Preparation and Purification of β-1,3-glucan-Linked Candida glabrata Cell Wall Proteases by Ion-Exchange Chromatography, Gel Filtration, and MDPF-Gelatin-Zymography Assay

Materials and reagents

Biological materials

1. C. glabrata T-1639, blood isolate (Helsinki University Central Hospital, Helsinki, Finland). Other Candida species may be studied by these methods if there are structural analogies with C. glabrata cell wall protein attachment molecules.

   
   

1. Sabouraud dextrose agar (Lab M, catalog number: LAB009)

2. Lyticase, 10 kU (Sigma-Aldrich, catalog number: 37340-57-1)

3. 2-Methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) (Sigma-Aldrich, catalog number: 50632-57-0)

4. Precision Plus Protein^TM standards (Bio-Rad, catalog number: 161-0373)

5. Tris base (Sigma, catalog number: T-1378)

6. ZnCl₂ (Sigma, catalog number: 208086)

7. CaCl₂ (Sigma, catalog number: 10035-04-8)

8. Tween 80, BioXtra viscous liquid (Sigma, catalog number: P8074)

9. Bacto^TM yeast extract (BD, catalog number: 212750)

10. Peptone, Bacto^TM peptone (BD, catalog number: 211677)

11. Glucose (Merck, catalog number: 108337)

12. Sodium dodecyl sulphate (SDS) (Fisher Scientific, catalog number: BP166-500)

13. Sodium azide (NaN₃) (Merck, catalog number: 6688), for long-term preservation of ion-exchange columns

14. Coomassie Brilliant Blue, Serva Blue R (Serva Electrophoresis GmbH, catalog number: 35051)

15. Sodium tetraborate (STB) (Merck, catalog number: 6308)

16. Gelatin (Merck, catalog number: 1.04070)

17. Acetone (Merck, catalog number: 1.00658)

18. Na₂HPO₄ (Sigma, catalog number: 137036)

19. KCl (Sigma, catalog number: PHR1329)

20. NaCl (Fisher Scientific, catalog number: FLS271500)

21. KH₂PO₄ (Merck, catalog number: 104871)

22. β-mercaptoethanol (Merck, catalog number: M6250)

23. TEMED (Merck, catalog number: 411019)

24. Acrylamide 30% (Merck, catalog number: A3574)

25. EtOH, Ethanolum anhydricum, Aa (Berner, catalog number: 13110124)

26. HCl 1 M (Riedel-de-Haen, catalog number: 30721)

27. HCl 37% (Sigma, catalog number: 258148)

28. NaOH 1 M (Acros Organics, catalog number: 15634890)

29. Methanol (Fisher, catalog number: M/4057/PB17)

30. Acetic acid (Fisher, catalog number: A/0400/PB17)

31. Ammonium persulfate (APS) 10% (Sigma, catalog number: A3678), store in 100 µL aliquots at -20 °C

32. 4x Laemmli Sample Buffer (Bio-Rad, catalog number 1610747)

33. Glycine (Merck, catalog number: G8898)

    
    

Solutions

1. Yeast extract peptone glucose (YPG) (see Recipes)

2. MDPF-gelatin (see Recipes)

3. Separating gel (lower) buffer for zymo gels (see Recipes)

4. Stacking gel (upper) buffer for zymo gels (see Recipes)

5. 8% MDPF-gelatin SDS-PAGE (see Recipes)

6. SDS-PAGE running buffer 5× (see Recipes)

7. Zymo buffer 10× (see Recipes)

8. Zymo gel incubation buffers (see Recipes)

9. Coomassie Brilliant Blue (CBB) 0.1% (see Recipes)

10. IEC start buffer (see Recipes)

11. IEC elution buffer I (see Recipes)

12. IEC elution buffer II (see Recipes)

13. Phosphate-buffered saline (PBS) (see Recipes)

14. 10% SDS (see Recipes)

15. Lyticase stock (see Recipes)

    
    

Recipes

Perform pH adjustments with 1 M NaOH or 1 M HCl. Prepare zymogels as needed and solutions according to Recipes beforehand. Preparing double amounts of Recipes 10–13 is advised.

1. Yeast extract peptone glucose (YPG)

   0.5% yeast extract

   1% peptone

   1% glucose

   Add Milli-Q water (MQ) up to 4 L

   Sterilize by autoclave at 121 °C for 15 min

   
   

2. MDPF-gelatin

   20 mM STB solution: 0.3814 g of STB per 50 mL of MQ, pH 9.2.

