Protein Syntheses in Cell Free Systems

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
  
  

  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 Aunits
  Viral mRNA or Globin 9S mRNA	2.0 to 5.0 µg
  or
  Poly U3	10.0 µg

Procedure4

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

   • 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.

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

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

   • 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.

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

   • 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.

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

   • 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.

   • 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. <