrifugation. The peak of Rubisco (B) is flanked by a descending absorbance due to nucleic acids (A) and an increasing absorption by the bulk of other (less weighty) proteins (C).

  3. Anion-exchange chromatography
    1. Dilute the pooled Rubisco-containing fractions obtained from the sucrose gradient three-fold with chromatography buffer A (CBA).
    2. Load the diluted fractions on a 6 ml Resource Q ion-exchange column mounted on a FPLC system and equilibrated with CBA at a 2 ml/min flux rate.
    3. Elute the column at a 6 ml/min flux with a programmed 120 ml (i.e., 20 column volumes) linear gradient ranging from 0% to 42% of chromatography buffer B (CBB) in CBA while monitoring the absorbance at 280 nm of the eluate. Collect the Rubisco, which will elute as a sharp and very intense peak around a 25% of CBB. Afterwards, the column can be thoroughly washed with 100% CBB, before re-equilibrating it with CBA for processing the next sample. Otherwise, follow the manufacturer's instructions for column storage.

  4. Rubisco desalting
    1. Load 2.5 ml of the collected Rubisco solution onto a Sephadex G-25 (PD-10) desalting column equilibrated with activation buffer (AB) and elute the protein with 3.5 ml of the same buffer. This step will eliminate salts [which inhibit Rubisco activity at concentrations higher than 0.2 M (Sudhani et al., 2013)], and provide Mg2+ and CO2, which promote the catalytically competent conformation of Rubisco.

Representative data

The final preparation should contain more than 95% of Rubisco protein being substantially free of nucleic acids (A280/A260 ratio higher than 1.9). Rubisco concentration may be calculated from the UV absorbance assuming an extinction coefficient of ε1% = 15.9 dl·g-1·cm-1. at 280 nm. Typical final concentration of Rubisco is about 1 mg/ml.
Figure 3 illustrates the degree of purity as ascertained by Coomassie Blue-stained SDS-PAGE. The described protocol leads to high purity Rubisco that might be used even for crystallization of the enzyme. In case that such a degree of purity is not needed, the protocol might be shortened omitting the anion exchange chromatography (section C). After the sucrose gradient centrifugation, Rubisco is already more than 80% of the total protein in the extract (see Figure 3B), although there is still a significant contamination of nucleic acids.


Figure 3. Coomassie-stained SDS-PAGE analysis of Rubisco purification from C. reinhardtii. Arrows indicate the position of the large (LS) and small (SS) subunits of Rubisco on a 14% acrylamide SDS-PAGE gel. A. Final preparation of purified Rubisco (0.25 μg, lane P) run in parallel to markers (MM) of molecular mass (in kDa). B. Fractions (0.7 ml each) collected from the bottom of the sucrose gradient after centrifugation. The red line above indicates the fractions to be pooled for further purification by anion exchange chromatography.

Notes

  1. We have successfully tested the same purification protocol starting from crude extracts obtained from many other species including leaves of higher plants (spinach, rice, mulberry and orange trees), fronds of Lemna, and cultures of Euglena. Only when purifying Rubisco from diatom species, the ammonium sulfate cuts had to be shifted (to 0-35% saturation) for better yield (Marín-Navarro et al., 2010).

