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Rubisco Extraction and Purification from Diatoms
硅藻中的Rubisco提取和纯化   

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Abstract

This protocol describes a method to extract ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) from diatoms (Bacillariophyta) to determine catalytic performance. This protocol has been adapted from use in cyanobacteria and higher plants (Andrews, 1988; Whitney and Sharwood, 2007). First part (steps A1-A3) of the extraction provides a crude extract of Rubisco that is sufficient for carboxylation assays to measure the Michaelis constant for CO2 (KC) and the catalytic turnover rate (kcatc). However, the further purification steps outlined (steps B1-B4) are needed for measurements of Rubisco CO2/O2 Specificity (SC/O, [Kane et al., 1994]).

Keywords: Rubisco(Rubisco), Diatoms(硅藻), Extraction(萃取), Phytoplankton(浮游植物), Carbon fixation(碳固定)

Background

Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco, EC 4.1.1.39) catalyzes the first step in the photosynthetic assimilation of CO2 and thus plays a fundamental role in photosynthesis and the global carbon cycle. Rubisco has been isolated from a wide range of organisms, from archaea, bacteria, algae to plants, and displays a diverse range of kinetics between organisms (Galmes et al., 2014; Tcherkez et al., 2006; Whitney et al., 2011). Knowledge of Rubisco kinetics is a key component for understanding how photosynthesis and thus the biological sink of carbon will respond to rising anthropogenic CO2. Diatoms are a group of unicellular algae responsible for ~20% of global photosynthesis (Falkowski and Raven, 2007) but as yet have been relatively poorly studied in terms of their Rubisco kinetics.

Isolation and purification of Rubisco is required before kinetic assays can be undertaken. Due to differences in cell structure and organic composition between organisms, the method for the purification of viable Rubisco enzyme needs to be continually optimized. This protocol describes a method to extract and purify Rubisco using size exclusion chromatography from diatoms in preparation for kinetic assays. The method is similar to Whitney and Sharwood (2007), used for the purification of Rubisco overexpressed in E. coli, in that a French press is used to mechanically rupture cells. The French press is necessary to obtain sufficient cell lysis as diatoms are unicellular with silica frustules, unlike plant tissue in which sufficient lysis is easily achieved by homogenizing frozen leaf tissue in a mortar and pestle. Furthermore, due to the low in vivo concentrations of Rubisco in diatoms (Losh et al., 2013), large diatom culture volumes concentrated via centrifugation, are needed to obtain enough biomass compared to plant tissue and E. coli lines with overexpressed Rubisco.

Materials and Reagents

  1. 15 ml centrifuge tubes with conical bottoms
  2. 50 ml centrifuge tubes with conical bottoms
  3. 500 ml centrifuge tubes with conical bottoms
  4. 1.5 ml microcentrifuge tubes
  5. 1 ml syringe
  6. 1 ml Bio-Scale mini Macro-Prep high Q ion exchange column (Bio-Rad Laboratories, catalog number: 7324120 )
  7. Amicon Ultra-4 centrifugal filter (30,000 NMWL) (EMD Millipore, catalog number: UFC803024 )
  8. Amicon Ultra-100 centrifugal filter (100,000 NWML) (EMD Millipore, catalog number: UFC910024 )
  9. Diatoms
  10. Liquid nitrogen
  11. Polyvinylpolypyrrolidone (PVPP; insoluble) (Sigma-Aldrich, catalog number: 77627 )
  12. Additional materials To test for Rubisco activity (Optional):
    Labelled CO2 (as NaH14CO3) (5 mCi) (PerkinElmer, catalog number: NEC086H005MC )
    Ribulose-1,5-bisphosphate (RuBP; synthesized, purified and stored anaerobically as described in Kane et al., 1998)
  13. Acetic acid (Sigma-Aldrich, catalog number: A9967 or 27225 )
    Note: The product acetic acid ( A9967 ) has been discontinued.
  14. Methanol (Sigma-Aldrich, catalog number: M1770 or 494437 )
    Note: The product Methanol ( M1770 ) has been discontinued.
  15. Soluble protein (as determined by Bradford assay) (Coomassie Plus Assay Kit) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23236 )
  16. Additional materials to test for protein using Native and SDS-PAGE (Optional):
    Gel code blue stain (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 24590 )
    4-12% Tris-glycine mini gels (Thermo Fisher Scientific, InvitrogenTM, catalog number: XV04120PK20 )
    4-12% Bis-Tris gels (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0321PK2 )
    SDS reducing buffer for SDS-PAGE (see Recipes)
    TBS buffer for SDS-PAGE (see Recipes)
    AttoPhos reagent (Astral Scientific, Gymea, NSW, Australia)
    Antisera raised against the large subunit holoenzyme of Rubisco in Phaeodactylum tricornutum
    Alkaline Phosphatase conjugated secondary antibody
  17. 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) (Sigma-Aldrich, catalog number: E9502 )
  18. Ethylenediaminetetraacetic acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  19. Dithiothreitol (Sigma-Aldrich, catalog number: D0632 )
  20. Plant protease inhibitor cocktail (Sigma-Aldrich, catalog number: P9599 )
  21. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  22. Triethanolamine (Sigma-Aldrich, catalog number: 90279 )
  23. Magnesium acetate (Sigma-Aldrich, catalog number: M5661 )
  24. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  25. Extraction buffer (see Recipes)
  26. Column buffer (see Recipes)
  27. Column elution buffer (see Recipes)
  28. Specificity (SC/O) buffer (see Recipes)

Note: All chemicals are of A.C.S. grade.

