Tetramers: Inclusion Body Preparation

This protocol describes isolation and washing of the inclusion bodies. These protocols are adapted from those originally described by David Garboczi.

Materials and Reagents

Equipment

Reagents and Chemicals

Resuspension Buffer (pH 8.0):

Wash Buffer (pH 8.0):

Wash Buffer without Triton X-100 (pH 8.0):

Urea Solution (pH 6.0):

Procedure

This protocol starts with a suspension of E. coli prepared according to the autoinduction protocol.

  1. Pool the resuspended bacteria by tranfer to a 100 ml polypropylene beaker containing a 1/2 inch stir bar.

    If you've ignored my pleas not to freeze the bacterial pellets, thaw the frozen pellets prior to transfer to the beaker.

  2. Stir the bacterial suspension on a stir plate at moderate speed.

    Resuspended bacteria are stirred during the addition of lysozyme, MgCls, DNase, Triton X-100; and DTT.

  3. To stirring mixture (usually 60 ml) add drop wise: 1.2 ml 50 mg/ml lysozyme (final = 1 mg/ml), 300 ul 1.0 M MgCl2 (final=5mM),1.0 ml of 2 mg/ml Dnase I in 50% glycerol containing 75 mM NaCl, 600 ul Triton-X 100 (final= 1%), 600 ul 1M DTT (final=10 mM).

    Adjust volumes accordingly if the volume of resuspended bacteria is not 60 ml.

    At some point in this process, usually after the addition of Triton X-100, the viscosity of the solution will increase dramatically, as the chromosomal DNA of the bacteria is released. As the DNase has a chance to digest the DNA, the viscosity will decrease.

    The subsequent sonication of the suspension serves a dual purpose: (1) to ensure that the bacterial are completely lysed; and (2) to further sheer the DNA that is not completely digested by the DNase. Sonication is also used throught the washing of the inclusion bodies, to ensure resuspension to homogeneity.

  4. Remove the stir bar from the bacterial suspension.
  5. Place the 100 ml beaker containing the bacterial lysate into a disposable 500 ml polypropylene beaker containing ice such that the smaller beaker is held firmly in place.
  6. Place the above on the lab jack inside the sonicator cabinet and insert the CL4 ultrasonic converter into the 100 ml beaker containing the bacterial lysate. Adjust the lab jack such that the ultrasonic converter is at the maximum depth without touching the bottom of the beaker.
  7. Sonicate the solution for 1.5 min at 0.5 sec alternations on power 4.
  8. Test the viscosity of the solution by drawing up an aliquot in a 1 ml pipette tip. If the solution flows freely without forming "strings", proceed to step 9. Otherwise, repeat step 7, taking care to ensure that the solution does not heat up.
  9. Transfer the lysates to three 25 X 89 mm Beckman polyallomer tubes and centrifuge in the Beckman CS-15R centrifuge with FO 630 rotor at Max RPM for 10 minutes at 4 °C.
  10. Take a 50 µl sample of the supernatant, label S1, and store at -20 °C.
  11. Decant the supernatant and add 1-2 ml wash buffer to the pellet.
  12. Fully dissociate the pellet using a plastic stir rod, add wash buffer to 15 ml, and continue to stir until the pellet is dispersed.
    Includion body disruption

    Inclusion bodies are disrupted with a Teflon stirring rod in a small amount of wash buffer before a larger amount of wash buffer is added and the suspension is sonicated.

  13. On ice, sonicate solution for 1.5 min at 0.5 sec alternations on power 4.
  14. Centrifuge the samples in the Beckman CS-15R centrifuge with FO 630 rotor at Max RPM for 10 minutes at 4 °C.
  15. Decant supernatant and take a 50 µl sample (S2).
  16. Repeat this wash step 3X and take supernatant samples (S3-S5).

    The number of wash steps depends upon the removal of contaminating material. Usually, this is obvious, as the pellets after each centrifugation step contain concentric rings of material. The inner ring is compact and lighter in color; it contains the desired protein. The outer rings are less compact, and contain contaminants. The size of the outer ring decreases with each washing step; you should wash repeatedly until the outer ring is nearly absent.

  17. Resuspend pellets in wash buffer without Triton X-100 as above.
  18. Centrifuge in a Beckman CS-15R centrifuge with FO 630 rotor at Max RPM for 10 minutes at 4 °C.
  19. Decant the supernatant and resuspend the pellet in 200 ul of ddH20. Once a white paste has formed resuspend the pellets in a total 10 ml urea solution. (ie-3 pellets in 10 ml M Urea).
  20. Centrifuge the 10 ml of inclusion bodies in a Beckman CS-15R centrifuge with FO 630 rotor at Max RPM for 10 minutes at 4 °C.
  21. Transfer the supernatant to a 15 ml polypropelene screw top centrifuge tube.
  22. Dilute 2 µl of the above inclusion bodies in 98 µl of urea solution and take a UV scan from 240 nm to 320 nm.

    In the Altman lab, our FileMaker databases perform this calculation for us, using extinction coefficients based upon the primary sequence of the protein (i.e. the number of tyrosine and tryptophan residues), using the method of Gill and von Hippel.

  23. Calculate the protein concentration. Aliquot 500 nMol fractions into eppendorf tubes.
  24. Quick freeze the aliquots in liquid nitrogen and store at -80 °C.
  25. Run pre-induction, post-induction, S1-S5, and a final inclusion body sample on a 10% or 12% SDS PAGE gel.

    We usually use 12% gels. When running b2m on the gel, it is essential to stop the gel as soon as the bromphenol blue dye front leaves the bottom of the gel, if not before.