   Add 500 mg of gelatin into STB in warm water bath (approximately 37 °C) with magnetic stirring.

   Dissolve 25 mg of MDPF into -20 °C acetone on ice and add MDPF into gelatin-STB at room temperature (RT) with magnetic stirring for 1 h protected from light. Divide into 500 µL aliquots and store at -20 °C protected from light.

   
   

3. Separating gel (lower) buffer for zymo gels

   800 mL of MQ

   187 g of Tris base

   Adjust pH to 8.8

   4 g of SDS

   Add MQ up to 1 L

   
   

4. Stacking gel (upper) buffer for zymo gels

   800 mL of MQ

   60.5 g of Tris base

   Adjust pH to 6.8

   4 g of SDS

   Add MQ up to 1 L

   
   

5. 8% MDPF (1 mg/mL)-gelatin SDS-PAGE (4 gels)

   Prepare gels in a fume hood:

   Bottom: separating gel

   Separating buffer 5 mL (Recipe 3)

   30% Acrylamide 5.3 mL

   MQ 7.3 mL

   MDPF-gelatin (1 mg/mL) 2 mL (Recipe 2)

   10% SDS 200 µL

   TEMED 20 µL

   10% APS 200 µL

   
   

   Top: stacking gel

   Stacking gel buffer 2.5 mL (Recipe 4)

   30% Acrylamide 1.35 mL

   MQ 5.95 mL

   10% SDS 100 µL

   TEMED 10 µL

   10% APS 100 µL

   
   

6. SDS-PAGE running buffer 5×

   Add 4 L of MQ into a Duran bottle

   723 g of glycine

   150 g of Tris base

   20 g of SDS

   Adjust pH to 8.3

   Add MQ up to 5 L

   
   

7. Zymo buffer 10×

   Add 800 mL of MQ into a Duran bottle

   60.55 g of Tris base

   2 g of NaN₃

   33 mL of 37% HCl

   Adjust pH to 7.5

   Add MQ up to 1 L

   
   

8. Zymo gel incubation buffers for 4 gels

   
   

   Zymo buffer I

   360 mL of 1× zymo buffer (dilute from 10× zymo buffer with MQ)

   40 mL of 25% Tween 80

   
   

   Zymo buffer II

   270 mL of 1× zymo buffer

   30 mL of 25% Tween 80

   30 µL of 10 mM ZnCl₂

   1530 µL of 100 mM CaCl₂

   
   

   Zymo buffer III

   300 mL of 1× zymo buffer

   30 µL of 10 mM ZnCl₂

   1530 µL of 100 mM CaCl₂

   
   

9. Coomassie Brilliant Blue (CBB), 0.1% (staining and destaining in fume hood)

   1 g of Coomassie Brilliant Blue, Serva Blue R

   600 mL of MQ

   300 mL of methanol

   100 mL of acetic acid

   Destaining: 40% MeOH + 10% acetic acid in MQ

   
   

10. IEC start buffer (20 mM Tris HCl, pH 8.2)

    2.4 g of Tris base

    Add 800 mL of MQ

    Adjust pH to 8.2

    Add MQ up to 1 L

    Sterile filter and degas

    
    

11. IEC elution buffer I (20 mM Tris HCl-0.5 M NaCl, pH 8.2)

    29.22 g of NaCl

    2.4 g of Tris base

    Add 800 mL of MQ

    Adjust pH to 8.2

    Add MQ up to 1 L

    Sterile filter and degas

    
    

12. IEC elution buffer II (20 mM Tris HCl- 1 M NaCl, pH 8.2)

    14.61 g of NaCl

    0.6 g of Tris base

    Add 200 mL MQ

    Adjust pH to 8.2

    Add MQ up to 250 mL

    Sterile filter and degas

    
    

13. Phosphate-buffered saline (PBS), pH 7.4

    800 mL of MQ

    8.0 g of NaCl

    1.44 g of Na₂HPO₄

    0.2 g of KCl

    0.24 g of KH₂PO₄

    Adjust pH to 7.4 using HCl

    Add MQ up to 1 L

    Filter and sterilize by autoclavation

    
    

14. 10% SDS

    80 mL of MQ

    10 g of SDS

    Add MQ up to 100 mL

    
    

