Jun 30, 2020

Public workspacePurification of 2,3-bisphosphate-dependent phosphoglycerate mutase (dPGM)

DOI
dx.doi.org/10.17504/protocols.io.bgawjsfe
  • 1Lancaster Environment Centre, Lancaster University, Library Avenue, Lancaster, LA1 4YQ, UK;
  • 2Biosystems & Integrative Sciences Institute (BioISI), Science Faculty of Lisbon University, Lisbon, 1749-016, Portugal
Cristina Rodrigues Gabriel Sales
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bgawjsfe
Protocol Citation: Cristina Rodrigues Gabriel Sales, Anabela Silva, Elizabete Carmo-Silva 2020. Purification of 2,3-bisphosphate-dependent phosphoglycerate mutase (dPGM). protocols.io https://dx.doi.org/10.17504/protocols.io.bgawjsfe
Manuscript citation:
Sales CRG, Silva AB, Carmo-Silva E. 2020. Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays. Journal of Experimental Botany, https://doi.org/10.1093/jxb/eraa289
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 12, 2020
Last Modified: June 30, 2020
Protocol Integer ID: 36918
Keywords: Protein purification, E. coli
Abstract
This protocol describes the purification of dPGM based on Fraser et al. (1999) and Scales et al. (2014) with modifications. Some steps of the purification protocol were also informed by van de Loo & Salvucci (1996), Schmidt & Skerra (2007), and White & Fothergill-Gilmore (1992).

Guidelines
  1. Check the "Materials" tab for a list of all the chemicals used in this protocol.
  2. In the "Steps" tab there is a brief description of the materials and equipment necessary for the protocol execution.
  3. The references cited are at the end of the "Materials" tab.
Note
The figures uploaded in this protocol, explaining about the columns, are from iba lifesciences (https://www.iba-lifesciences.com/isotope/2/2-1002-100-Manual_Strep-Tactin-Purification.pdf)

Materials
MATERIALS
ReagentSodium hydroxide (NaOH)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5881
Reagent2-Mercaptoethanol Merck MilliporeSigma (Sigma-Aldrich)Catalog #M6250
ReagentPhenylmethanesulfonyl fluoride (PMSF)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P7626
ReagentTryptoneNeogenCatalog #MC005
ReagentYeast extractNeogenCatalog #MC001
ReagentCarbenicillinMelfordCatalog #C0109
ReagentIsopropyl-β-D-thiogalactopyranoside (IPTG)Fisher ScientificCatalog #10356553
ReagentGlycerolAlfa AesarCatalog #36646
ReagentPotassium phosphate monobasic Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9791
ReagentSodium chloride (NaCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #31434-M
ReagentTris baseMerck MilliporeSigma (Sigma-Aldrich)Catalog #T1503
ReagentHydrochloric acid (HCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #H1758
Reagent10x Buffer R; Strep-Tactin® Regeneration Buffer with HABAibaCatalog #2-1002-100
ReagentLeupeptin hemisulfateAG ScientificCatalog #L-1165
ReagentSephadex® G-25 fineMerck MilliporeSigma (Sigma-Aldrich)Catalog #G2580
ReagentStrep-Tactin Sepharose 50% suspension ibaCatalog #2-1201-010
ReagentAmmonium sulfate Merck MilliporeSigma (Sigma-Aldrich)Catalog #A4418
ReagentDesthiobiotinibaCatalog #2-1000-002
ReagentEthanol absolute 99.8 %Fisher ScientificCatalog #10437341


CITATION
IBA Life Sciences (2020). Expression and purification of proteins using Strep-Tactin. A comprehensive manual.
LINK
https://www.iba-lifesciences.com/isotope/2/2-1002-100-Manual_Strep-Tactin-Purification.pdf

CITATION
Fraser HI, Kvaratskhelia M, White MF (1999). The two analogous phosphoglycerate mutases of Escherichia coli. FEBS Letters 455: 344-348.
LINK
10.1016/s0014-5793(99)00910-2

