His-Tag recombinant protein purification

[qpwoei4756]

Hello,

I am purifying a six-His tagged 50kDa hnRNP K protein from E coli cells using IMAC with Cobalt TALON resin. I am trying to send the proteins for crystallography and it requires a good quality of sample. My problem is that smaller bands are visible on the gel and I don't know how to get rid of them.
What might the problem and how can I fix it?
Can using a whole range of gradients (50, 60, 70, 80, 90, 100, 110mM, etc.), or adding imidazole to the wash buffer improve the specificity of the resin-bound proteins?

Attached is one sample of my purification on an SDS-PAGE gel. This was done after growing E coli on 1 liter of liquid media. From left to right: protein standards, lysis buffer mix, resin flow-through, wash 1, wash 2, wash 3, 50mM imidazole, 100mM, 200mM, 300mM.
Please Note that the wash buffer didn't elute out anything, and 100, 200, 300mM on the right side has a thick band with smaller contaminating proteins. I tried higher concentration of tris-HCl (but not NaCl)on the wash buffer but it did not improve the results for the washing step.

Thank you so much and appreciate your help!

qpwoei4756



P.S. Here is a short hand note of my reagents/protocol:

lysis buffer: 50MmTris-HCl (ph7.5), 10% glycerol, 0.5M NaCl;
Washing buffer: 50MmTris-HCl (ph7.5), 10% glycerol, 0.3M NaCl;
Elution buffer: 50MmTris-HCl (ph7.5), 10% glycerol, 0.3M NaCl, made 20ml each then add imidazole to final concentration of 50mM, 100mM, 200mM, 300mM to each.
Used 1L LB+drug to grow E coli up to OD600 of 0.6. Then induced the recombinant protein on a plasmid vector using IPTG for 4 hours 37C. Centrifuged and collected cell pellets. Went to 4C room, added lysis buffer with PMSF, sonicated, centrifuged, obtained supernatants, poured it onto cobalt-resin,
washed three times, and collected using imidazole elution buffer. SDS gel.

[mmorgan]

Hi,
To get your protein totally pure you will probably need to use a second purification step. You could try size exclusion chromatography or experiment with various types of columns. It's possible that those bands are cleavage products of your protein that contain the His tag, so you may never be able to wash them off without also eluting your protein.

Another possibility is to add a second tag to your construct and to a tandem affinity purification. However, seeing as how you already seem to have good expression of your construct, it might be faster to try to clean up the sample that you have. Go make friends with somebody with an AKTA purifier!

Best of luck!

[qpwoei4756]

Thanks mmorgan,

Before doing size exclusion to filter out the smaller proteins, I can try to optimize my extraction conditions. I just found out that the PMSF protease inhibitor was water-sensitive and I've been storing it in water this whole time! Maybe that's the problem.
I will also use a gradient of elution buffer in a whole range of imidazoles, like 50, 75, 100, 150, 200, 250, 300, instead of 50, 100, 200, 300mM. I'm also going to try to pre-treat the wash buffer with 5mM imidazole to elute out endogenous proteins that have the ability to bind my resins. But, SEC will be my last resort.

Cheers,

qpwoei4756

[admin]

HI QP, I am not sure you can do this with your protein crystallography method but to get rid of small kDA proteins I would dialyze in buffer with a membrane that keeps proteins in larger than for example 20 or 30kDa. Would get rid of all your small protein issues.

As mentioned there are size exclusion collumns but the only issue is sometimes they bind the protein of interest very well and can make it difficult for you to retain a good amount. Another caveat for dialysis of protein is you have to be very sure to use a fresh membrane, of good quality well sealed at the ends as you can lose your entire sample if you are not careful.

best wishes

[qpwoei4756]

To admin,

Well, I did use a dialysis tubing after doing the gel, but I haven't checked its effectiveness yet. I only thought its purpose was to desalt my eluted sample. I will try it next time on the gel, but that's a good point.
The nominal MWCO for the cellulose dialysis tubing I used was 6,000-8,000, so I guess I can get one with a higher (up to 30,000?) mw cut off and try it.
Thanks for your advice!

qpwoei4756