Equipment

  1. French pressure cell press (Thermo Fisher Scientific, model: FA-078 )
  2. Coulter Counter Z Series (Beckman Coulter, model: Z Series Coulter Counter )
  3. Centrifuge (large volumes [1 L] at 2,000 x g, small volumes [< 15 ml] at 17, 600 x g, 4 °C)
  4. Fume hood
  5. Superdex 200 (GE Healthcare, catalog number: 17517501 )
  6. FPLC (Äkta Pure 25) setup at 4 °C for size-exclusion chromatography using Superdex 200/30 (GE Healthcare, model: Äkta Pure 25 )
  7. To confirm purification and activity of Rubisco:
    1. Protein transfer apparatus and immunoblot imagining equipment – to check abundance and purity of extracted Rubisco
    2. Radioisotope laboratory and associated septum capped vials and syringes

Procedure

  1. Total soluble cellular (crude) protein extraction – suitable for measurement of the Michaelis constant for CO2 (KC), catalytic turnover rate (kcatc) and the first step for CO2/O2 specificity assays (SC/O).
    1. Diatoms are grown in 2 L batch cultures at 20 °C under continuous light (c. 150 μmol photons m-2 sec-1) in sterile seawater to which 0.2 μM filtered nutrients, vitamins and trace metals were added to give the final concentrations as defined in the Aquil medium. For a full recipe and guidelines for making Aquil recipe, see Sunda et al., 2005. Growth rates are monitored by cell counting using a Coulter Counter. During exponential growth, cells are concentrated to a pellet via gentle centrifugation (2,000 x g for 10 min) at 15 °C and the supernatant discarded. Approximately 1 g of a cell pellet is required to purify Rubisco for measuring SC/O. To limit storage space, collect cells into 1.5 ml microcentrifuge tubes and then snap freeze in liquid nitrogen and store at -80 °C until further analysis.
    2. The cell pellets are re-suspended in a total of 5 ml ice cold extraction buffer in a 15 ml polypropylene tube. The cells are ruptured using a pre-chilled French press at 140 MPa (see Figure 1 for photo and Video 1 of French press) and the cell lysate collected into the same tube.


      Figure 1. Cell lysis using a French press. Photo of a French Pressure Cell that can efficiently lyse microalgae cells, including diatoms. Algae cell extracts in ice cold buffer are placed in the ice-cold stainless steel French Pressure Cell and hydraulic pressure applied. Once at 140 MPa the outlet to the Cell is slowly opened. The cells lyse as they emerge from high to ambient pressure and the cell extract is collected into a 15 ml or 50 ml polypropylene tubes. A slow flow rate of sample out of the Cell (~1 to 2 drops sec-1) is maintained to ensure the pressure is maintained (see Video 1, acknowledgement Bratati Mukherjee). 

      Video 1. Use of French press to lyse cells

    3. To the cell lysate, add polyvinylpolypyrrolidone (1%, w/v) to bind secondary metabolites, which are then removed by centrifugation (17,600 x g, 4 °C, 5 min).
    4. After centrifugation Rubisco remains fully intact as confirmed by native PAGE (see Data analysis, Figure 2) and fully soluble, remaining in the cell lysis supernatant as confirmed by SDS-PAGE (see Data analysis, Figure 3).
    5. Rubisco kcatc (and KC) is quantified by 14CO2 fixation assays (see Data analysis).
      1. Rubisco in the soluble protein extract is activated for 10 to 15 min with 15 mM NaH14CO3 and 15 mM MgCl2.
      2. In 7 ml septum capped glass scintillation vials 20 μl of the extract is assayed for 60 sec in 0.5 ml of 50 mM EPPS-NaOH pH 8.2, 10 mM MgCl2, 0.6 mM RuBP, 10 μg ml-1 carbonic anhydrase and varying NaH14CO3 concentrations (1.2 to 12 mM which equates to ~15 to 150 μM 14CO2 at pH 8.0 at 25 °C) (Sharwood et al., 2016). The buffer and vials are equilibrated with the appropriate O2/N2 gas mixture prior to adding the NaH14CO3 and sample.
      3. The reaction is stopped with 0.2 ml 0.5 N formic acid and non-fixed 14CO2 is vented by heating the vials at 85 °C in the fume hood.
      4. The dried residue is dissolved in 0.5 ml double distilled H2O and vortexed with 1 ml Scintillant (Packard, Ultima Gold XR) before the labelled 3-phosphoglycerate (14C) is measured in a scintillation counter. This crude extraction is suitable for measurements of Rubisco maximum carboxylation rate (Vmax) and Rubisco Michaelis constant for CO2 under N2 (KC) and 21:79% O2:N2 (Kcair) (Sharwood et al., 2008; Sharwood et al., 2016). To quantify kcatc the Vmax value is divided by the Rubisco content in the assay. Measuring Rubisco content is achieved using the 14C-CABP binding method or by immunoblot analysis. The accuracy and experimental limitations of both approaches are detailed in Whitney and Sharwood, 2014. Purification of Rubisco is necessary for measuring SC/O (see next step).

