TITLE: Purification of 2,3-bisphosphate-dependent phosphoglycerate mutase (dPGM)
AUTHORS: Cristina Rodrigues Gabriel Sales, Anabela  Silva, Elizabete Carmo-Silva

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">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).</div></div></div>

[STEPS]
?. [REAGENTS & SOLUTIONS]
REAGENTS & SOLUTIONS TO PREPARE BEFOREHAND
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LB mediumTryptone  Yeast extractAdd ultrapure H2O and mix until dissolved; check if  and adjust with NaOH if required.
Add   agar if making solid LB agar.
To pour LB agar plates:Melt agar in the microwave if required (loosen the lid).Place into a water bath at   and allow agar to equilibrate to this temperature (at least  ).In the flow hood, add   carbenicillin and mix gently.Poor approximately   into each plate.Leave with lids off to set for approximately   .Store plates for up to 2-3 weeks in a sealed bag in the fridge (no parafilm).
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.
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CarbenicillinDissolve in ultrapure H2O; filter through a 0.25 µm sterile syringe filter.
[(storage)]
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IPTGDissolve in ultrapure H2O; dispense in  aliquots.
[(storage)]
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Glycerol
[(storage)]
?. [REAGENTS & SOLUTIONS]
Buffer A Potassium phosphate NaCl Dissolve in ultrapure H2O; adjust to  with NaOH; filter.
[(storage)]
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Buffer W (Washing buffer) Tris base NaClDissolve in ultrapure H2O; adjust to  with HCl; filter.
[(storage)]
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Buffer F (Final buffer) Tris baseDissolve in ultrapure H2O; adjust to  with HCl; filter.
[(storage)]
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10x Buffer R, Strep-Tactin Regeneration Buffer with HABA
[(storage)]
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Leupeptin hemisulfateTo the bottle received from AG Scientific (0.025 g) add  ethanol.Dispense in aliquots.
5.25 mL
[(storage)]
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2-Mercaptoethanol
[(storage)]
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PMSFDissolve in ethanol.
[(storage)]
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Sephadex G-25 finePlace  of resin in buffer A (from step 1.5) and allow to swell for at least  at Fill the glass column with  of bed volume and equilibrate with buffer A (3 times column volume).
5 g
4 Room temperature
20 mL
1 g swell to 4-6 mL.
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Strep-Tactin Sepharose 50% suspension Bed volume of .  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.
20 mL
[(storage)]
Always work at  . If it is not possible to perform chromatography at  and column needs to be transferred to   air bubbles may form since cold storage buffer is able to take up more gas than buffers at  . Therefore, it is recommended to equilibrate the columns immediately after exposure to higher temperatures with buffer that is equilibrated at such temperature.
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Ammonium sulfateAdjusted to  with NH4OH; let precipitate.
[(storage)]
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Desthiobiotin
[(storage)]
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SOLUTIONS TO PREPARE JUST BEFORE USE
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Lysis buffer
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Complete Buffer W
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Buffer E (Elution buffer)
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Buffer R (Regeneration buffer)
?. [PROCEDURE]
CELL GROWTH & COLLECTIONDay 11. Inoculate the cultures with a small volume of cells using a sterile tip onto the plate containing LB medium with  carbenicillin.2. Incubate plates upside down  (approx. 9h) at .Day 23. Add  carbenicillin into  LB in a 50 mL Falcon tube.4. Pick a single colony from the plate and add into the LB.5. Shake  (approx. 9h) at .Day 36. Prepare a glycerol stock of the culture (for long term storage): combine  culture + 30% glycerol in a 2 mL screw-cap tube, mix gently by inversion.
37 °C
Transformed E. coli cells stored as frozen glycerol stocks are used as inoculums for starter cultures. Keep glycerol stock  .
10 mL
37 °C
For large cultures do the above procedure in 4 Falcon tubes to get 40 mL of culture.
300 µl
300 µl
[(storage)]
?. [PROCEDURE]
INDUCTIONDay 37. Dilute 1:50 in 1 L Erlenmeyer flask ( of culture in a final volume of ). 8. Grow cells at  under continuous stirring at 225 rpm.9. Check optical density at 600 nm after  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 ~).10. Remove  aliquot before adding IPTG for SDS-Page of uninduced and induced expression. Centrifuge, discard supernatant and keep at  for later.11. Induce dPGM expression by adding  IPTG to the cell culture ( of  IPTG in ).12. Maintain the culture at  under continuous stirring.Day 413. Transfer  culture to two 500 mL centrifuge bottles on ice.14. Collect the cells by centrifuging at . 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  of buffer A. Pool the cell suspension and distribute into 2x 50 mL screw cap tubes (). Rinse the 500 mL bottles with about   of buffer A and add in the 50 mL tubes as well.16. Centrifuge at  Discard supernatant and dry the tubes as much as possible. The wet cell pellets are stored at  until extraction.
20 mL
1 L
37 °C
1 mL
-20 °C
1 mL
1 L
29 °C
Tests were done at  and  . Both had good results but at   more protein was produced.