CITATION
van de Loo FJ, Salvucci ME (1996). Activation of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) involves Rubisco activase Trp16. Biochemistry 35: 8143-8148.
LINK
10.1021/bi9604901

CITATION
Scales JC, Parry MA, Salvucci ME (2014). A non-radioactive method for measuring Rubisco activase activity in the presence of variable ATP: ADP ratios, including modifications for measuring the activity and activation state of Rubisco. Photosynthesis Research 119: 355-365.
LINK
https://doi.org/10.1007/s11120-013-9964-5

CITATION
Schmidt TGM, Skerra A (2007). The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nature Protocols 2: 1528-1535.
LINK
https://doi.org/10.1038/nprot.2007.209

CITATION
White MF, Fothergill-Gilmore LA (1992). Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Journal of Biochemistry 207: 709-714.
LINK
10.1111/j.1432-1033.1992.tb17099.x


Safety warnings
Before using the protocol always check the Safety Data Sheet (SDS) for each chemical.
Before start
MATERIAL & EQUIPMENTS (for list of chemicals check "Materials" tab)
  • E. coli cells stored at -80°C as frozen glycerol stock: BL21Star(DE3)pLysS E. coli transformed with dPGM cDNA cloned into pET23a
  • Shaking incubator(s) for 30°C and 37°C
  • Vortex
  • Microcentrifuge
  • Bench top centrifuge
  • Plate reader & LVis plate
  • UV-Vis spectrophotometer
  • Sonifier with standard tip
REAGENTS & SOLUTIONS
REAGENTS & SOLUTIONS
REAGENTS & SOLUTIONS TO PREPARE BEFOREHAND
LB medium
Concentration10 mg/mL Tryptone
Concentration5 mg/mL Yeast extract
  • Add ultrapure H2O and mix until dissolved; check if Ph7 and adjust with NaOH if required.
  • Top volume up to the final volume with ultrapure H2O; autoclave.

Note
Add Concentration10 mg/mL agar if making solid LB agar.


Note
To pour LB agar plates:
  • Melt agar in the microwave if required (loosen the lid).
  • Place into a water bath at Temperature50 °C and allow agar to equilibrate to this temperature (at least Duration00:30:00 ).
  • In the flow hood, add Concentration100 ng/ml carbenicillin and mix gently.
  • Poor approximately Amount25 mL into each plate.
  • Leave with lids off to set for approximately Duration00:25:00 .
  • Store plates for up to 2-3 weeks in a sealed bag in the fridge (no parafilm).

Safety information
CAUTION – always watch the microwave when melting agar and never leave the area. The agar will become super-heated and can boil over very easily. Wear protective gloves.
CAUTION – never place anything cold (e.g. magnetic stirrer) into super-heated molten agar.

Concentration100 mg/mL Carbenicillin
  • Dissolve in ultrapure H2O; filter through a 0.25 µm sterile syringe filter. Temperature4 °C (storage)
Concentration0.1 Molarity (M) IPTG
  • Dissolve in ultrapure H2O; dispense in aliquots. Temperature-20 °C (storage)

Glycerol
TemperatureRoom temperature (storage)

Buffer A
Concentration0.1 Molarity (M) Potassium phosphate
Concentration75 millimolar (mM) NaCl
  • Dissolve in ultrapure H2O; adjust to Ph8 with NaOH; filter. Temperature4 °C (storage)

Buffer W (Washing buffer)
Concentration100 millimolar (mM) Tris base
Concentration150 millimolar (mM) NaCl
  • Dissolve in ultrapure H2O; adjust to Ph8 with HCl; filter. Temperature4 °C (storage)

Buffer F (Final buffer)
Concentration60 millimolar (mM) Tris base
  • Dissolve in ultrapure H2O; adjust to Ph7.9 with HCl; filter. Temperature4 °C (storage)
10x Buffer R, Strep-Tactin Regeneration Buffer with HABA
Temperature4 °C (storage)