Recipes

  1. Extraction buffer (EB) (100 mM Tris-sulfate, 10 mM MgSO4, 20 mM 2-mercaptoethanol, pH 8.0)
    For 200 ml:
    Dissolve 2.42 g of Tris and 0.493 g of MgSO4.7H2O in about 150 ml of dH2O
    Adjust the pH to 8.0 with diluted (e.g., 0.5 M) H2SO4
    Bring the final volume to 200 ml and store at 4 °C
    Dissolve 1 tablet of protease inhibitors per 50 ml, and add 1.4 μl of 2-mercaptoethanol per ml immediately before use.
  2. Sucrose gradient buffer (SGB) (10 mM Tris-H2SO4, 10 mM MgSO4, 10 mM NaHCO3, 1 mM β-mercaptoethanol, pH 8)
    For 200 ml:
    Dissolve 2.42 g of Tris, 0.493 g of MgSO4.7H2O and 0.168 g of NaHCO3 in about 150 ml of dH2O
    Add 140 μl of 2-mercaptoethanol
    Adjust the pH to 8.0 with diluted (e.g., 0.5 M) H2SO4
    Bring the final volume to 200 ml
    This buffer should be prepared fresh for each experiment shortly before dissolving the sucrose and preparing the gradients.
  3. Preparing the sucrose density gradient
    For 4 gradients:
    0.2 M sucrose solution: Dissolve 2.55 g of sucrose in 35 ml of SGB
    0.8 M sucrose solution: Dissolve 9.30 g of sucrose in 28.2 ml of SGB
    Fill the mixing (stirred) chamber of a gradient mixer with 8 ml of the 0.8 M sucrose solution and the auxiliary chamber with 8 ml of 0.2 M sucrose solution.
    Connect the two chambers and direct the outflow through a peristaltic pump to a thick wall ultracentrifuge tube, letting the mixed solution drop along the wall to the bottom of the tube (see Figure 4).
    Repeat this procedure four times to get four gradients in four separate centrifuge tubes.
    Cool down the gradients to 4 °C by leaving them undisturbed (i.e., no vibrations) in the cold room for several hours or overnight.


    Figure 4. Preparing the sucrose density gradients

  4. Chromatography buffer A (CBA) (20 mM Tris-chloride, pH 7.5)
    For 1 L:
    Dissolve 2.42 g of Tris in about 0.8 L of dH2O
    Carefully adjust the pH to 7.5 using diluted (e.g., 0.2 M) HCl
    Bring the volume to 1 L and filter through a sterilizing 0.22 μm disposable filtration system connected to an aspirator vacuum pump
    Shortly before using, degas the buffer by placing the bottle (losely open) in an ultrasonic water bath for 10 min
  5. Chromatography buffer B (CBB) (20 mM Tris-chloride, 1 M NaCl, pH 7.5)
    For 0.5 L:
    Dissolve 1.21 g of Tris and 29.58 g of NaCl in about 0.4 L of dH2O
    Carefully adjust the pH to 7.5 using diluted (e.g., 0.2 M) HCl
    Bring the volume to 0.5 L, and filter and degas as for CBA
  6. Activation buffer (AB) (100 mM Tris-HCl, 10 mM MgCl2, 10 mM NaHCO3, pH 8.2)
    For 100 ml:
    Dissolve 1.210 g of Tris, 0.203 g of MgCl2.6H2O and 0.084 g of NaHCO3 in some 80 ml of dH2O
    Adjust the pH to 8.2 using diluted (e.g., 1 M) HCl
    Bring the final volume to 100 ml and transfer to a 100 ml bottle leaving little headspace. This buffer should be prepared shortly before use to avoid loss of bicarbonate as CO2.

Acknowledgments

This work was supported by a grant (UV-INV-AE14-269247) of the University of Valencia. The protocol is a detailed and expanded version of the one published as supplementary information in Sudhani and Moreno (2015).

References

  1. Goldwaithe, J. and Bogorad, L. (1975). Ribulose-1,5-diphosphate carboxylase from leaf. Meth Enzymol 42: 481-487.
  2. Marín-Navarro, J., García-Murria, M. J. and Moreno, J. (2010). Redox properties are conserved in Rubiscos from diatoms and green algae through a different pattern of cysteines. J Phycol 46: 516-524.
  3. Sudhani, H. P. and Moreno, J. (2015). Control of the ribulose 1,5-bisphosphate carboxylase/oxygenase activity by the chloroplastic glutathione pool. Arch Biochem Biophys 567: 30-34.
  4. Sudhani, H. P., Garcia-Murria, M. J. and Moreno, J. (2013). Reversible inhibition of CO2 fixation by ribulose 1,5-bisphosphate carboxylase/oxygenase through the synergic effect of arsenite and a monothiol. Plant Cell Environ 36(6): 1160-1170.