400 mL
Centrifuge: 4000 33, 10 min, 4 10
20 mL
on ice
5 mL
Centrifuge: 5000 34, 10 min, 4 10
-80 °C
?. [PROCEDURE]
LYSISDay 5 (long day)17. Thaw pellets  (freeze-thaw cycle).18. Suspend the cell pellets to about  of wet cell pellet weight to lysis buffer.19. Vortex and keep . 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 . Pause for 30s between each burst.21. Centrifuge at . 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).
on ice
Transfer tube with pellets to   in the evening before.  , keep pellet upside down, not directly touching ice to thaw faster.
E.g. if pellet weighs 2.694 g, add 13.47 mL buffer.
on ice
on ice
Centrifuge: 26000 33, 20 min, 4 10
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).
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PRECIPITATION 23. Measure the volume of supernatant and add saturated ammonium sulfate to 24. Add the saturated ammonium sulfate drop-wise to the supernatant while stirring continuously using a separatory funnel in a ring stand. Stir for .25. Collect precipitated material by centrifugation at  (slow brake).
Vol saturated ammonium sulfate for 80% saturation = (Vol supernatant / 0.2) - Vol supernatant
Centrifuge: 20000 34, 13 min, 4 10
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RESUSPENSION26. Suspend pellets in a total volume of  of buffer A , mixing well using a 1 mL pipette. 27. Centrifuge at to clean any remaining debris and be easier to flow through Sephadex G-25 fine column
3 mL
on ice
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.
Centrifuge: 20000 34, 20 min, 4 10
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DESALTING28. 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.30. Once desalting of the sample is complete, wash column with 2 column volumes of  NaOH, rinse with water, and re-equilibrate with 2-3 column volumes of buffer A.
Start adding   of buffer A and collecting the aliquots. Check TSP “by eye” and when it gets blue reduce to  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.
For storage, antimicrobial agents should be added to the column to prevent contamination (  phenyl mercuric salts,   thimerosal,   chlorobutanol,   chlorhexine,   sodium azide, or   ethanol are acceptable). When necessary, the gel can be removed from the column and sterilized by autoclaving.
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PURIFICATION & ELUTION31. Purification and elution of dPGM uses a Strep-tactin Sepharose,  bed column, as detailed in the manufacturer’s instructions:
20 mL
For purification and regeneration of Strep-tactin resin, see Appendices 3 and 4.
?. [PROCEDURE]
PRECIPITATION32. Measure the volume of supernatant and add saturated ammonium sulfate to  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  (slow brake).35. Ressuspend pellet with   buffer F.
Centrifuge: 20000 34, 13 min, 4 10
3 mL
?. [PROCEDURE]
DESALTING36. 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).37. Pool samples with highest protein content.38. Once desalting of the sample is complete, wash column with 2 column volumes of  NaOH, rinse with water, and re-equilibrate with 2-3 column volumes of buffer A. 39. Store pooled fractions at .
It is not necessary to add proteases inhibitors as now it is a pure protein.
Start adding  of buffer F and collecting   aliquots. Check TSP “by eye”. In our hands, samples pooled were from fractions 3 to 6.
For storage, antimicrobial agents should be added to the column to prevent contamination (  phenyl mercuric salts,   thimerosal,   chlorobutanol,   chlorhexine,   sodium azide, or   ethanol are acceptable). When necessary, the gel can be removed from the column and sterilized by autoclaving.
-80 °C
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 1. Purification of plasmid DNA, aka minipreps for sequencingFollowing Day 3Prepare 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.Spin the remaining cell culture down for 10 min at 4000 rpm, room temperature.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.Resuspend the pellets in 250 μl Resuspension Solution (stored at 4°C) and transfer to labelled 1.5 ml tubes.Vortex briefly to eliminate all clumps of bacteria.Add 250 μl Lysis Solution, invert gently 4-6 times to mix, then incubate at RT for 4 min.Add 350 μl Neutralisation Solution, invert gently 4-6 times to mix.Spin in the microcentrifuge at 13,400 rpm for 5 min. Transfer the supernatant to a labelled spin column.Spin in the microcentrifuge at 13,400 rpm for 1 min. Discard the flow-through.Wash the column by adding 500 μl Wash Solution.Spin in the microcentrifuge at 13,400 rpm for 1 min. Discard the flow-through.Wash the column for a second time by adding another 500 μl Wash Solution.Spin in the microcentrifuge at 13,400 rpm for 1 min. Discard the flow-through.Spin the empty column in the microcentrifuge at 13,400 rpm for 1 min.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.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 fractionsUNINDUCED Sample (from Induction step 10.) Thaw pellet. Resuspend in about    of Lysis Buffer. Vortex and incubate on ice. 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.Centrifuge at 4000 rpm for 10 min at 4°C. Keep SN and pellet. 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).Resuspend pellet in   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.) After sonication, centrifuge at 4000 rpm for 10 min at 4°C. Keep supernatant and pellet. 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).
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							APPENDIX 2. SDS-Page for checking protein in different fractionsUNINDUCED Sample (from Induction step 10.) Thaw pellet. Resuspend in about   of Lysis Buffer. Vortex and incubate on ice. 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.Centrifuge at 4000 rpm for 10 min at 4°C. Keep SN and pellet. 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).Resuspend pellet in   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.) After sonication, centrifuge at 4000 rpm for 10 min at 4°C. Keep supernatant and pellet. 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).Resuspend pellet in  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.) After sonication, centrifuge at 4000 rpm for 10 min at 4°C. Keep supernatant and pellet. 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).Resuspend pellet in  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.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:
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100 µl
100 µl
100 µl
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.