Concentration10 millimolar (mM) Leupeptin hemisulfate
  • To the bottle received from AG Scientific (0.025 g) add Amount5.25 mL ethanol.
  • Dispense in aliquots. Temperature-20 °C (storage)

2-Mercaptoethanol
TemperatureRoom temperature (storage)

Concentration100 millimolar (mM) PMSF
  • Dissolve in ethanol. Temperature4 °C (storage)

Sephadex G-25 fine
  • Place Amount5 g of resin in buffer A (from step 1.5) and allow to swell for at least Duration03:00:00 at TemperatureRoom temperature
  • Fill the glass column with Amount20 mL of bed volume and equilibrate with buffer A (3 times column volume).
Note
1 g swell to 4-6 mL.

Strep-Tactin Sepharose 50% suspension
  • Bed volume of Amount20 mL . Temperature4 °C (storage)
  • Fill the glass column with the resin [it comes in 50% suspension in buffer W (from step 1.6), so it is already in the buffer that will be used later for purification (check below)].
  • Equilibrate the column with 2 times column bed volumes of buffer W.
Note
Always work at Temperature4 °C . If it is not possible to perform chromatography at Temperature4 °C and column needs to be transferred to TemperatureRoom temperature air bubbles may form since cold storage buffer is able to take up more gas than buffers at TemperatureRoom temperature . Therefore, it is recommended to equilibrate the columns immediately after exposure to higher temperatures with buffer that is equilibrated at such temperature.



Concentration4.5 Molarity (M) Ammonium sulfate
  • Add the powder in ultrapure H2O; warm up with mixing to dissolve (mild heat).
  • Adjusted to Ph7 with NH4OH; let precipitate. Temperature4 °C (storage)

Desthiobiotin
Temperature4 °C (storage)

SOLUTIONS TO PREPARE JUST BEFORE USE
  • Prepared with reagents/solutions described in step 1.
Lysis buffer



Complete Buffer W



Buffer E (Elution buffer)



Buffer R (Regeneration buffer)



PROCEDURE
PROCEDURE
CELL GROWTH & COLLECTION

Day 1
1. Inoculate the cultures with a small volume of cells using a sterile tip onto the plate containing LB medium with Concentration100 ng/ml carbenicillin.
2. Incubate plates upside down DurationOvernight (approx. 9h) at Temperature37 °C .
Note
Transformed E. coli cells stored as frozen glycerol stocks are used as inoculums for starter cultures. Keep glycerol stock TemperatureOn ice .


Day 2
3. Add Concentration100 ng/ml carbenicillin into Amount10 mL LB in a 50 mL Falcon tube.
4. Pick a single colony from the plate and add into the LB.
5. Shake DurationOvernight (approx. 9h) at Temperature37 °C .
Note
For large cultures do the above procedure in 4 Falcon tubes to get 40 mL of culture.


Day 3
6. Prepare a glycerol stock of the culture (for long term storage): combine Amount300 µL culture + Amount300 µL 30% glycerol in a 2 mL screw-cap tube, mix gently by inversion. Temperature-80 °C (storage)

INDUCTION

Day 3
7. Dilute 1:50 in 1 L Erlenmeyer flask (Amount20 mL of culture in a final volume of Amount1 L ).
8. Grow cells at Temperature37 °C under continuous stirring at 225 rpm.
9. Check optical density at 600 nm after Duration02:00:00 incubation, and keep checking regularly until the cells reach an optical density of approx. 0.8 at 600 nm (for the large culture it took ~Duration04:00:00 ).
10. Remove Amount1 mL aliquot before adding IPTG for SDS-Page of uninduced and induced expression. Centrifuge, discard supernatant and keep at Temperature-20 °C for later.
11. Induce dPGM expression by adding Concentration0.1 Molarity (M) IPTG to the cell culture (Amount1 mL of Concentration0.1 Molarity (M) IPTG in Amount1 L ).
12. Maintain the culture at Temperature29 °C DurationOvernight under continuous stirring.
Note
Tests were done at Temperature37 °C and Temperature29 °C . Both had good results but at Temperature29 °C more protein was produced.