15. Lyticase stock

    1.6 mL of MQ

    Add 10 kU lyticase

    Store at -20 °C in 400 µL aliquots

    
    

Laboratory supplies

1. Sterile plastic inoculation loops (Greiner Bio-One, catalog number: 731171)

2. Millipore Express^TM Plus or Filtration unit Stericup^® with MILLIPORE Express^® PLUS (PES), 0.22 µm (Merck Millipore, catalog number: X342.1, optional method: centrifugation)

3. Millipore Millex, 0.22 µm (Merck, catalog number: 051581)

4. Agar plates, plastic Petri dishes, PS (Sigma, catalog number: P5731)

5. Glass chromatography column (9.5 mL) with stopcock and lower silicon tubing (or Econo Alpha column, Bio-Rad, catalog number: 12009430) and column stand

6. Glass Erlenmeyers, 2× 3 L, 2× 200 mL

7. Amicon^® Ultra-15 Centrifugal Filter Devices, 10 K and 50 K (Merck Millipore, catalog numbers: UFC901024, UFC905024)

8. Glass decanters 500 mL

9. Eppendorf^TM Snap-Cap Microcentrifuge Safe-Lock^TM Tubes [Fisher, catalog number: 05-402-18 (1.5 mL) and 05-402-18 (0.5 mL)]

10. Falcon^TM tubes, 15 mL, 50 mL (Fisher, catalog numbers: 14-959-53A, 14-432-22)

11. Micropipettes and sterile pipette tips, 10 µL–5 mL

12. Aluminum foil

13. Tube racks for Falcon^TM and Eppendorf^TM tubes

14. HiTrap^® IEX Selection Kit (Merck, GE17-6002-33)

15. Sephadex G-100 and G-200 (Pharmacia Fine Chemicals, catalog numbers: 01-900-1-1861-03, 01-900-1-1863-03) 1 g/40 mL of buffer

16. 10 mL sterile plastic syringes (Fisher, catalog number: 14955459)

17. Ice, 2 × 5 L plastic dish

18. Duran bottles, 1 L, 5 L

19. Funnel that fits gel filtration column

Equipment

1. Heating cabinet, 31 °C, 37 °C, optional: Melag Incubat^® 85 (or Merck My Temp Mini Digital Incubator, catalog number: Z763357) or SureTemp^® Dual Convection Incubator (Merck, catalog number: Z742696)

2. Nanodrop 1000 (or NanoDrop Lite Plus Spectrophotometer, catalog number: NDLPLUSGL)

3. pH meter (VWR^® phenomenal^® MU 6100 H, catalog number: 665-0311)

4. PAGE-running device, 20 µL well volume, 4–6 combs of 10 wells (Bio-Rad, model: Mini-Protean II, catalog number: 165-2940)

5. Plastic containers (500 mL) for zymo buffer incubation, disposable PP, flat bottom (Marjukka, catalog number: 6410416249018)

6. Light box for gel visualization, LX104, Lammex (or X-ray viewer; Quirumed, catalog number: 482-6001.S)

7. Gel scanner, GS-700 densitometer (Bio-Rad, catalog number: 170-7601 with QUANTITY ONE PROGRAM)

8. Light microscope, 10–40× magnification

9. Vacuum suction system for degassing buffers and Sephadex gel

10. UV light for gel visualization and photography (Uvidoc, model: M03 0103 or GelDoc Go Gel Imaging System with Image Lab Touch Software, catalog number: 12009077)

11. Level mixer (Heidolph Vibramax 100, catalog number: 544-21200-00)

12. Centrifuge SL16R and adapters for 15 mL tubes (Thermo Scientific, catalog number: 75004030)

13. Autoclave (Systec, model: D45)

14. Fume hood (Visukaluste Ltd, model: VISU ProFocus EN 14175-2)

Procedure

1. Preparation of yeast cells

   1. Transfer C. glabrata cells from -80 °C onto a Sabouraud dextrose agar (SDA) plate with a sterile inoculation loop. Incubate the plate at 37 °C for 24 h. This incubation time is optimal to obtain enough stationary-phase yeast cells (expected OD > 2), as C. glabrata does not form true hyphae. However, the 24 h incubation time might be a problem in hyphae-forming species, such as C. albicans [try overnight (o/n) culturing].

      Check purity of growth visually from the culture plate and with light microscopy (10–40× magnification) regarding colony and cell morphology. Prepare a pure culture from this by picking one colony and incubate as previously described.