  2. Rubisco purification for measuring SC/O
    The soluble cellular protein needs to be further purified before quantifying SC/O (CO2/O2 specificity).
    1. Soluble cellular protein from step A3 above is manually passed by syringe through a 1 ml Bio-Scale mini Macro-Prep high Q ion exchange (IEX) column equilibrated with column buffer. All steps are performed at 4 °C. The method involves: Pre-washing the IEX column is with 2 ml of column buffer. Soluble cellular protein is passed through the column by syringe (flowrate at ~3 to 5 ml min-1) and then the column is washed with 8 ml of column buffer. Bound Rubisco is eluted in 1.5 ml column elution buffer, collecting in three 500 μl elution fractions. The last two fractions contain > 90% of the Rubisco activity and are therefore pooled before concentrating to ~0.4 ml by centrifugation (4,000 x g, 10 min, 4 °C) using an Amicon Ultra-100 centrifugal filter (i.e., a 100 kDa MW cut-off filter).
    2. The concentrated Rubisco is injected into the 200 μl sample loop of the Äkta purification system onto a Superdex 200 column pre-equilibrated with SC/O buffer at 4 °C. The sample is loaded onto the column using the flow rate of 0.5 ml/min (max delta column pressure alarm set to 1.5 MPA) and fractions (0.5 ml) collected after the void volume (8 ml). Absorbance at 280 nm and conductance are continuously monitored. RuBP dependent 14CO2-fixation activity in the fractions is assayed and those with peak Rubisco activity (typically fractions 5 to 7) are pooled and concentrated to ~0.1 ml by centrifugation (4,000 x g, 20 min, 4 °C) using an Amicon Ultra-100 centrifugal filter.
    3. The concentrated and purified Rubisco is now ready for the SC/O assay. Alternatively, glycerol can be added to 20% (v/v) final concentration and the enzyme then frozen in liquid nitrogen and stored at -80 °C.
    4. Optional: the SC/O assay (not part of this protocol on Rubisco extraction)
      1. SC/O assays are carried out at 25 °C (or other temperature) according to the method of (Kane et al., 1994). The purified Rubisco (10-50 μl) is injected into 20 ml glass, septum sealed glass vials containing 1 ml assay buffer (see Recipes) that has been equilibrated for 0.5 to 1 h with 500 ppm CO2 mixed with O2 using Wostoff gas-mixing pumps. After 15 min, further equilibration the reactions are initiated with the injection of 1 nmol of 2-3H-RuBP. Alkaline phosphatase is injected after 30 min to convert the 3-3H-phosphoglycerate (carboxylation product) and 2-3H-phosphoglycolate (oxygenation product) into 3H-3-glycerate and 3H-2-glycolate.
      2. The reactions are passed through a 0.4 ml AGI-X8 (10% formate) anion-exchange column and then the bound 3H-3-glycerate and 3H-2-glycolate eluted in 0.5 ml 20% (v/v) H2SO4 before separating them by HPLC using an isocratic gradient of 0.012 M H2SO4 through a Aminex HPX-87H column at 65 °C at a flow rate of 0.4 ml min-1.
      3. The SC/O at 25 °C is then calculated from the radioactivity in the 3H-3-glycerate and 3H-2-glycolate using equation:



        Where,
        represents the molar ratio of O2 and CO2 (99.95% and 0.05% [v/v] respectively),
        0.037 is the proportion of the CO2 solubility relative to O2 in H2O at 25 °C (Kane et al., 1994). 

Data analysis

  1. Intact Rubisco remains in the soluble fraction during extraction
    It was determined that full cell lysis is routinely obtained using a French Pressure cell and that Rubisco remains intact and within the soluble fraction during the crude extraction (steps A1-A3). Native PAGE (Figure 2) shows Rubisco remains as an intact L8S8 enzyme (comprising 8 large [L] and 8 small [S] subunits), even following electrophoresis for 16 h at 4 °C and 60 V. The differing mobility of the Rubsico band between species occurs due to subunit sequence differences and subtle variations in quaternary structural properties (i.e., slight differences in amino acid sequence and length). SDS-PAGE (Figure 3) demonstrates that comparable levels of Rubisco L- and S-subunit are detected in the whole cell lysis (after mechanical rupture but prior to centrifugation, step A2) and soluble cell protein (after centrifugation, step A3) indicating the extracted Rubisco is fully soluble.
  2. Rubisco retains activity after extraction
    The activity of Rubisco within the crude extracts (steps A1-A3) of 11 diatom species were tested and published (Young et al., 2016). Full activation of Rubisco was obtained after 10 min of extraction at 25 °C and the activity remained stable during a further 10 min testing. Measurements of KC and kcatc for the diatom Rubiscos are published in (Young et al., 2016).
  3. Quantifying SC/O (steps B1-B4). Figure 4 shows the 3H-elution profile of the HPLC separated 3H-glycerate and 3H-glycolate peaks which are used to calculate SC/O as described above in step B4c.


    Figure 2. Native-PAGE blot of crude extract to show fully intact Rubisco protein. Total soluble cell extract (5 μg as determined by Bradford assay, Coomassie plus) was separated at 4 °C through a precast 4-12% Tris-glycine gel overnight (16 h) at 60 V. Gel was rinsed with deionized water, fixed for 30 min with 45% (v/v) H2O, 5% (v/v) acetic acid and 50% (v/v) methanol the extensively rinsed with multiple changes if deionized water. The proteins were visualized using Gelcode blue Coomassie stain (Invitrogen). Arrows indicate where the ~520 kDa Rubisco complex (L8S8) locates on the gel. Lane numbers indicate extract from different diatom species: (1) Thalassiosira weissflogii CCMP 1336, (2) Thalassiosira oceania CS-427, (3) Skeletonema marinoi CCMP 1332, (4) Chaetoceros calcitrans CCMP 1315, (5) Chaetoceros calcitrans CS-178, (6) Chaetoceros muelleri CCMP 1316, (7) Phaeodactylum tricornutum CCMP 642, (8) Phaeodactylum tricornutum UTEX 630, (9) Phaeodactylum tricornutum CS-29, (10) Bellerochea sp. CS-874/01, (11) Isochrysis sp. CS-177, (12) Pleurochrysis cartera CS-287. Last three lanes contain 5, 10 and 20 μl of crude cell extract from tobacco as a control. Shown are the Rubisco active site contents quantified for each sample by 14C-CABP binding (Whitney and Sharwood, 2014; Sharwood et al., 2008).