简介

衣藻衣原体是叶绿体研究的模式生物体。 除了其他方便的特点,叶绿体基因组转化的可行性区分这种单细胞藻理想的操纵叶绿体基因表达瞄准生物技术目标,如改善生物燃料和生物质生产。 核酮糖1,5-二磷酸羧化酶/加氧酶(EC 4.1.1.39,Rubisco)是光合作用碳固定酶,其被认为对藻类培养物中的生物量累积是至关重要的。 Rubisco的野生型和定点突变体的纯化。 通常进行研究其催化性质并评估菌株的碳固定潜力。 在该方案中,通过细胞沉淀的超声处理提取Rubisco,并通过硫酸铵沉淀,蔗糖梯度离心(Goldwaithe和Bogorad,1975)和阴离子交换层析纯化。

关键字:二磷酸核酮糖的羧化酶/加氧酶, 蛋白质纯化, 莱茵衣藻, Rubisco, 密度梯度离心法

材料和试剂

  1. 真空驱动一次性过滤系统(Stericup0.22μm)(Merck Millipore Corporation,目录号:P25628X1)
  2. 量筒(50ml)
  3. Sephadex G-25柱(PD-10)(GE Healthcare,目录号:17-0851-01)
  4. 玻璃杆
  5. 用于超速离心的厚壁聚碳酸酯塑料管(Beckman Coulter,目录号:355631)
  6. 玻璃一次性微量移液管(Corning Inc.,目录号:7099S-20)
  7. Resource Q阴离子交换柱6ml(GE Healthcare,目录号:17-1179-01)
  8. (从2-3升的1.2×10 7细胞/ml光异养培养物中收集的约8g湿重)的细胞。
  9. 三(2-氨基,2-羟基甲基,1,3-丙二醇)(Trizma碱)(Sigma-Aldrich,目录号:T-1503)
  10. 硫酸镁七水合物(MgSO 4,7H 2 O)(vidraFOC,Panreac,目录号:131404-1210)
  11. 氯化镁六水合物(MgCl 2·6H 2 O·6H 2 O)(Merck Millipore Corporation,目录号:1.05833)
  12. 氯化钠(NaCl)(AppliChem GmbH,Panreac,目录号:121659.1211)
  13. 碳酸氢钠(NaHCO 3)(Merck Millipore Corporation,目录号:1.06329)
  14. 2-巯基乙醇(Merck Millipore Corporation,目录号:8.05740)
  15. 硫酸(H 2 SO 4)(Sigma-Aldrich,Fluka,目录号:84718)
  16. 盐酸(HCl)(VWR International,J.T.Baker ,目录号:6081)
  17. 蔗糖(Sigma-Aldrich,Fluka,目录号:84100)
  18. cOmplete(蛋白酶抑制剂片剂)(Roche Diagnostics,目录号:11697498001)
  19. 聚(乙烯基聚吡咯烷酮)(Sigma-Aldrich,目录号:P-6755)
  20. 硫酸铵[(NH 4)2 SO 4](AppliChem GmbH,Panreac,目录号:141140)
  21. dH 2 O(去离子水)(由来自Merck Millipore Corporation的Milli-Q系统处理)
  22. 考马斯蓝染SDS-PAGE(可选)
  23. 提取缓冲液(EB)(参见配方)
  24. 蔗糖梯度缓冲液(SGB)(参见配方)
  25. 蔗糖密度梯度(参见配方)
  26. 色谱缓冲液A(CBA)(参见配方)
  27. 色谱缓冲液B(CBB)(参见配方)
  28. 激活缓冲区(AB)(参见配方)

