Materials and Reagents

1. Protein purification
   1. pET28-a(+) (EMD Millipore, catalog number: 69864 ) or similar IPTG-inducible T7-based protein expression plasmid incorporating a 6x His tag
      
   2. Standard reagents and tools for molecular cloning (see Hasmann et al., 2011)
      
   3. E. coli BL21 (DE3) (New England BioLabs, catalog number: C2527I , or other source), or similar E. coli T7-based protein overexpression strain
      
   4. Kanamycin sulfate (50 mg/ml in dH₂O, filter sterilized) or appropriate antibiotic if using different plasmid
      
   5. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (100 mM in dH₂O, filter sterilized)
      
   6. Ni-NTA Agarose (QIAGEN, catalog number: 30210 )
      
   7. Imidazole (2 M in dH₂O)
      
   8. Lysogeny broth (LB) (see Recipes)
      
   9. Lysis buffer (see Recipes)
      
      
2. SDS-PAGE and zymogram
   1. SDS-PAGE gel (12% separation and 4% stacking layer, He, 2011)
      
   2. Laemmli buffer/sample loading buffer (He, 2011)
      
   3. 2-propanol
      
   4. Renaturation buffer (see Recipes)
      
   5. Coomassie brilliant blue protein stain solution (see Recipes)
      
   6. YPS broth (see Recipes)
      
   7. Destain solution (see Recipes)
      
   8. Phosphate buffer (see Recipes)
      

Equipment

1. 250 ml culture flasks
   
2. Incubator with shaker for culture flask (temperature adjustable) (or similar)
   
3. French press for cell lysis (see Note on alternative lysis methods)
   
4. Rotor JA-20 (Beckman Coulter) or equivalent
   
5. Centrifuge suitable for rotor (Beckman Coulter, model: J2-HS or similar)
   
6. Gravity column for Ni-NTA agarose
   
7. SDS-PAGE apparatus with power supply (Mini-Protean and PowerPac, Bio-Rad Laboratories or equivalent)
   
8. Microtube centrifuge (table-top)
   
9. Microwave (optional)
   
10. Flat (roux, or tissue culture) bottles (1 L for photoheterotrophic growth)
    
11. Light-emitting incubator for photoheterotrophic growth (see Note 6)
    
12. Erlenmeyer flask (500 ml) (pyrex, or other heat resistant glass)
    
13. Glass beaker (1 L or large enough to encompass a 500 ml flask) (pyrex, or other heat resistant glass)
    
14. Bunsen burner (or similar heat source)
    
15. Vacuum concentrator (Savant SpeedVac SC110A concentrator with UVS400 vacuum system, or similar) (optional)
    

Procedure