    Figure 3. SDS-PAGE analysis of Rubisco solubility, integrity and complete extraction by French Pressure Cell lysis. A. Coomassie stain and B. Diatom Rubisco antibody blot (see Whitney et al., 2001 for details) of total cellular lysate (L, following French Pressure Cell lysis) and soluble cellular protein (S, following centrifugation) from the diatom species: (1) P. tricornutum CS-29, (2) Skeletonema ardens CS-348, (3) Pavlova lutheri CS-182, (4) Fragilariopsis cylindrus CCMP 1102, (5) Cylindrotheca fusiformis CS-13, (6) Thalassiosira oceania CS-427, and (7) Thalassiosira weissflogii CCMP 1336. 5 μl of tobacco soluble leaf protein was loaded for comparison. The equal intensity of the L-subunit in both the L and S protein fractions indicate all the Rubisco was extracted (complete cell lysis) and fully soluble with no L-subunit degradation evident in the Western blot. Sample preparation and electrophoresis: protein (L or S) extracts (150 μl) were added to 4x SDS-reducing buffer (50 μl) and boiled for 5 min then centrifuged (16,000 x g, 5 min) before separating by 4-12% Bis-Tris SDS-PAGE at 200 V for 45 min in MES buffer (50% methanol, 40% H2O and 10% glacial acetic acid). Duplicate gels were either (A) fixed and Coomassie stained (see Figure 2) or (B) the proteins transferred onto nitrocellulose membrane and probed with an antibody to P. tricornutum Rubisco (see Whitney et al., 2001 for further experimental details).


    Figure 4. 3H-Elution profile of the HPLC fractions that are used to calculate SC/O from the amount of 3H incorporated into 3H-glycerate and 3H-glycolate (peaks 1 and 2 respectively) as described in step B4c. Shown here are results of an SC/O assay using purified Rubisco from the diatoms, Thalassiosira weissflogii (T.w., red) and Phaeodactylum tricornutum (P.t., blue) showing two technical replicates (rep) for each.

Recipes

  1. Extraction buffer
    50 mM EPPS-NaOH, pH 8.0
    1 mM EDTA
    2 mM dithiothreitol (DTT)
    1% (v/v) plant protease inhibitor cocktail
    Dissolve EPPS and EDTA in deionized water to the final desired concentrations
    Bring pH to 8.0 with NaOH
    Notes:
    1. Add DTT just prior to starting extractions.
    2. Immediately prior to use, add protease inhibitor cocktail to the aliquot of buffer being used for extraction.
  2. Column buffer
    50 mM EPPS-NaOH, pH 8.0
    1 mM EDTA
    10 mM NaCl
    Dissolve all salts in deionized water to the final desired concentrations
    Bring pH to 8.0 with NaOH
  3. Column elution buffer
    50 mM EPPS-NaOH, pH 8.0
    1mM EDTA
    0.8 M NaCl
    Dissolve all salts in deionized water to the final desired concentrations
    Bring pH to 8.0 with NaOH
  4. Specificity (SC/O) buffer
    30 mM triethanolamine
    15 mM magnesium acetate, pH 8.3
    Dissolve all salts in deionized water to the final desired concentrations
    Bring pH to 8.3 with acetic acid
    Store at 4 °C
  5. Assay buffer (1 ml)
    30 mM triethanolamine
    15 mM magnesium acetate, pH 8.3
    10 μg ml-1 carbonic anhydrase
  6. 4x SDS reducing buffer for SDS-PAGE (Optional)
    125 mM Tris-HCl pH 6.8
    4% (w/v) SDS
    0.01% (w/v) bromophenol blue
    20% (v/v) glycerol
    75 mM 2-mercaptoethanol
    Dissolve all salts in deionized water to the final desired concentrations
    Add glycerol and 2-mercaptoethanol before use
  7. BS buffer for SDS-PAGE (Optional)
    10 mM Tris-HCl, pH 7.5
    150 mM NaCl
    Dissolve all salts in deionized water to the final desired concentrations 

Acknowledgments

We thank the reviewers for helpful comments that improved the article. Funding for J. N.Y. was through ANU visiting scholar (CE140100015) and NSF Grant 1040965. A.H. was funded through a Clarendon Scholarship, Oxford and ANU visiting scholar (CE140100015). R.E.S was funded through ARC DECRA scheme (DE13010760). R.E.M.R. was funded through ERC Starting Grant (SP2-GA-2008-200915), F.M.M.M. was funded through NSF Grant 104095 and S.M.W was funded through Australian Research Council Grant CE14010001. We would like to thank Bratati Mukherjee for assistance in the video of the French Press.

