Proteolytic Fragment Isolation and Analysis (ex. N-terminal GST-tagged CLAVATA3 Protein GST-CLV3)

Materials and Reagents

1. Glutathione Sepharose 4B (Amersham biosciences , catalog number: 17-0756-01 )
   
2. Glutathione (Thermo Fisher Scientific, catalog number: 78259 )
   
3. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P9599 )
   
4. Triton X-100 (Pierce, catalog number: 85111 )
   
5. GST-mCLV3 proteins
   
6. Tris-HCl
   
7. Hepes (Sigma-Aldrich, catalog number:  H3375 )
   
8. EDTA (Sigma-Aldrich, catalog nummber: ED100g )
   
9. Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number:  78830-1G )
   
10. Aprotinin (Sigma-Aldrich, catalog number: A4529-1MG )
    
11. Chymostatin (Sigma-Aldrich, catalog number:  C7268-1MG )
    
12. Leupeptin (Sigma-Aldrich, catalog number: L2884-5MG )
    
13. Eluting buffer (see Recipes)
    
14. Cauliflower extraction buffer (see Recipes)
    

Equipment

1. Rotor
   
2. Microcon YM-10 centrifugal filter (EMD Millipore)
   
3. Tabletop centrifuge
   
4. Mass spectrometer (4700 proteomics analyzer)
   

Procedure

1. Cauliflower (Brassica oleracea) meristem protein extracts were prepared as described before (Trotochaud et al., 1999) with or without 0.1% Triton X-100. Note that 1 ml of protease inhibitor cocktail for use with plant cell extracts was added in the extraction buffer per 300 x g of tissues. Before use, the extracts were centrifuged at 40,000 x g for 30 min at 4 °C. It is very important to use fresh cauliflower. We usually try to get cauliflower from local farms. The meristem tissues were collected using razor blade “shaving” the head of the cauliflower.
   
   
2. Incubate ~100 μg purified GST-mCLV3 proteins (mature CLV3 protrein) with ~400 μl cauliflower protein extracts for 2 h at room temperature (RT) on a rotor.
   
   
3. For N-terminal tagged fragment (~ 32-34 kD) isolation.
   1. Bound protein mixtures to Glutathione Sepharose 4B.
      
   2. Elute the N-terminal GST containing fragments from the beads with buffer containing 10 mM Glutathione, 50 mM Tris-HCl (pH 8.0).
      
   3. If needed, concentrate protein elution using Microcon YM-50 centrifugal filter at 14,000 x g, 4 °C for 30 min and elute in desired volume and buffer (e.g. 0.1% TFA trifluoroacetic acid/water) for MS analysis.
      
   
   
4. For C-terminal untagged fragment (~ 2-4 kD) isolation.
   1. Subject protein mixtures to centrifuge through Microcon YM-10 centrifugal filter at 14,000 x g, 4 °C for 30 min.
      
   2. Collect the flow-through fractions for intact MS analysis.
      
   3. Further characterize peaks of interest by MS/MS analysis (control: Cauliflower protein extracts only; GST-CLV3 incubated with cauliflower extraction buffer).
      

Recipes

1. Eluting buffer
   50 mM Tris-HCl
   10 mM reduced glutathione (pH 8.0)
   
2. Cauliflower extraction buffer
   50 mM Hepes (pH 7.4)
   10 mM EDTA
   0.1% Triton X-100
   1 mM phenylmethylsulfonyl fluoride
   5 mg/ml aprotinin
   10 mg/ml chymostatin
   1 mg/ml leupeptin
   For extracts without membrane proteins, exclude Triton X-100 from the buffer.
   

Acknowledgments

This protocol has been adapted from previous publications including Ni and Clark (2006), Ni et al. (2011) and Trotochaud et al. (1999).

References

1. Ni, J. and Clark, S. E. (2006). Evidence for functional conservation, sufficiency, and proteolytic processing of the CLAVATA3 CLE domain. Plant Physiol 140(2): 726-733.
   
2. Ni, J., Guo, Y., Jin, H., Hartsell, J. and Clark, S. E. (2011). Characterization of a CLE processing activity. Plant Mol Biol 75(1-2): 67-75.
   
3. Trotochaud, A. E., Hao, T., Wu, G., Yang, Z. and Clark, S. E. (1999). The CLAVATA1 receptor-like kinase requires CLAVATA3 for its assembly into a signaling complex that includes KAPP and a Rho-related protein. Plant Cell 11(3): 393-406.