设备

  1. 配有19 mm高增益探头的超声波处理器(Sonics Vibracell,型号:VCX 500)
  2. 光学显微镜(Microscope Central,Nikon,型号:Alphaphot 2 YS2)
  3. 磁力搅拌器(Bibby Scientific,Stuart,型号:SB161-3)和磁棒
  4. 装备有固定角转子(Beckman Coulter,型号:JA-20)的预制离心机(GMI,Beckman Coulter,型号:J2-HS)
  5. 装备有固定角转子(Beckman Coulter,型号:55.2Ti)的超速离心机(GMI,Beckman Coulter,型号:L-70)
  6. 两室(2×8ml)梯度混合器(自制)
  7. UV监视器(GE Healthcare,Amersham-Pharmacia Biotech,型号:具有Rec112寄存器的UV-1)
  8. 快速液相色谱设备(FPLC)(GE Healthcare,Amersham-Pharmacia Biotech,型号:?ktaPrime)
  9. 蠕动泵(GE Healthcare,Pharmacia,型号:P-1)
  10. UV-V分光光度计(Shimadzu Scientific Instruments,型号:UV-1603)
  11. 水喷射真空泵(吸气器)(KARTELL SPA VIA DELLE INDUSTRIE,型号:1395)
  12. 超声水浴(Scientific Support,Branson,型号:2200)

程序

  1. Rubisco的提取和硫酸铵沉淀
    1. Rubisco可以从新鲜收获的培养物中纯化(沉淀 ?通过在2,000×g离心5分钟)或从冷冻丸粒(其通过离心 ?如果立即在液体中冷冻,可以无限期保守 氮)。在后一种情况下,冷冻的细胞应该在冰上解冻 在具有蛋白酶抑制剂的提取缓冲液(EB)的存在下 下一步)。
    2. 将约8g(湿重)的细胞沉淀重悬在32ml冰冷的EB(含蛋白酶抑制剂)中。
    3. 超声处理细胞悬浮液在冰浴中用6个脉冲(30秒, ?每个,间隔60秒的等待间隔)的19mm高增益探头 ?传送100W。细胞壁破裂的程度可以通过 光学显微镜
    4. 加入0.64g不溶物 将聚乙烯聚吡咯烷酮加入到超声处理的悬浮液中, 磁条在冷室(4℃)中5分钟
    5. 离心机 25,000×g 在4℃温育15分钟,并收集上清液(避免 深绿色层在颗粒的顶部)测量其体积在a 量筒
    6. 每毫升加入0.194克固体硫酸铵 的上清液(这将使溶液达到35%盐饱和) 同时用磁棒搅拌。在冷却下保持搅拌30分钟 ?房间(4℃)。
    7. 在4℃下以25,000×g离心10分钟 收集澄清的上清液,再次测量其体积在刻度 ?缸。
    8. 加入0.151g固体硫酸铵/ml 上清液(以使溶液达到60%盐饱和) 在冷室(4℃)中再次搅拌30分钟
    9. 离心机 25,000×g在4℃温育10分钟,弃去上清液。重新悬挂 ?沉淀的蛋白质在小于3ml的蔗糖梯度缓冲液(SGB) ?通过与玻璃棒小心混合(不要涡旋,因为这将 形成耐久的泡沫)
    10. 使溶液体积为5 ml 并加载两个Sephadex G-25(PD-10)脱盐柱(2.5ml 每个)预先在4℃下用SGB平衡。用每个柱洗脱 3.5 ml相同的SGB,最后汇集洗脱液。

  2. 蔗糖梯度离心
    1. 通过使用双室梯度混合器,准备四个塑料厚壁 离心管含有0.2至0.8M的16ml线性梯度 蔗糖溶于SGB
    2. 分配7 ml脱盐洗脱液 在四个16ml蔗糖梯度的顶部并平衡管 等于重量。
    3. 在4℃下在固定角转子中将管在132,000×g下超速离心4小时。选择慢初始 加速度以避免梯度的扰动
    4. 后 离心,使用玻璃分级每个管的内容物 插管(例如,一次性微型移液管)粘在底部 管,连接到符合a的有向蠕动泵 UV监视器调整到280 nm(图1)。


      图1.超速离心后从蔗糖梯度收集馏分的装配

    5. 单独收集出现在2内的不同Rubisco峰 并从管的底部取5ml(参见图2)。结合 从四个管收集峰,并存储在-20°C冷冻,除非 随后立即进行色谱法。梯度 馏分可无限期储存在-20℃