References

  1. Andrews, T. J. (1988). Catalysis by cyanobacterial ribulose-bisphosphate carboxylase large subunits in the complete absence of small subunits. J Biol Chem 263(25): 12213-12219.
  2. Falkowski, P. G. and Raven, J. A. (2007). Aquatic photosynthesis. Princeton University Press.
  3. Galmes, J., Kapralov, M. V., Andralojc, P. J., Conesa, M. A., Keys, A. J., Parry, M. A. and Flexas, J. (2014). Expanding knowledge of the Rubisco kinetics variability in plant species: environmental and evolutionary trends. Plant Cell Environ 37(9): 1989-2001.
  4. Kane, H. J., Viil, J., Entsch, B., Paul, K., Morell, M. K. and Andrews, T. J. (1994). An improved method for measuring the CO2/O2 specificity of ribulosebisphosphate carboxylase-oxygenase. Aust J Plant Physiol 21(4): 449-461.
  5. Kane, H. J., Wilkin, J. M., Portis, A. R. and John Andrews, T. (1998). Potent inhibition of ribulose-bisphosphate carboxylase by an oxidized impurity in ribulose-1,5-bisphosphate. Plant Physiol 117(3): 1059-1069.
  6. Losh, J. L., Young, J. N. and Morel, F. M. M. (2013). Rubisco is a small fraction of total protein in marine phytoplankton. New Phytol 198(1): 52-58.
  7. Sharwood, R. E., Ghannoum, O., Kapralov M. V., Gunn L. H. and Whitney, S. M. (2016). Variation in response of C3 and C4 Paniceae Rubisco to temperature provides opportunities for improving C3-photosynthesis. Nat Plants 2: 16186
  8. Sharwood, R. E., von Caemmerer, S., Maliga, P. and Whitney, S. M. (2008). The catalytic properties of hybrid Rubisco comprising tobacco small and sunflower large subunits mirror the kinetically equivalent source Rubiscos and can support tobacco growth. Plant Physiol 146(1): 83-96.
  9. Sunda, W. G., Price, N. M. and Morel, F. M. M. (2005). Trace metal ion buffers and their use in culture studies. In: Anderson, R. A. (Ed.). Algal culturing techniques. Elevier Academic Press, pp: 35-63.
  10. Tcherkez, G. G., Farquhar, G. D. and Andrews, T. J. (2006). Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proc Natl Acad Sci U S A 103(19): 7246-7251.
  11. Whitney, S. M., Baldet, P., Hudson, G. S. and Andrews, T. J. (2001). Form I Rubiscos from non-green algae are expressed abundantly but not assembled in tobacco chloroplasts. Plant J 26(5): 535-547.
  12. Whitney, S. M. and Sharwood, R. E. (2014). Plastid transformation for Rubisco engineering and protocols for assessing expression. In Maliga, P. (Ed.). Chloroplast Biotechnology. Humana Press, pp 245-262.
  13. Whitney, S. M., Houtz, R. L. and Alonso, H. (2011). Advancing our understanding and capacity to engineer nature’s CO2-sequestering enzyme, Rubisco. Plant Physiol 155(1): 27-35.
  14. Whitney, S. M. and Sharwood, R. E. (2007). Linked Rubisco subunits can assemble into functional oligomers without impeding catalytic performance. J Biol Chem 282(6): 3809-3818.
  15. Young, J. N., Heureux, A. M., Sharwood, R. E., Rickaby, R. E., Morel, F. M. M. and Whitney, S. M. (2016). Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms. J Exp Bot 67(11): 3445-3456.

简介

该方案描述了从硅藻(“芽孢杆菌”)提取核酮糖-1,5-二磷酸羧化酶加氧酶(Rubisco)的方法以确定催化性能。该方案已经在蓝细菌和高等植物中得到了应用(Andrews,1988; Whitney and Sharwood,2007)。提取的第一部分(步骤A1-A3)提供Rubisco的粗提取物,其足以用于羧化测定以测量CO 2(K 3 C)的Michaelis常数和催化更换率( k c )。然而,为了测量Rubisco CO 2 / O 2特异性(S C / O )需要进一步的纯化步骤(步骤B1-B4) >,[Kane等人,1994])。

背景 核酮糖-1,5-二磷酸羧化酶加氧酶(Rubisco,EC 4.1.1.39)催化了CO 2光合同化的第一步,因此在光合作用和全球碳循环中起着重要的作用。 Rubisco已经从古菌,细菌,藻类和植物的各种生物体中分离出来,并且在生物体之间显示出各种各样的动力学(Galmes等人,2014年; Tcherkez等人,2006; Whitney等人,2011)。 Rubisco动力学的知识是了解光合作用以及因此碳的生物沉积物对人为CO 2升高的响应的关键组成部分。硅藻是一组单细胞藻类,占全球光合作用的约20%(Falkowski和Raven,2007),但在Rubisco动力学方面尚未得到相对较差的研究。
&nbsp;在进行动力学测定之前,需要分离和纯化Rubisco。由于生物之间的细胞结构和有机组成的差异,需要不断优化用于纯化可行Rubisco酶的方法。该协议描述了一种使用硅藻的尺寸排阻色谱法提取和纯化Rubisco的方法,用于动力学测定。该方法类似于Whitney和Sharwood(2007),用于纯化在E中过表达的Rubisco。大肠杆菌,因为法国压机用于机械破裂细胞。法国压力机是必需的,以获得足够的细胞裂解,因为硅藻与二氧化硅截骨体是单细胞的,不像植物组织,其中通过使研钵和研杵中的冷冻叶组织均匀化容易地实现足够的裂解。此外,由于硅藻中Rubisco的低浓度(Losh等人,2013),需要通过离心浓缩的大硅藻培养体积以获得足够的生物量与植物组织相比,E。大肠杆菌线与过表达的Rubisco。