Day 4
13. Transfer Amount400 mL culture to two 500 mL centrifuge bottles on ice.
14. Collect the cells by centrifuging at Centrifigation4000 rpm, 4°C, 00:10:00 . Discard supernatant. Repeat using the same 500 mL bottles until the entire culture content is collected in the two bottles.
15. Suspend the accumulated cell pellets in about Amount20 mL of buffer A. Pool the cell suspension and distribute into 2x 50 mL screw cap tubes (TemperatureOn ice ). Rinse the 500 mL bottles with about Amount5 mL of buffer A and add in the 50 mL tubes as well.
16. Centrifuge at Centrifigation5000 x g, 4°C, 00:10:00 Discard supernatant and dry the tubes as much as possible. The wet cell pellets are stored at Temperature-80 °C until extraction.
LYSIS

Day 5 (long day)
17. Thaw pellets TemperatureOn ice (freeze-thaw cycle).
Note
Transfer tube with pellets to Temperature-20 °C in the evening before. TemperatureOn ice , keep pellet upside down, not directly touching ice to thaw faster.


18. Suspend the cell pellets to about Concentration5 mg/g of wet cell pellet weight to lysis buffer.
Note
E.g. if pellet weighs 2.694 g, add 13.47 mL buffer.


19. Vortex and keep TemperatureOn ice .
20. Transfer the cells to a beaker. Rupture the cells and DNA by sonicating the suspension until solution becomes easy to pipette. Insert sonicator half way into the solution and apply 3 x 45s bursts, maintaining the beaker always TemperatureOn ice . Pause for 30s between each burst.
21. Centrifuge at Centrifigation26000 rpm, 4°C, 00:20:00 . Keep supernatant (and pellet for SDS-Page checking).
22. Analyse the expression of dPGM as soluble polypeptides in the uninduced and induced samples by SDS-PAGE (Appendix 2).
Note
It is useful to verify the expression of dPGM as insoluble and soluble polypeptides in the uninduced and induced samples by SDS-PAGE. It is expected that the protein will be in the soluble fraction but occasionally it may be in the insoluble fraction (see Appendix 2 for SDS-PAGE).

PRECIPITATION

23. Measure the volume of supernatant and add saturated ammonium sulfate to Concentration80 % (v/v)
Note
Vol saturated ammonium sulfate for 80% saturation = (Vol supernatant / 0.2) - Vol supernatant

24. Add the saturated ammonium sulfate drop-wise to the supernatant while stirring continuously using a separatory funnel in a ring stand. Stir for Duration00:30:00 .
25. Collect precipitated material by centrifugation at Centrifigation20000 x g, 4°C, 00:13:00 (slow brake).
RESUSPENSION

26. Suspend pellets in a total volume of Amount3 mL of buffer A TemperatureOn ice , mixing well using a 1 mL pipette.
Note
If more than one tube, resuspend one and do the resuspension of the next one with the content of the first one, to reduce the volume produced. If needed, add more buffer.


27. Centrifuge at Centrifigation20000 x g, 4°C, 00:20:00 to clean any remaining debris and be easier to flow through Sephadex G-25 fine column
DESALTING

28. After equilibrating Sephadex G-25 fine column with buffer A (2 x column volumes) and with lysis buffer (1 x column volume), load the sample and elute with lysis buffer, taking aliquots and checking the amount of protein with Bradford reagent (monitor the development of blue colour by eye for a qualitative assessment, not necessary to read absorbance).

29. Pool samples with highest protein content.
Note
Start adding Amount5 mL of buffer A and collecting the aliquots. Check TSP “by eye” and when it gets blue reduce to Amount2 mL elutions, until the aliquot has low protein content. The objective is to collect as much protein as possible without diluting the sample more than is necessary.