      图2.典型 记录蔗糖梯度在280nm的光密度 超速离心后从管的底部收集。 Rubisco(B)的峰侧面是由于核酸的下降吸光度 ?酸(A)和增加吸收的其他(较少 重量)蛋白(C)。

  3. 阴离子交换色谱
    1. 用层析缓冲液A(CBA)稀释从蔗糖梯度获得的合并的含Rubisco的级分三次。
    2. 将稀释的级分装载在6ml Resource Q离子交换柱上 安装在FPLC系统上并用CBA以2ml/min通量平衡 率。
    3. 用编程的120以6ml/min的流量洗脱柱子 ml(,20柱体积)线性梯度,范围为0%至42% 色谱缓冲液B(CBB)在CBA中,同时监测吸光度 280nm的洗脱液。收集Rubisco,这将洗脱作为一个锋利 和在25%的CBB附近的非常强的峰。之后,列可以 ?用100%CBB彻底洗涤,然后用CBA重新平衡 用于处理下一个样品。否则请按照制造商 列存储的说明。

  4. Rubisco脱盐
    1. 将2.5ml收集的Rubisco溶液装载到Sephadex G-25上 (AB)平衡的(PD-10)脱盐柱 用3.5ml相同的缓冲液洗脱蛋白质。这一步将会 消除盐[其在更高的浓度下抑制Rubisco活性 ?比0.2M(Sudhani等人,2013)],并提供Mg 2+和CO 2 +,其中 促进Rubisco的催化能力的构象。

代表数据

最终制剂应含有大于95%的Rubisco蛋白质,其基本上不含核酸(A280/A260比率高于1.9)。可以从假定消光系数ε1%= 15.9dl·g -1 ·cm <-1> 的UV吸光度计算Rubisco浓度。 。 Rubisco的典型最终浓度为约1mg/ml 图3说明了通过考马斯蓝染色的SDS-PAGE确定的纯度。所描述的方案导致高纯度Rubisco,其甚至可以用于酶的结晶。在不需要这种纯度的情况下,可以省略阴离子交换层析(C部分)来缩短方案。在蔗糖梯度离心后,Rubisco已经是提取物中总蛋白的80%以上(见图3B),尽管仍然存在核酸的显着污染。


图3.来自C的Rubisco纯化的考马斯染色的SDS-PAGE分析。 reinhardtii 。 箭头表示在14%丙烯酰胺SDS-PAGE凝胶上Rubisco的大(LS)和smal(SS)亚基的位置。 A.纯化的Rubisco(0.25μg,泳道P)的最终制备与分子量(以kDa计)的标记(MM)平行进行。 B.离心后从蔗糖底部收集级分(每次0.7ml)。上面的红色线表示待合并的级分,用于通过阴离子交换色谱法进一步纯化。

笔记

  1. 我们已经成功地测试了相同的纯化方案,从从许多其他物种,包括高等植物的叶子(菠菜,水稻,桑树和橙树),叶子的Lemna和Euglena的文化获得的粗提取物。只有当从硅藻种中纯化Rubisco时,硫酸铵馏分必须移动(至0-35%饱和度),以获得更好的产率(Marín-Navarro等人,2010)。