关键字:Rubisco, 硅藻, 萃取, 浮游植物, 碳固定

材料和试剂

  1. 15 ml带圆锥底部的离心管
  2. 50ml锥形底部的离心管,
  3. 500 ml带圆锥底部的离心管
  4. 1.5 ml微量离心管
  5. 1 ml注射器
  6. 1 ml Bio-Scale mini Macro-Prep高Q离子交换柱(Bio-Rad Laboratories,目录号:7324120)
  7. Amicon Ultra-4离心过滤器(30,000 NMWL)(EMD Millipore,目录号:UFC803024)
  8. Amicon Ultra-100离心过滤器(100,000 NWML)(EMD Millipore,目录号:UFC910024)
  9. 硅藻
  10. 液氮
  11. 聚乙烯吡咯烷酮(PVPP;不溶性)(Sigma-Aldrich,目录号:77627)
  12. 附加材料要测试Rubisco活动(可选):
    标记的CO 2(作为NaH 14 O 3(5mCi))(PerkinElmer,目录号:NEC086H005MC)
    核酮糖-1,5-二磷酸盐(RuBP;如Kane等人,1998)所述合成,纯化和无菌储存)
  13. 乙酸(Sigma-Aldrich,目录号:A9967或27225)
    注意:产品乙酸(A9967)已停产。
  14. 甲醇(Sigma-Aldrich,目录号:M1770或494437)
    注意:产品甲醇(M1770)已停产。
  15. 可溶性蛋白质(通过Bradford测定法测定)(考马斯加分析试剂盒)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23236)
  16. 使用天然和SDS-PAGE(可选)测试蛋白质的其他材料:
    凝胶代码蓝色染色(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:24590)
    4-12%Tris-甘氨酸微凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:XV04120PK20)
    4-12%Bis-Tris凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:NP0321PK2)
    SDS-PAGE的SDS还原缓冲液(参见食谱)
    用于SDS-PAGE的TBS缓冲液(参见食谱)
    AttoPhos试剂(Astral Scientific,Gymea,NSW,Australia)
    Antisera针对三角褐指尖中Rubisco的大亚基全酶提取
    碱性磷酸酶共轭二抗
  17. 4-(2-羟乙基)-1-哌嗪丙磺酸(EPPS)(Sigma-Aldrich,目录号:E9502)
  18. 乙二胺四乙酸二钠盐(EDTA)(Sigma-Aldrich,目录号:E5134)
  19. 二硫苏糖醇(Sigma-Aldrich,目录号:D0632)
  20. 植物蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P9599)
  21. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  22. 三乙醇胺(Sigma-Aldrich,目录号:90279)
  23. 醋酸镁(Sigma-Aldrich,目录号:M5661)
  24. 甘油(Sigma-Aldrich,目录号:G5516)
  25. 提取缓冲液(见配方)
  26. 列缓冲区(见配方)
  27. 柱洗脱缓冲液(参见食谱)
  28. 特异性(S/C/O)缓冲区(见配方)

注意:所有化学品均为A.C.S.等级。

设备

  1. 法国压力容器(Thermo Fisher Scientific,型号:FA-078)
  2. 库尔特计数器Z系列(Beckman Coulter,型号:Z系列库尔特计数器)
  3. 离心机(大体积[1L],2,000xg,小体积[<15ml],17,600×g,4℃)
  4. 通风柜
  5. Superdex 200(GE Healthcare,目录号:17517501)
  6. FPLC(ÄktaPure 25)在4°C下使用Superdex 200/30进行尺寸排阻色谱(GE Healthcare,型号:ÄktaPure 25)
  7. 确认Rubisco的纯化和活性:
    1. 蛋白质转移装置和免疫印迹想象设备 - 检查提取的Rubisco的丰度和纯度
    2. 放射性同位素实验室和相关隔膜盖的小瓶和注射器

程序

  1. 总可溶性细胞(粗)蛋白质提取 - 适用于测量CO 2(KCl)的Michaelis常数,催化更换率(k o) c ),而CO 2 /O 2特异性测定(S C/O))。
    1. 将硅藻在无菌海水中在20℃下在连续光(c.150μmol光子m sec -1 )在20℃下在2L批次培养物中生长,其中过滤0.2μM添加营养素,维生素和微量金属,以得到如Aquil培养基中所定义的最终浓度。有关制作Aquil食谱的完整食谱和指南,请参阅Sunda等人,2005年。通过使用库尔特计数器进行细胞计数来监测生长速率。在指数生长期间,在15℃下通过温和离心(2,000×g×10分钟)将细胞浓缩至沉淀,并弃去上清液。需要大约1g细胞沉淀来纯化Rubisco以测量S C/O 。为了限制储存空间,将细胞收集到1.5ml微量离心管中,然后在液氮中快速冷冻并储存在-80°C直到进一步分析。
    2. 将细胞沉淀物重新悬浮在15ml聚丙烯管中的总共5ml冰冷萃取缓冲液中。使用预冷法国压力机在140MPa下破裂细胞(参见图1和法国印刷机的视频1),并将细胞裂解物收集到相同的管中。


      图1.使用法国印刷机进行细胞裂解法国压力计的照片可以有效地裂解微藻细胞,包括硅藻。将冰冷缓冲液中的藻细胞提取物置于冰冷的不锈钢法国压力池中并施加液压。一旦在140MPa,电池的出口被缓慢打开。当细胞从高到环境压力出现时,细胞裂解,并将细胞提取物收集到15ml或50ml聚丙烯管中。保持样品从细胞的缓慢流速(〜1〜2滴sec -1 ),以确保压力保持(见视频1,确认Bratati Mukherjee)。 >
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      视频1.使用法国新闻界裂解细胞
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    3. 向细胞裂解物中加入聚乙烯吡咯烷酮(1%,w/v)以结合次级代谢物,然后通过离心(17,600×g,4℃,5分钟)除去。
    4. 离心后,Rubisco通过天然PAGE(参见数据分析,图2)证实完全完整,完全可溶,保留在细胞裂解上清液中,通过SDS-PAGE证实(参见数据分析,图3)。
    5. )通过 14 2 2 固定分析定量(参见数据分析)。
      1. 可溶性蛋白质提取物中的Rubisco通过15mM NaH 14和30mM MgCl 2活化10至15分钟。
      2. 在7ml间隔盖玻璃闪烁瓶中,将20μl提取物在0.5ml 50mM EPPS-NaOH pH8.2,10mM MgCl 2,0.6mM RuBP,10μg/sup> -1 碳酸酐酶和不同的NaH浓度(1.2〜12mM,相当于〜15〜150μM±14%在25℃,pH 8.0下)(Sharwood等,2016)。将缓冲液和小瓶用适当的O 2 N 2 N 2气体混合物平衡,然后加入NaH 4 SO 3气体混合物, sub>和sample。
      3. 用0.2ml 0.5N甲酸停止反应,并在通风橱中通过加热85℃的小瓶来排出非固定的14"CO 2"。
      4. 将干燥的残余物溶解在0.5ml双蒸馏的H 2 O中,并在标记的3-磷酸甘油酸( 14℃)之前用1ml Scintillant(Pakard,Ultima Gold XR)涡旋,在闪烁计数器中测量。这种粗提取适用于测定Rubisco最大羧化率( max )和对于CO 2的Rubisco Michaelis常数 C)和21:79%O 2 > air )(Sharwood等人,2008; Sharwood等人,2016)。要量化 sub> max 值除以测定中的Rubisco含量。使用 14 C-CABP结合方法或通过免疫印迹分析测量Rubisco含量。这两种方法的准确性和实验限制在Whitney和Sharwood于2014年详述。Rubisco的纯化对于测量S/O 是必需的(见下一步)。