30. Once desalting of the sample is complete, wash column with 2 column volumes of Concentration0.2 Molarity (M) NaOH, rinse with water, and re-equilibrate with 2-3 column volumes of buffer A.
Note
For storage, antimicrobial agents should be added to the column to prevent contamination (Concentration0.001 % (v/v) phenyl mercuric salts, Concentration0.005 % (v/v) thimerosal, Concentration0.05 % (v/v) chlorobutanol, Concentration0.002 % (v/v) chlorhexine, Concentration0.02 % (v/v) sodium azide, or Concentration20 % (v/v) ethanol are acceptable).
When necessary, the gel can be removed from the column and sterilized by autoclaving.

PURIFICATION & ELUTION

31. Purification and elution of dPGM uses a Strep-tactin Sepharose, Amount20 mL bed column, as detailed in the manufacturer’s instructions:




Note
For purification and regeneration of Strep-tactin resin, see Appendices 3 and 4.

PRECIPITATION

32. Measure the volume of supernatant and add saturated ammonium sulfate to Concentration80 % (v/v)
33. Add the saturated ammonium sulfate drop-wise to the supernatant while stirring continuously using a separatory funnel in a ring stand. Stir for 30 min.
34. Collect precipitated material by centrifugation at Centrifigation20000 x g, 4°C, 00:13:00 (slow brake).
35. Ressuspend pellet with Amount3 mL buffer F.

DESALTING

36. After equilibrating Sephadex G-25 fine column with 3 x column volume buffer F, load the sample and elute with buffer F, taking aliquots and checking the amount of protein with Bradford reagent (monitor the development of blue colour by eye for a qualitative assessment, not necessary to read absorbance).
Note
It is not necessary to add proteases inhibitors as now it is a pure protein.

37. Pool samples with highest protein content.
Note
Start adding Amount5 mL of buffer F and collecting Amount2 mL aliquots. Check TSP “by eye”. In our hands, samples pooled were from fractions 3 to 6.

38. Once desalting of the sample is complete, wash column with 2 column volumes of Concentration0.2 Molarity (M) NaOH, rinse with water, and re-equilibrate with 2-3 column volumes of buffer A.
Note
For storage, antimicrobial agents should be added to the column to prevent contamination (Concentration0.001 % (v/v) phenyl mercuric salts, Concentration0.005 % (v/v) thimerosal, Concentration0.05 % (v/v) chlorobutanol, Concentration0.002 % (v/v) chlorhexine, Concentration0.02 % (v/v) sodium azide, or Concentration20 % (v/v) ethanol are acceptable).
When necessary, the gel can be removed from the column and sterilized by autoclaving.

39. Store pooled fractions at Temperature-80 °C .
Note
Before freezing samples check the activity (3-PGA comsumption in presence of increasing concentrations of 2,3dPGA using the PK-LDH protocol in this series). It is important to keep an aliquot of previous dPGM preparation to compare results obtained.

APPENDIX
APPENDIX
APPENDIX 1. Purification of plasmid DNA, aka minipreps for sequencing
Following Day 3

  1. Prepare a glycerol stock of the culture (for long term storage): combine 300 μL culture + 300 μL 30% glycerol in a 2 mL screw-cap tube, mix gently by inversion. Store at -80°C.
  2. Spin the remaining cell culture down for 10 min at 4000 rpm, room temperature.
  3. Pour off the supernatant into the bacterial waste bottle and leave tubes upside down and open on blue roll for a few minutes to drain any excess supernatant.
  4. Resuspend the pellets in 250 μl Resuspension Solution (stored at 4°C) and transfer to labelled 1.5 ml tubes.
  5. Vortex briefly to eliminate all clumps of bacteria.
  6. Add 250 μl Lysis Solution, invert gently 4-6 times to mix, then incubate at RT for 4 min.
  7. Add 350 μl Neutralisation Solution, invert gently 4-6 times to mix.
  8. Spin in the microcentrifuge at 13,400 rpm for 5 min. Transfer the supernatant to a labelled spin column.
  9. Spin in the microcentrifuge at 13,400 rpm for 1 min. Discard the flow-through.
  10. Wash the column by adding 500 μl Wash Solution.
  11. Spin in the microcentrifuge at 13,400 rpm for 1 min. Discard the flow-through.
  12. Wash the column for a second time by adding another 500 μl Wash Solution.
  13. Spin in the microcentrifuge at 13,400 rpm for 1 min. Discard the flow-through.
  14. Spin the empty column in the microcentrifuge at 13,400 rpm for 1 min.
  15. Transfer the column to a clean labelled 1.5 ml tube, add 55 μl SDW to the middle of the column and incubate at RT for 2 min. Spin in the microcentrifuge at 13,400 rpm for 1 min. Discard the column.
  16. Determine the concentration and purity of pDNA using a nanodrop (DNA concentration, 260:230 nm ratio)

