Rubisco Extraction and Purification from Diatoms

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 Scientific^TM, catalog number: 23236 )
    
16. Additional materials to test for protein using Native and SDS-PAGE (Optional):
    Gel code blue stain (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 24590 )
    4-12% Tris-glycine mini gels (Thermo Fisher Scientific, Invitrogen^TM, catalog number: XV04120PK20 )
    4-12% Bis-Tris gels (Thermo Fisher Scientific, Invitrogen^TM, 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 (S_C/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 (K_C), catalytic turnover rate (k_cat^c) and the first step for CO₂/O₂ specificity assays (S_C/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 S_C/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 k_cat^c (and K_C) 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 (V_max) and Rubisco Michaelis constant for CO₂ under N₂ (K_C) and 21:79% O₂:N₂ (K_c^air) (Sharwood et al., 2008; Sharwood et al., 2016). To quantify k_cat^c the V_max 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 S_C/O (see next step).
      
      
2. Rubisco purification for measuring S_C/O
   The soluble cellular protein needs to be further purified before quantifying S_C/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 S_C/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 S_C/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 S_C/O assay (not part of this protocol on Rubisco extraction)
      
      1. S_C/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 S_C/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 K_C and k_cat^c for the diatom Rubiscos are published in (Young et al., 2016).
   
3. Quantifying S_C/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 S_C/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 S_C/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 S_C/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 (S_C/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 C_3-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.