  2. 用于测量S/O
    的Rubisco纯化 在量化S C/O(CO 2/2/O 2特异性)之前需要进一步纯化可溶性细胞蛋白。
    1. 通过注射器通过用柱缓冲液平衡的1ml Bio-Scale mini Macro-Prep高Q离子交换(IEX)柱手动通过来自上述步骤A3的可溶性细胞蛋白。所有步骤都在4°C进行。该方法包括:预先清洗IEX柱是用2ml柱缓冲液。可溶性细胞蛋白通过注射器通过柱(流速为〜3至5ml/min),然后用8ml柱缓冲液洗涤柱。结合的Rubisco在1.5ml柱洗脱缓冲液中洗脱,收集三个500μl洗脱级分。最后两个分数含有> 90%的Rubisco活性,因此通过使用Amicon Ultra-100离心过滤器(即<! - SIPO - >)离心(4,000xg,10分钟,4℃)浓缩至约0.4ml之前,/em> 100kDa MW截止滤光片)
    2. 将浓缩的Rubisco注射到Äkta纯化系统的200μl样品环中,在4℃下用S/C/O缓冲液预平衡的Superdex 200柱上。将样品加载到柱上,使用0.5ml/min(最大三柱柱压力报警设定为1.5MPA)的流速和在空隙体积(8ml)之后收集的级分(0.5ml)。连续监测280nm处的吸光度和电导率。测定级分中的RuBP依赖性 CO 2 2 2 2-活性,并将具有峰Rubisco活性(通常为5至7级)的那些合并,并通过离心浓缩至约0.1ml (4,000xg,20分钟,4℃),使用Amicon Ultra-100离心过滤器。
    3. 浓缩和纯化的Rubisco现在已经准备好进行S/C/O检测。或者,甘油可以加入到20%(v/v)终浓度,然后酶在液氮中冷冻并储存在-80℃。
    4. 可选:S/C/O检测(不是本协议中Rubisco提取的一部分)
      1. 在25℃(或其他温度)下,根据(Kane e)的方法进行S/O ,1994)。将纯化的Rubisco(10-50μl)注射到含有1ml测定缓冲液(参见食谱)的20ml玻璃,隔膜密封的玻璃小瓶中,该缓冲液已经用500ppm CO 2平衡了0.5至1小时使用Wostoff气体混合泵与O 2混合。 15分钟后进一步平衡,注入1nmol的2- H-RuBP引发反应。在30分钟后注入碱性磷酸酶,将3-磷酸甘油酸(羧化产物)和2-叔丁氧羰基乙酸酯(氧合产物)转化成3' H-3-甘油酸酯和H 3 - 羟基乙酸酯。
      2. 将反应物通过0.4ml AGI-X8(10%甲酸盐)阴离子交换柱,然后将结合的3-H-3-甘油酸酯和3-sup-3-H-2-乙醇酸在0.5ml 20%(v/v)H 2 SO 4中洗脱,然后通过HPLC分离它们,使用0.012Mg 2 /通过Aminex HPX-87H柱在65℃下以0.4ml /分钟的流速通入SO 4。
      3. 然后根据3-H 3 H-甘油酸酯和3-H-3-甘油酸酯的放射性计算25℃下的S/C/O,乙醇酸使用公式:



        哪里,
        表示O 2和CO 2 (99.95%和0.05%[v/v]),
        0.037是在25℃下H 2 O 2中的CO 2 <2>的溶解度的比例(Kane等,/em>。,1994)。 

数据分析

  1. 提取过程中完整的Rubisco保留在可溶性部分中 确定使用法国压力池常规获得全细胞裂解,并且Rubisco在粗提取期间保持完整并在可溶性级分内(步骤A1-A3)。天然PAGE(图2)显示Rubisco保留为完整的L 8 S 8 N酶(包含8个大的[L]和8个小[S]亚基)),即使在电泳在4℃和60V下16小时。由于亚基序列差异和四分体结构性质的微妙变化,物种之间Rubiesico带的不同迁移率发生,氨基酸序列的轻微差异和长度)。 SDS-PAGE(图3)表明,在全细胞裂解(机械破裂后但离心前,步骤A2)和可溶性细胞蛋白(离心后步骤A3)中检测到相当水平的Rubisco L和S-亚单位,表明提取的Rubisco是完全可溶的
  2. Rubisco在提取后保留活动
    在11种硅藻种类的粗提物(步骤A1-A3)中Rubisco的活性进行了测试和公布(Young等人,2016)。在25℃提取10分钟后获得Rubisco的完全活化,并且在进一步的10分钟测试期间活性保持稳定。 c
    硅藻Rubiscos发表于(Young等人,2016)。
  3. 量化S C/O(步骤B1-B4)。图4显示了用于计算S的HPLC分离的3-H-甘油酸酯和3-OH-羟基乙酸酯峰的 3 H-洗脱曲线如上文步骤B4c所述。