   2. Transfer a loopful of cells with a sterile inoculation loop from the pure culture SDA plate (check purity as above) into 2 × 2 L of YPG (see Recipe 1) divided into two Erlenmeyers. Cap the Erlenmeyers loosely with aluminum foil, place onto a level mixer, and incubate at low speed (65 rpm) for 24 h at 37 °C.

   3. Filter the cell suspension into 2 L aliquots with a Stericup with MILLIPORE Express PLUS 0.22 µm filter with vacuum suction. Use a second filter unit for the rest of the cell suspension, as the filter may clog. Recover the cells from the filter membranes with 150 mL of PBS/filter into eight 50 mL Falcon tubes in equal volumes.

   4. Centrifuge at 1,753× g for 10 min at RT. Discard supernatant.

   5. Add 40 mL of PBS onto the cells in each tube and suspend to even composition. Centrifuge at 1,753× g for 10 min at RT. Discard supernatants, add 40 mL of PBS to each tube, centrifuge at 1,753× g, and discard supernatants.

   6. Recover all cells from the bottom of each Falcon tube with 2.5 mL of PBS by suspending cells with a pipette and transfer them into a 100 mL Erlenmeyer. Add 40 mL of PBS (total suspension volume = 80 mL).

      
      

2. Lyticase treatments

   1. Add 400 µL of prepared lyticase solution onto the cell suspension (final concentration approximately 30 U/mL), cap loosely with aluminum foil, and incubate on a level shaker (at 65 rpm) for 22 h at 31 °C. (Here, the heating cabinet was placed and strapped onto a level mixer; optionally, place level mixer into a larger incubator cabinet.)

   2. Divide cell suspension equally into two 50 mL Falcon^TM tubes and centrifuge at 1,753× g for 10 min at RT. Recover supernatants (total volume = 35 mL) and store at -20 °C.

   3. Recover cells from Falcon tubes with a total of 40 mL of PBS into a 200 mL Erlenmeyer. Add 400 µL of lyticase solution, cap loosely with aluminum foil, and incubate on a level shaker (65 rpm) for 22 h at 31 °C.

   4. Repeat step B3. Freeze supernatants or continue pooling with supernatants from the freezer. Take a 500 µL sample for zymography and protein concentration measurement and store the rest at -20 °C.

   5. Resuspend cells in residual volume in the Falcons and verify that cells are intact with light microscopy (see Figure 1 ).

      
      

      
      Figure 1. Intact C. glabrata spheroplasts after lyticase treatments. Light microscopy, 40× magnification.

      
      

3. Separation of 10–50 kDa and > 50 kDa fractions

   1. Place PBS on ice. Thaw and filter pooled supernatants (V = 70 mL) with a Filtration unit Stericup with MILLIPORE Express PLUS 0.22 µm filter with vacuum suction or, optionally, by centrifugation. Take a 500 µL sample for protein concentration analysis and zymography. Divide the supernatant into eight Amicon^® Ultra-15 Centrifugal Filter Devices 10 K. Centrifuge at 1,753× g for 20 min at 4 °C. Remove and pool flowthroughs.

   2. Add 10 mL of cold PBS to each tube and centrifuge at 1,753× g for 20 min at 4 °C.

   3. Pool flowthroughs with earlier flowthroughs. Take a 500 µL sample for zymography of the pooled total flowthrough and store it and the rest in 50 mL Falcon tubes at -20 °C (< 10 kDa fraction). Transfer upper supernatants equally into two Amicon^® Ultra-15 Centrifugal Filter Devices 50 K. Centrifuge at 1,753× g for 20 min at 4 °C.

   4. Recover flowthrough (10–50 kDa fraction).

   5. Add 12 mL of PBS/filter and centrifuge at 1,753× g for 20 min at 4 °C. Pool flowthrough with the 10–50 kDa fraction (approximately 40 mL). Take a 500 µL sample and store it and the rest in 10 mL aliquots in Falcon tubes at -20 °C.

   6. Recover > 50 kDa fraction by suspending 3 × 5 mL of PBS to obtain approximately 30 mL of > 50 kDa fraction. Take a 500 µL sample and store it and the rest in 8 mL aliquots in Falcon tubes at -20 °C.

   7. Measure protein concentrations (A₂₈₀ nm) with Nanodrop 1000 and run 8% MDPF-gelatin zymography to evaluate protein recovery. See Figure 2.

