One-step Affinity Purification of the Chloroplast ClpP Complex from the Green Alga Chlamydomonas reinhardtii Using the Strep-tagII Epitope Tag

Materials and Reagents

1. Strain: The strain ClpP1-Strep has been described in (Derrien et al., 2012) and can be obtained from the ChlamyStation culture collection (http://chlamystation.free.fr)
   
2. StrepTrap HP 1 ml (GE Healthcare, catalog number: 28-9075-46 )
   
3. EDTA free protease inhibitors (Roche, catalog number: 05 056 489 001 )
   
4. d-Desthiobiotin ≥98% (IBA, catalog number: 2-1000-005 )
   
5. 1 M Tris HCl pH 8.0 at 4 °C
   
6. Ammonium sulphate saturated solution (>4.5 M; undissolved salt will settle to the bottom)
   
7. 0.4 M EDTA (pH 8.0)
   
8. 5 M NaCl 
9. DTT
   
10. 1 M MgCl₂
    
11. Glycerol
12. Buffer A (see Recipes)
13. Buffer B (see Recipes)
14. Elution buffer (see Recipes)
    

Equipment

1. Beckman centrifuge, rotors ( JA-10 , JA-20 , JA-12 and Type 70ti ) and polycarbonate centrifuge tubes (Beckman Coulter)
   
2. Semi-continuous centrifuge (e.g. Firlabo)
   
3. FPLC apparatus (BioLogic DuoFlow, Bio-Rad Laboratories)
   
4. French Press (Aminco)
   
5. Rotary wheel
   

Procedure