February 28, 2014

A quick technical note/tip for folks doing recombinant protein expression in E.coli

When doing large-ish scale expression of proteins in E.coli, it is common to freeze the cell pellet post-harvesting and prior to cell lysis and protein purification. Typically this is achieved by re-suspending the cell pellet in a small about of Luria Broth or PBS and then re-pelleting the cells in a 50ml falcon tube, and freezing them at -80ยบC.

Whilst this is convenient, the down side of this is that it introduces another centrifugation step into your harvesting protocol, and a solid lump of cell paste can take a while to thaw out fully, and can be difficult to get completely homogenous and lump-free prior to whatever lysis technique you choose to use.

A colleague of mine who recently joined the lab from AstraZeneca (after they closed much of the Alderley Park research facility) has brought with him an ingenious way of speeding both harvesting and thawing out considerably: BUG SPLATS.

Poo in a bag?

Revolutionary bacteria pellet freezing protocol!

Rather than re-suspend your pellet in LB/PBS and re-pellet it, just scoop it out with a spatula (or similar) and place the bug pellet into a small (~10x20cm) press-lock bag.

Collect all the pellet into the bottom of the bag and then smooth it out so that your bug splat is nice and thin (<5mm is ideal), and stick it in the freezer.

The thin bug splat will freeze faster than a pellet in the bottom of a falcon tube.

It will also thaw out much faster. For thawing – snap the brittle, frozen bug splat into small chunks, and empty them into a suitable container (50ml falcon or similar), and add the required amount of lysis/sonication buffer. It should thaw out rapidly, and it should be easier to obtain a smooth, homogenous solution before lysis than a large ~25ml lump of frozen paste in the bottom of a falcon tube.

As the pellet is brittle, you can also snap smaller piece off if you don’t want to thaw the whole thing out.

So there you go – a ludicrously simple way of saving time in the lab, and making the freeze/thaw process faster, which is (anecdotally) better for your protein.