Protein Synthesis in Cell Free Systems

Materials

• Suspension culture of fibroblast cells (1 liter)\
• 35 mM of Tris-HCl, pH 7.4, 140 mM NaCl (TBS buffer)
• 10 mM of Tris-HCl, pH 7.5, 10 mM KCl, and 1.5 mM magnesium acetate (TBS-M)
• 10X TBS-M: 200 mM of Tris-HCl, pH 7.5, 1200 mM KCl, 50 mM magnesium
• acetate and 70 mM β--mercaptoethanol
• 10X solution of 20 amino acids
• Teflon homogenizer
• Refrigerated preparative centrifuge
• Saturated (NH₄)₂SO₄
• TBS-M plus 20% (v/v) glycerol
• 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
  
  

  Tris-HCl, pH 7.5	1.5 µm
  Mg acetate	0.15-0.20 µm
  KCl	4.0-5.0 µm
  β--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
  14C-leucine (150 µCi/mmol)	0.125 µCi
  Ribosome fraction	1 to 2 A260 units
  Viral mRNA or Globin 9S mRNA	2.0 to 5.0 µg
  or
  Poly U	10.0 µg

Procedure

1. Chill the suspension culture (~10⁹ 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 3 times with cold TBS buffer.
2. Suspend the final pellet in 2 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 10000 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 concentrations:
   
   • 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 10000 xg for 10 minutes at room temperature. Cool the supernatant and pass it 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 (NH₄)₂SO₄ to yield a final 60% (NH₄)₂SO₄. 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 M sucrose into the bottom of a centrifuge tube and layer the suspended ribosomes on top. Centrifuge at 216000 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 A₂₆₀. 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 synthesis.
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 3-MM 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.
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 mutants known to alter the structure of either rRNA or any of the ribosome structural proteins.