What's the best way to purify my His tagged protein? Supernatant super viscous after first sonication?

Question

I am trying to purify my his-tagged protein of interest, disulfide isomerase. It is about 40kDa and is cloned in pET28a vector, at XholI and NdelI, and expressed in BL21.

I'm having issues with my protein purification.

My current protocol:

1. 1L culture incubated at 37°C for 6 hours until OD600 reaches 1.5-2.0.
2. IPTG is added to a final concentration of 1mM and is then further incubated for another 3-4 hours until OD600 reaches 2.2-2.7.
3. I then lyse the cells using 1X binding buffer with 1X Protease inhibitor, no urea, 0.1mg/ml lysozyme, and DNase1.
4. solubilize/sonicate with 1X binding buffer WITH 8M urea and 1X protease inhibitor.
5. purify my protein by doing affinity chromatography using a nickel column with 1mL of Ni-Nta resin.
6. I washed twice with 1XBB with 8M urea, 15-20mM imidazole, and 1X protease inhibitor.
7. elute with 1XBB with 8M urea, 250mM imidazole, and 1X protease inhibitor.

During the solubilization step, I sonicate twice. After my first sonication and spinning down in the centrifuge for about 30min, my supernatant comes out super viscous and when I run it through my ni-nta column, it ends up clogging it and causing it to run super slow (1 drip = 5min). What can I do to fix this issue?

Should I try using 6M urea instead of 8M? Would that help with the viscosity?

Also, my elutions come out really dirty still with a lot of contaminant protein. How can I clean up my elutions?

My protocol for my wash calls for protease inhibitor, but I feel like that's a bit wasteful. Can I wash without using protease inhibitor first and then do another wash with the protease inhibitor? Or should I be using protease inhibitors in every wash?

Answer 1

As Chris said in the comments, make sure you have sufficient DNase in your lysis buffer. That combined with good sonication will definitely shear nucleic acids and make your solution less viscous. If you don't feel the lysate starting to warm up when you're sonicating, you might want to increase the power. To that end, keep the lysate on ice while sonicating so the heat doesn't denature your protein of interest irreversibly.

You may also want to increase the amount of lysis buffer you're using to dilute everything a little bit, as you'll be concentrating and eluting it later. Sometimes lysates can be so thick that even harsh sonication won't completely eliminate it, and you run the risk of damaging your protein.

As far as the dirty eluate is concerned, a couple tips. First, after binding, make sure you wash the column extensively before eluting. You may find with experience that washing with a little bit of imidazole helps clean up non-specific binding as well. If you have one available, use an automated fraction collector (or even collect small fractions by hand) instead of doing a bulk elution. This way you can test each fraction separately and get rid of the ones with the most contamination and least specific product. Keep in mind that very concentrated samples, even if they are quite pure, will look dirty if you load too much protein on the gel, so keep a protein quantitation kit that's compatible with your buffers handy.

Another option to doing a bulk, single-concentration imidazole elution (say, at 250 mM like you're doing currently) is to do what's called a gradient elution. Say you're using a 10 ml column. After washing with 10-20 column-volumes (CV), start eluting with 10 ml of perhaps 50 mM imidazole, let it run out (but not totally dry), then add 10 ml of 100 mM, and so on. This is obviously much easier if you have an FPLC or similar setup, but it can be done reproducibly by hand if you're careful. Collect 5 ml fractions, and test them individually before pooling the good ones.

Protease inhibitors aren't strictly necessary in the wash, but I'd include them at least in the first wash, as that's when proteases are most likely to be non-specifically associated with your column beads and acting on any available substrates. Keeping everything cold is helpful, as well.