      
      

      
      Figure 2. UV photograph of 8% MDPF-gelatin zymography after separation of 10–50 kDa and > 50 kDa proteases with centrifugation devices. Lanes: 1. Molecular weight standard in kDa. 2. Lyticase-treated fraction. 3. Lyticase-treated fraction with 1 µL of β-mercaptoethanol. 4. Lyticase-treated 10–50 kDa fraction. 5. Lyticase-treated > 50 kDa fraction. 6. Lyticase-treated > 50 kDa fraction with 1 µL of β-mercaptoethanol. Fraction in lane 5 was chosen for ion-exchange chromatography because of desired strong gelatinolytic activity of > 50 kDa sized proteases. The activating effect of β-mercaptoethanol as a reducing agent breaking disulfide bonds in the > 50 kDa fraction in lane 6 may be seen.

      
      

4. Preparation for gel filtration

   1. Measure 1 g of Sephadex G-200 and swell in 40 mL of 20 mM Tris HCl, 0.5 M NaCl, pH 8.2 at RT for 3 days in a sterile decanter (covered with aluminum foil).

   2. Degas gel and buffers with vacuum suction until no bubbling of gases. Store these at 4 °C until used.

      
      

5. MDPF-gelatin zymography

   1. Prepare 8% MDPF-gelatin-labeled zymogels. Prepare bottom separating gel. Add 4.45 mL of this solution between glasses in the casting device and add MQ on top of the gel to prevent drying and to obtain a linear top level of the gel. Let it settle for 30 min. Remove MQ by pouring and gently absorbing with cellulose blotting paper before casting the stacking gel. Prepare top stacking gel. Add stacking gel and comb and cover from light and drying while setting of gels (30 min). Store at 4 °C in MQ-moistened paper wrap and plastic bag covered from light until used. Shield from light during and after setting. Gels may be stored at 4 °C wrapped in MQ-moistened paper in a plastic bag shielded from the light up to one week.

   2. Incubate 15 µL of samples with 5 µL of 4× Laemmli sample buffer in 0.5 mL Eppendorf tubes at RT for 2 h in non-shaking conditions.

   3. Fill the running chamber using 1× SDS-PAGE running buffer (diluted from 5× running buffer with MQ). Use pre-stained SDS–polyacrylamide gel electrophoresis standards (10 µL, Precision Plus Protein^TM standards) for evaluation of protein size, pipette the standard, and incubated samples into gel wells.

   4. Perform electrophoresis at 110 V with the device shielded from light and on ice water.

   5. Incubate gels in zymo buffer I for 30 min at RT and then in zymo buffer II for 30 min at RT on a platform swing. Then, incubate in zymo buffer III o/n or for 1–7 days at 37 °C in a heat cabinet, all protected from light with aluminum foil.

   6. Follow the gelatinolytic reaction for 1–2 days with UV light (photograph), stop at the desired gelatin degradation stage, and stain the gel with 0.1 CBB, destain, scan image, and analyze. Gels may be stored in MQ until scanned.

   7. Choose fractions with the highest gelatinolytic activity and protein concentration. Pool and/or concentrate fractions if needed for the next step (see Figure 2).

      
      

6. Ion-exchange chromatography (IEC)

   Evaluate cation and anion exchangers with pH optimization (tested pH for the 10–50 kDa fraction was 6.4; for the > 50 kDa fraction, pH was 7.5 and 8.2) with both weak and strong anion and cation exchangers to find the best combination to capture desired proteins (HiTrap^® IEX Selection Kit). This process depends on the isoelectric point of the proteins and affects the bonding to and elution from the column depending on the net charge of the proteins. Aggregation of proteins is maximal at the isoelectric point (pI). We had prior knowledge of the estimated pIs from earlier studies using isoelectric focusing and 2D-MDPF-gelatin zymography. DEAE FF (weak anion exchanger) with buffer pH 8.2 is described in the rest of the protocol as an example for obtaining best recovery of the > 50 kDa fraction.

   1. Prepare IEC start buffer, IEC elution buffer I, and IEC elution buffer II (see Recipes). Filter buffers (0.22 µm), degas, and place on ice.

   2. Centrifuge 8 mL of > 50 kDa fraction at 1753× g for 10 min at 4 °C (fraction C6). Discard supernatant and recover volume to 8 mL with IEC start buffer. Concentrate sample as needed by centrifugation at 1753× g for 20 min at 4 °C. Keep sample on ice.