    图2.原始提取物的天然PAGE印迹显示完全完整的Rubisco蛋白总可溶性细胞提取物(通过Bradford测定法测定的5μg,考马斯加)通过预制物4在4℃下分离-12%Tris-甘氨酸凝胶在60V下过夜(16小时)。用去离子水漂洗凝胶,用45%(v/v)H 2 O,5%(v/v)乙酸和50%(v/v)甲醇,如果去离子水,则多次漂洗。使用Gelcode blue考马斯染色(Invitrogen)显现蛋白质。箭头表示〜520kDa的Rubisco复合物(L 8 S 8 S 8)位于凝胶上。泳道数字表示来自不同硅藻种类的提取物:(1)海水沙门氏菌CCMP 1336,(2)海洋细胞沙门氏菌CS-427,(3)骷髅马蹄毒 CCMP 1332,(4)ae os rans rans>> CC 16 16 16 16>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> ,(7)Ph> um um>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>贝勒诺卡 sp。 CS-874/01,(11)等sis sis>>。。 CS-177,(12) CS-287。最后三条通道含有5,10和20μl来自烟草的粗细胞提取物作为对照。显示了通过 14 C-CABP结合定量的每个样品的Rubisco活性位点内容(Whitney和Sharwood,2014; Sharwood等人,2008)。


    Fi gure 3。通过法国压力池裂解产生Rubisco溶解度,完整性和完全提取的SDS-PAGE分析。A.考马斯染色和B.硅藻Rubisco抗体印迹(参见Whitney总细胞溶胞产物(L,以下法国压力细胞裂解法)的细胞裂解物(L, )和可溶性细胞蛋白(S,离心后),从硅藻种类:(1) ricornutum >

    (5)Cylindroheca fusiform 我 CS-13,(6) CS> 427和(7) CSL 1336.加载5μl烟草可溶性叶蛋白进行比较。在L和S蛋白级分中,L亚单位的强度相等,表明所有的Rubisco都被提取(完全细胞裂解),并且在Western印迹中没有明显的L-亚基降解而完全溶解。样品制备和电泳:将蛋白质(L或S)提取物(150μl)加入到4×SDS-缓冲液(50μl)中并煮沸5分钟,然后离心(16,000×g Rubisco(参见惠特尼 ,2001,进一步的实验细节)。


    4。 3用于 计算S 的HPLC部分的H-洗脱曲线 3 H的量 3 3根据 中描述的H-乙醇酸(峰 1和2分别 )步骤B4c。这里显示了使用来自硅藻的纯化Rubisco的测定结果,所述测定使用来自硅藻的纯化的Rubisco(例如,Halassiosira weissflogii Tw,red)和 P haeodactylum tricornutu (Pt,blue),显示每个技术复制(rep)。

食谱

  1. 提取缓冲区
    50mM EPPS-NaOH,pH 8.0
    1 mM EDTA
    2mM二硫苏糖醇(DTT)
    1%(v/v)植物蛋白酶抑制剂混合物
    将EPPS和EDTA溶于去离子水中至最终所需浓度 用NaOH将pH调至8.0 注意:
    1. 在开始提取之前添加DTT。
    2. 在使用之前,将蛋白酶抑制剂混合物加入用于提取的缓冲液等分试样 。
  2. 列缓冲区
    50mM EPPS-NaOH,pH 8.0
    1 mM EDTA
    10 mM NaCl
    将去离子水中的所有盐溶解至最终所需浓度
    用NaOH将pH调至8.0
  3. 柱洗脱缓冲液
    50mM EPPS-NaOH,pH 8.0
    1mM EDTA
    0.8 M NaCl
    将去离子水中的所有盐溶解至最终所需浓度
    用NaOH将pH调至8.0
  4. 特异性(S/C/O)缓冲区
    30mM三乙醇胺
    15mM乙酸镁,pH8.3
    将去离子水中的所有盐溶解至最终所需浓度
    用乙酸将pH调至8.3 储存于4°C
  5. 测定缓冲液(1 ml)
    30mM三乙醇胺
    15mM乙酸镁,pH8.3
    10μgml 碳酸酐酶
  6. 用于SDS-PAGE的4x SDS还原缓冲液(可选)
    125mM Tris-HCl pH 6.8
    4%(w/v)SDS
    0.01%(w/v)溴酚蓝
    20%(v/v)甘油 75mM 2-巯基乙醇
    将去离子水中的所有盐溶解至最终所需浓度
    使用前加入甘油和2-巯基乙醇
  7. 用于SDS-PAGE的BS缓冲液(可选)
    10mM Tris-HCl,pH7.5
    150 mM NaCl
    将去离子水中的所有盐溶解至最终所需浓度的

致谢

我们感谢审稿人提供有用的意见,改进了文章。通过ANU访问学者(CE140100015)和NSF Grant 1040965为J. N.Y.提供资金。A.H.通过Clarendon奖学金,牛津大学和澳大利亚国立大学访问学者(CE140100015)资助。 R.E.S是通过ARC DECRA计划(DE13010760)资助的。 R.E.M.R.通过ERC起始授权(SP2-GA-2008-200915),F.M.M.M.通过NSF Grant 104095资助,S.M.W由澳大利亚研究委员会授予的CE14010001资助。我们要感谢Bratati Mukherjee在法国新闻录像带上的协助。

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  13. Young,JN,Heureux,AM,Sharwood,RE,Rickaby,RE,Morel,FMM和Whitney,SM(2016)。  硅藻的Rubisco动力学的巨大变化揭示了它们的碳浓缩机制之间的多样性。 Exp Bot 67(11):3445-3456。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Young, J. N., Heureux, A. M., Rickaby, R. E., Morel, F. M., Whitney, S. M. and Sharwood, R. E. (2017). Rubisco Extraction and Purification from Diatoms. Bio-protocol 7(6): e2191. DOI: 10.21769/BioProtoc.2191.
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