食谱

  1. 提取缓冲液(EB)(100mM Tris-硫酸盐,10mM MgSO 4,20mM 2-巯基乙醇,pH8.0)
    对于200毫升:
    将约2.42g的Tris和0.493g的MgSO 4·7H 2 O 2溶解在约150ml的dH 2/O
    用稀释的(例如0.5M)H 2 SO 4 4调节pH至8.0/
    使最终体积为200 ml,并在4℃下保存
    每50 ml溶解1片蛋白酶抑制剂,在使用前立即加入1.4μl2-巯基乙醇/ml。
  2. 蔗糖梯度缓冲液(SGB)(10mM Tris-H 2 SO 4,10mM MgSO 4,10mM NaHCO 3, 1mMβ-巯基乙醇,pH 8) 对于200毫升:
    将2.42g Tris,0.493g MgSO 4·7H 2 O和0.168g NaHCO 3溶解在二氯甲烷中,约150ml的dH 2 O 2 / 加入140μl的2-巯基乙醇 用稀释的(例如0.5M)H 2 SO 4 4调节pH至8.0/
    使最终体积为200 ml
    对于每个实验,在溶解蔗糖和制备梯度之前不久应该新鲜制备该缓冲液
  3. 准备蔗糖密度梯度
    对于4个渐变:
    0.2M蔗糖溶液:将2.55g蔗糖溶于35ml SGB中 0.8M蔗糖溶液:将9.30g蔗糖溶于28.2ml SGB中 用8ml 0.8M蔗糖溶液填充梯度混合器的混合(搅拌)室,辅助室用8ml 0.2M蔗糖溶液填充。
    连接两个室,并通过蠕动泵将流出物引导至厚壁超速离心管,使混合溶液沿着壁滴落到管的底部(参见图4)。
    重复此过程四次,以在四个独立的离心管中获得四个梯度 将冷却室中的温度保持不变(即无振动)数小时或过夜,将梯度冷却至4°C。


    图4.准备蔗糖密度梯度

  4. 色谱缓冲液A(CBA)(20mM Tris-氯化物,pH7.5) 对于1 L:
    将2.42g的Tris溶解在约0.8L的dH 2 O中 使用稀释的(例如,0.2M)HCl
    小心地调节pH至7.5 使体积为1 L,并通过连接到吸气真空泵的灭菌0.22μm一次性过滤系统过滤 使用前,在超声波水浴中脱气10分钟(缓缓打开)瓶子
  5. 色谱缓冲液B(CBB)(20mM Tris-氯化物,1M NaCl,pH7.5) 对于0.5 L:
    将1.21g Tris和29.58g NaCl溶于约0.4L dH 2 O中。
    使用稀释的(例如,0.2M)HCl
    小心地调节pH至7.5 使体积达到0.5升,过滤和除气与CBA一样
  6. 活化缓冲液(AB)(100mM Tris-HCl,10mM MgCl 2,10mM NaHCO 3,pH8.2)
    对于100 ml:
    将1.210g的Tris,0.203g的MgCl 2·6H 2 O,6H 2 O和0.084g的NaHCO 3溶解在约80ml dH 2 O 2 / 使用稀释的(例如,1mM)HCl
    将pH调节至8.2 使最终体积为100毫升,并转移到一个100毫升的瓶子,留下很少的顶空。该缓冲液应在使用前不久制备以避免碳酸盐作为CO 2的损失。

致谢

这项工作是由瓦伦西亚大学的赠款(UV-INV-AE14-269247)支持的。该协议是在Sudhani和Moreno(2015)中作为补充信息发布的一个详细和扩展版本。

参考文献

  1. Goldwaithe,J。和Bogorad,L。(1975)。 叶中的核酮糖-1,5-二磷酸羧化酶 Meth Enzymol 42:481-487。
  2. Marín-Navarro,J.,García-Murria,M.J.and Moreno,J。(2010)。 氧化还原性质在Rubiscos中通过不同的模式保存在硅藻和绿藻中的半胱氨酸。 46:516-524
  3. Sudhani,H. P.和Moreno,J.(2015年)。 叶绿体谷胱甘肽库控制核酮糖1,5-二磷酸羧化酶/加氧酶活性。 Arch Biochem Biophys 567:30-34。
  4. Sudhani,H.P.,Garcia-Murria,M.J.and Moreno,J。(2013)。 由核酮糖1,5-二磷酸羧化酶对CO 2定位的可逆抑制/氧化酶通过亚砷酸盐和单硫醇的协同作用。植物细胞环境36(6):1160-1170。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sudhani, H. P., García-Murria, M. J., Marín-Navarro, J., García-Ferris, C., Peñarrubia, L. and Moreno, J. (2015). Purification of Rubisco from Chlamydomonas reinhardtii. Bio-protocol 5(23): e1673. DOI: 10.21769/BioProtoc.1673.
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