ProteinSynthesesinCellFreeSystems

LEVEL III

Materials

• Suspension culture of fibroblast cells (1 liter)
  

• 35 mM Tris-HCl, pH 7.4, 140 mM NaCl (TBS buffer)
  

• 10 mM Tris-HCl, pH 7.5, 10 mM KCl, and 1.5 mM magnesium acetate (TBS-M)
  

• 10X TBS-M
  

• 200 mM Tris-HCl, pH 7.5, 1200 mM KCl, 50 mM magnesium acetate




• and 70 mM

 

• -mercaptoethanol




• 10X solution of 20 amino acids
  

• Teflon homogenizer
  

• Refrigerated preparative centrifuge
  

• Sat. (NH)SO
  

• TBS-M plus 20% (v/v) glycerol
  

• 1X TBS-M buffer containing 0.25 M sucrose
  

• 1X TBS-M buffer containing 1.0 M sucrose
  

• Sephadex G-25 column equilibrated with 1X TBS-M buffer
  

• Liquid nitrogen storage
  

• Reaction mixture for protein synthesis, containing the following in a total volume of 50 µl 2

  
  

  Tris-HCl, pH 7.5	1.5 µm
  Mg acetate	0.15 - 0.20 µm
  KCL	4.0 - 5.0 µm
  -Mercaptoethanol	0.25 µm
  ATP	0.05 µm
  GTP	0.005 µm
  Creatine phosphate	0.50 µm
  Creatine kinase	8.0 µg
  Each of 19 amino acids(-leucine)	2.0 nmol
  C-leucine (150 mCi/mmol)	0.125 µCi
  Ribosome fraction	1 to 2 A units
  Viral mRNA or Globin 9S mRNA	2.0 to 5.0 µg
  or
  Poly U3	10.0 µg

Procedure 4

1. Chill the suspension culture (app. 10 cells) rapidly in an ice bath. Collect the cells as a pellet, by centrifugation at 600 xg for 10 minutes at 4° C. Resuspend the cells in TBS buffer and wash them three times with cold TBS buffer.

   
   

2. Suspend the final pellet in two volumes of TBS-M for 5 minutes at 0° C and homogenize the cells with 10 to 20 strokes in a tight fitting Teflon homogenizer.

   
   

3. For each 0.9 ml of homogenate, add 0.1 ml of concentrated 10X TBS-M buffer. Centrifuge the mixture at 10,000 xg for 10 minutes at 4° C.

   
   

4. Decant and collect the supernatant extract and adjust the extract such that the following are added to yield final concentration:

   
   

   
   

• ATP to 1.0 mM ATP




• GTP to 0.1 mM GTP




• Creatine phosphate to 10 mM




• Creatine kinase to 160 µg/ml




• Amino acids to 40 µm each




5. Incubate the mixture for 45 minutes at 37° C.

   
   

6. Centrifuge the mixture at 10,000 xg for 10 minutes at room temperature. Cool the supernatant and pass the supernatant through a Sephadex G-25 column at 4° C.

   
   

7. Turn on a UV spectrophotometer and adjust the wavelength to 260 nm. Blank the instrument with TBS buffer.

   
   

8. Centrifuge the filtrate excluded from the Sephadex column at 165,000 xg for 90 minutes at 4° C.

   
   

9. Precipitate the proteins within the supernatant by the addition of saturated (NH)SO to yield a final 60% (NH)SO. Collect the precipitate by centrifugation.

   
   

10. Dissolve the precipitate in TBS-M buffer and dialyze it against the same buffer containing glycerol.

    
    

11. Suspend the resulting ribosome pellet in 1X TBS-M buffer containing 0.25 M sucrose. Place 5 ml of TBS buffer with 1.0 M sucrose into the bottom of a centrifuge tube and layer the suspended ribosomes on top. Centrifuge at 216,000 xg for 2.5 hours at 4° C.

    
    

12. Wash the resulting pellet with TBS-M buffer, and resuspend it in the same buffer with 0.25 M sucrose.

    
    

13. Determine the ribosome concentration using a UV spectrophotometer to measure the A. The extinction coefficient for ribosomes is 12 A units per mg per ml at 260 nm.

    
    

14. The ribosomes may be frozen and stored in liquid nitrogen, or used for in vitro protein synthesis. If frozen, they should be thawed only once prior to use.

    To test for protein synthesis, prepare the reaction mixture for protein syntheis.

    
    

15. Incubate the reaction mixture at 37° C for 60 minutes. Terminate the reaction by pipetting 40 µl of the mixture onto a 2.5 cm disk of Whatman 3MM filter paper. Dip the disk into cold 10% TCA for 15 minutes and then in 5% TCA at 90° C for 15 minutes.

    
    

16. Rinse the disk twice in 5% TCA for 5 minutes, once in alcohol:ether (1:1), and then dry it.

    
    

17. Place the disk into a scintillation vial and add a toluene-based fluor. Refer to Appendix H for details on the use of a scintillation counter.

    
    

18. Measure the amount of radioactively labeled amino acid incorporated into protein.

    
    

19. Graph the protein synthesized versus time.

Optional

For advanced work, compare the activity of ribosomes isolated from the fibroblast cultures to those isolated from a prokaryote culture, a plant (yeast or pea seedlings) and from genetic µ tants known to alter the structure of either rRNA or any of the ribosome structural proteins.

If sources of mRNA are available (or if the time is taken to extract RNA and electrophoretically purify it), then comparisons of the rate of protein synthesis can be made within this system. Finally, once the proteins are synthesized, they can be separated on SDS-PAGE and rates of synthesis can be determined by both time of incorporation of leucine and by increasing molecular weights. The latter will also demonstrate the possible presence of µ ltiple RNA''s with the system, whereas the radioactivity will mask the presence of heterogenous RNA.