Using the Bugbuster 10X cell lysis reagent in order to purify our honeybee silk protein via inclusion bodies.
Based protocol off of the Novagen manufacturer's protocol which can be found [http://wolfson.huji.ac.il/purification/PDF/Protein_Expression_Extraction/NOVAGEN_BugBuster_protein_extraction.pdf here].
Expressing honey bee silk in E. Coli, from [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001]
See 5/18 and 5/19 for details on the protein expression.
Step by Step Protocol
Determine wet wet weight of cell pellet after spinning liquid culter at 10000 x g for 10 min.
2.5 g
Resuspend in 5 ml/g Bug Buster (1x) by pipetting and gently vortexing.
This is 12.5 ml in our case
Put on shaker or rotating mixer for 15 min at RT
Took our first fraction at this point of the full cell lysate (F1)
Centrifuge 16000 g 20 min at 4 degrees C
Took next fraction at this point of the supernatant, labeled (S1)
Resuspend pellet in same volume of 1X bugbuster as before
12.5 ml
Pipett up and down and vortex gently to get an even suspension.
Took third fraction at this point (F2)
I did not go to great lengths to get an even suspension here, but I noticed in retrospect that the protocol emphasized the importance of this in order to get pure inclusion bodies.
Add dry lysozyme to final concentration of 200 ug / ml
For 12.5 ml solution I added around 2.5 mg of lysozyme
I did not add any DNAse because it was not mentioned on the manufacturer's protocol, but in the future I will try it with 1 ul of DNAse because chromosomal DNA might have been a problem in subsequent steps.