   3. Wash the DEAE FF column (HiTrap^® IEX Selection Kit) with 5 mL of IEC start buffer at 1 mL/min, then with 5 mL of IEC elution buffer II, and then with 5 mL of IEC start buffer again. Avoid air bubbles.

   4. Apply sample with syringe at 1 mL/min. Start gathering 0.5 mL fractions into 1.5 mL Eppendorf tubes. Wash the column with 5 mL of IEC start buffer and then add 5 mL of IEC elution buffer I.

   5. Store the column according to manufacturer’s instructions.

   6. Measure protein concentration with Nanodrop 1000 and run zymography gels to locate fractions with gelatinolytic activity and highest protein concentration. See Figure 3.

      
      

      
      Figure 3. UV photograph of 8% MDPF-gelatin zymography (A) and after Coomassie brilliant blue stain (B) for evaluation of ion-exchange chromatography. Lanes: 1. Molecular weight standard in kDa. 2. Protease sample before ion-exchange chromatography. 3 and 5. Flowthroughs. 4 and 6. Elution fractions. Elution fraction in lane 6 was chosen for gel filtration because of strongest gelatinolytic activity and highest A ₂₈₀ in the desired protein sizes.

      
      

7. Gel filtration

   The setup of the equipment for gel filtration is shown in Figure 4.

   
   

   
   Figure 4. Example of setting up the equipment for gel filtration. Keep samples on ice. Remember the safety loop in silicon tubing.

   
   

   1. Fill a 500 mL decanter with IEC elution buffer I. Enable buffer flow by placing the decanter so high that its silicon tubing end is at a higher level than the silicon tubing end attached to the lower part of the glass column. Ensure there is a safety loop in the tubing to avoid gel dehydration if buffer runs out (see Figure 4).

   2. Take a 9.5 mL column (15 cm length, radius = 4.5 cm) with lower silicon tubing. Open the lower valve of the column and, with a funnel, pour gel until the column is 4/5 full in one continuous motion to avoid air bubbles. Let it settle so that the upper level of the gel does not drop. Fill the column halfway from its lower end with 10% EtOH with a syringe to exclude air bubbles.

   3. Adjust the flow rate of the buffer with 2 × 10 mL of IEC elution buffer I to one drop per 30 s by adjusting decanter position or lower tube length. Drain the buffer to just above the top level of the gel. Shut the lower valve. The column may be stored at 4 °C for approximately one week if not immediately used. For longer storage, add 0.02% sodium azide and wash with approximately 2× column volumes before use.

   4. Recommended sample volume is 1%–5% of bed volume. Choose best elution fraction from ion exchange chromatography (see Figure 3) and concentrate 5 mL to 500 µL with Amicon^® Ultra-15 Centrifugal Filter Device 50 K.

   5. Apply 500 µL of the > 50 kDa fraction gently along the edge, avoiding splashing onto the top of the gel bed. Open the lower valve and let the sample sink in. Fill the column all the way to the top with IEC elution buffer I, close upper cap of the column, and attach the silicon tubing from the decanter to the top of the column. Open the lower valve and observe if buffer flow is continuous.

   6. Collect approximately 24 × 0.5 mL fractions. Number the tubes. Measure protein concentration and select the highest concentration (pool fractions if necessary). Run zymography gels to detect highest enzymatic activity for further analyses. See Figure 5. Chosen fraction may be further purified with Sephadex G-100 (as described above) and run on traditional 8% SDS-PAGE and parallel silver-stained gel for LC−MS/MS analyses (Figure 6).

      
      
      Figure 5. UV photograph of 8% MDPF-gelatin zymography after gel filtration. Lanes: 1. Molecular weight standard in kDa. 2. > 50 kDa fraction after ion-exchange chromatography. 3–8. Elution fractions 1–6 after G-200 gel filtration. Gel filtration of > 50 kDa fraction with Sephadex G-100 did not show any activity with zymography. Fraction 3 in lane 5 was further used in LC−MS/MS analyses.
      
      
      Figure 6. Overview of the purification results, 8% SDS-PAGE gel, silver stained. Lanes: 1. Molecular weight standard in kDa. 2. > 50 kDa fraction before IEC. 3. > 50 kDa fraction before gel filtration. 4−10. > 50 kDa gel filtration fractions.