APPENDIX 2. SDS-Page for checking protein in different fractions

UNINDUCED Sample (from Induction step 10.)
  1. Thaw pellet. Resuspend in about Amount100 µL of Lysis Buffer.
  2. Vortex and incubate on ice.
  3. Rupture the cells and DNA by sonicating the suspension until solution becomes easy to pipette. Insert sonicator half way into the solution and apply 3 x 5 s bursts, with the tube always on ice. Pause for some seconds between each burst.
  4. Centrifuge at 4000 rpm for 10 min at 4°C. Keep SN and pellet.
  5. Quantify the total soluble portein (TSP) in the supernatant. Add Loading Buffer (proportion of 4:5 LB:Sample), incubate at 95°C for 4 min. Dilute to 0.5 mgTSP/mL with blank (Lysis buffer + Loading Buffer).
  6. Resuspend pellet in Amount100 µL of Lysis Buffer. Add Loading Buffer (proportion of 4:5 LB:Sample), incubate at 95°C for 4 min.

INDUCED Sample (from Lysis step #21.)
  1. After sonication, centrifuge at 4000 rpm for 10 min at 4°C. Keep supernatant and pellet.
  2. Quantify TSP in the supernatant. Add Loading Buffer (proportion of 4:5 LB:Sample), incubate at 95°C for 4 min. Dilute to 0.5 mgTSP/mL with blank (Lysis buffer + Loading Buffer).
  3. Resuspend pellet in Amount100 µL of Lysis Buffer. Add Loading Buffer (proportion of 4:5 LB:Sample), incubate at 95°C for 4 min.

Run samples on SDS-Page and stain with coomassie blue to visulaise proteins.
Note
In our hands, loading 2 mg TSP/lane provides clear protein bands. The protein is expected to be in the soluble fraction, but some protein can be seen in the insoluble fraction. Note that the gel is just qualitative for the insoluble protein fraction.


APPENDIX 3. Step-tactin purification process, as detailed in the manufacturer’s instructions:


APPENDIX 4. Regeneration of Strep-tactin resin, as detailed in the manufacturer’s instructions:





Citations
Scales JC, Parry MA, Salvucci ME. A non-radioactive method for measuring Rubisco activase activity in the presence of variable ATP: ADP ratios, including modifications for measuring the activity and activation state of Rubisco
https://doi.org/10.1007/s11120-013-9964-5
IBA Life Sciences. Expression and purification of proteins using Strep-Tactin. A comprehensive manual
https://www.iba-lifesciences.com/isotope/2/2-1002-100-Manual_Strep-Tactin-Purification.pdf
van de Loo FJ, Salvucci ME. Activation of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) involves Rubisco activase Trp16
10.1021/bi9604901
Fraser HI, Kvaratskhelia M, White MF. The two analogous phosphoglycerate mutases of Escherichia coli
10.1016/s0014-5793(99)00910-2
Schmidt TGM, Skerra A. The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins
https://doi.org/10.1038/nprot.2007.209
White MF, Fothergill-Gilmore LA. Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase
10.1111/j.1432-1033.1992.tb17099.x