Materials and Reagents

1. Protein expression
   
   1. pGAPZ A, B, & C Pichia pastoris Expression Vectors (Life Technologies, catalog number: V200-20 )
      Note: Currently, it is “Thermo Fisher Scientific, Invitrogen^TM, catalog number: V200-20”.
      
   2. Zeocin^TM (InvivoGen, catalog number: ant-zn-5b ) for selection
      
   3. 1 % yeast extract (Carl Roth GmbH + Co., catalog number: 2363.3 )
      
   4. 2 % peptone (BD, catalog number: 211677 )
      
   5. D(+)-Glucose (Carl Roth GmbH + Co., catalog number: X997.3 )
      
   6. Yeast Peptone Dextrose (YPD) media (see Recipes)
   
   
2. Protein purification
   
   1. Acid washed glass beads (Sigma-Aldrich, catalog number: G 8772 )
      
   2. GFP-Trap^® (ChromoTek GmbH, catalog number: gta-20 )
      
   3. NanoOrange Protein Quantification Kit (Invitrogen, catalog number: N-6666 )
      Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes^TM, catalog number: N-6666”.
      
   4. 10 mM Tris (pH 7.5)
      
   5. 50 mM NaCl
      
   6. 0.5 mM EDTA (pH 8.0)
      
   7. 100 mM Glycine (pH 2.5)
      
   8. 1 M Tris (pH 10.4)
      
   9. Dilution buffer (see Recipes)
      
   10. Wash buffer I (see Recipes)
       
   11. Wash buffer II (see Recipes)
       
   12. Elution buffer (see Recipes)
   
   
3. GTPase activity assay
   
   1. GTPase Assay Kit (Innova Biosciences Ltd., catalog number: 602-0121 )
      
   2. Negative control: expressed and purified GFP using P. pastoris as expression system (plasmid pGAPZ::eGFP)

Equipment

1. 300 ml flasks
   
2. Shaker (160 rpm)
   
3. Centrifuge for 50 ml Falcon tubes (3,220 x g)
   
4. Vortex
   
5. Tube rotator at 4 °C and room temperature
   
6. Plate Reader (BioTek Instruments, model: Synergy HTX Multi-Mode Reader)
   
7. Tabletop centrifuge (cooling)
   

Procedure