Validation of protocol

This protocol or parts of it has been used and validated in the following research articles:

1. Pärnänen, P., Sorsa, T., Tervahartiala, T., and Nikula-Ijäs, P. (2020). Isolation, characterization, and regulation of moonlighting proteases from Candida glabrata cell wall. Microb. Pathogen. 149: 104547. https://doi.org/10.1016/j.micpath.2020.104547 (Figures 1 and 2).

2. Pärnänen, P., Meurman, J.H., and Nikula-Ijäs, P. (2015). A novel Candida glabrata cell wall associated serine protease. Biochem. Biophys. Res. Commun. 457: 676–680. https://doi.org/10.1016/j.bbrc.2015.01.047 (Figures 1 and 2).

3. Pärnänen, P., Kari, K., Virtanen, I., Sorsa, T., and Meurman, J. (2008). Human laminin-332 degradation by Candida proteinases. J. Oral Pathol. Med.37: 329-35. https://doi.org/10.1111/j.1600-0714.2008.00638.x (Figure 1).

General notes and troubleshooting

General notes

1. Keep fractions and samples on ice when appropriate to not lose activity.

2. Sterile filter or autoclave and degas buffers and store at 4 °C.

3. Ion exchange chromatography and gel filtration may be conducted also for the 10–50 kDa proteins in the sample with the same principles as for the > 50 kDa fraction.

4. Use sterile laboratory equipment to avoid contaminations.

5. Getting to know the principles of the methods in advance is advised.

   
   

Troubleshooting

1. Preparation of yeast cells

   Twenty-four hours has proven to be the best incubation time to ensure enough cells for the study. C. glabrata does not form true hyphae, but in other Candida species the incubation time should be shorter to keep cells in non-hyphal form; anti-hyphae substances may have to be used in addition. The OD ₆₀₀ should be well over two. Too long incubation periods often cause cells to start breaking or transforming.

   
   

2. Lyticase treatment

   1. If yeast cells are not intact when visualized by light microscopy, the lyticase concentration may be too high or treatment time too long. In this case, the intracellular proteins have leaked out and new cells must be cultured.

   2. If there is no or low gelatinolytic activity (zymography) left after lyticase treatment, check if incubation temperature is too low /too high. Other Candida species than C. glabrata may have a different bonding of the cell wall gelatinolytic proteases, which are not hydrolyzed by lyticase.

      
      

3. Separation of 10–50 kDa and > 50 kDa fractions

   1. If fractions show no or low gelatinolytic activity (zymography) left after centrifugal filtering devices, the proteins might not have been recovered from the filter. Try to gently suspend the buffer with a pipette tip (avoiding foaming) on the filter to detach stuck proteins.

   2. Too high salt concentration may precipitate proteins.

   3. Keep fractions on ice to avoid gradual loss of enzymatic activity.

      
      

4. MDPF-gelatin zymography

   1. If CBB-stained gels are not blue, check MDPF-gelatin concentration or expiration date. Keep MDPF-gelatin in the dark; otherwise, fluorescence is lost.

   2. Do not boil zymography samples prior to electrophoresis or enzymatic activity will be lost.

   3. Proteins < 50 kDa may be seen in Figures 3, 4, 6, and 7. This is assumed to be caused by autodegradation or dissociation of smaller subunits of proteins.

      
      

5. IEC

   1. Filter and degas buffers to avoid clogging of column by impurities or air bubbles.

   2. No or low activity in zymography: protein concentration too low (concentrate sample but remember that too much concentration aggregates proteins); wrong pH in buffer or unsuitable ion-exchange column matrix.

      
      

6. Gel filtration

   1. If proper size-dependent separation does not occur, the filtration column height or diameter may be wrong. You may also have to pack the column with matrix carefully and wait until the matrix settles to a horizontal position on the top part of the column. Gentle application of the sample into the column (not disturbing the settled matrix) ensures sharper fraction separation.

   2. If there is no buffer flowing through the column: impurities or air bubbles in gel matrix (caused by non-continuous packing of the column) or buffer vial located too low relative to the column (no hydrostatic pressure).

   3. Collect enough fractions and run zymograms of all fractions to recover/detect desired proteins.

Competing interests

The authors declare no competing interests.

References

1. Gow, N. A. R., Latge, J. P. and Munro, C. A. (2017). The Fungal Cell Wall: Structure, Biosynthesis, and Function. Microbiol. Spectr. 5. https://doi.org/10.1128/microbiolspec.FUNK-0035-2016
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