Purification and Detection of a PDGA Depolymerase from Pusillimonas noertemannii

Materials and Reagents

1. Pusillimonas noertemannii BS8 (isolated by soil enrichment)
   
2. Pseudomonas fluorescens BS2 (isolated by soil enrichment)
   
3. Poly-γ-D-glutamic acid >250 kDa [prepared in-house; protocol for production can be found in Negus and Taylor (2014)]
   
4. Protein assay dye reagent (Bio-Rad Laboratories, catalog number: 500-0006 )
   
5. 1.5 ml Microcentrifuge tubes (Eppendorf, catalog number: 0030120086 )
   
6. Laemmli sample buffer (Sigma-Aldrich, catalog number: S3401-10VL )
   
7. IPG strips (immobilized pH 3-10, 7 cm) (Bio-Rad Laboratories, catalog number: 163-2000 )
   
8. Mineral oil (Bio-Rad Laboratories, catalog number: 163-2129 )
   
9. Rehydration trays (Bio-Rad Laboratories, catalog number: 165-4035 )
   
10. Acrylamide solution (Bio-Rad Laboratories, catalog number 161-0156 )
    
11. TEMED (Sigma-Aldrich, catalog number T9281 )
    
12. Agarose overlay solution (Bio-Rad Laboratories, catalog number 163-2111 )
    
13. Zymogram renaturation buffer (Bio-Rad Laboratories, catalog number: 161-0765 )
    
14. Zymogram development buffer (Bio-Rad Laboratories, catalog number: 161-0766 )
    
15. Molecular biology grade water (Merck KGaA, catalog number: H20MB05010 )
    
16. 30% ethanol
    
17. 50% ethanol
    
18. Minimal salts medium broth (see Recipes)
    
19. Anion exchange binding buffer (see Recipes)
    
20. Anion exchange elution buffer (see Recipes)
    
21. Equilibration buffer (see Recipes)
    
22. Non-reducing sample buffer (see Recipes)
    
23. SDS buffer (see Recipes)
    
24. Resolving gel buffer (see Recipes)
    
25. Stacking gel buffer (see Recipes)
    
26. 10% SDS (see Recipes)
    
27. 10% APS (see Recipes)
    
28. 10x TGE running buffer (see Recipes)
    
29. Coomassie-based dye (see Recipes)
    
30. Coomassie destain solution (see Recipes)
    
31. Methylene blue stain (see Recipes)
    
32. 70% ethanol (see Recipes)
    

Equipment

1. Universal tube (Sterilin^®, catalog number: 128B/P )
   
2. Erlenmeyer conical flask narrow neck 2 L (Thermo Fisher Scientific, catalog number: 11577422 )
   
3. Cuvettes (1.6 ml semi-micro) (Thermo Fisher Scientific, catalog number: 10594175 )
   
4. Syringe driven filters (0.22 µm, polyethersulfone) (Millex^®, catalog number: SLGP033RS )
   
5. Electrode paper wicks (Bio-Rad Laboratories, catalog number: 165-4071 )
   
6. Shaking incubator (Infors AG, model: Multitron standard )
   
7. Spectrophotometer (PerkinElmer, model: Lambda 25 )
   
8. Benchtop centrifuge (Sigma-Aldrich, model: 4-16K )
   
9. Sonicator (MSE, model: Soniprep 150 )
   
10. Exponential sonicator probe (MSE, catalog number: 38121-114A )
    
11. Centrifugal protein concentrators (Sartorious, model: Vivaspin 20, 3 kDa MWCO, catalog number: VS2092 )
    
12. Centrifugal protein concentrators (Sartorious, model: Vivaspin 6, 3 kDa MWCO, catalog number: VS0692 )
    
13. Static incubator (Unitemps, discontinued) (B&T)
    
14. ÄKTAprime plus chromatography system (GE Healthcare, Prime plus, catalog number: 11-0013-13 )
    
15. Anion-exchange chromatography column (GE Healthcare, model: Hi-Trap Q XL, catalog number: 17-5158-01 )
    
16. Orbital rocking platform (Stuart, model: SSM1 )
    
17. Thermal cycler (TECHNE, discontinued, model: FTGENE 2D )
    
18. Electrophoresis cell (Bio-Rad Laboratories, Mini-PROTEAN^® Tetra Cell, catalog number: 165-8003 )
    
19. Isoelectric focusing cell (Bio-Rad Laboratories, Protean, catalog number: 165-4000 )
    
20. Vortex mixer (Appleton Woods, Clifton cyclone, catalog number: AA6500 )
    

Procedure