purifying proteins with repeats - (Jul/12/2013 )
I'm trying to purify a His tagged TALE fused to GFP in bacteria using nickel affinity chromatography. Notably, this protein contains 18x 200 bp direct repeats.
Unfortunately, I get a ladder of bands (including my specific band of 147 kDa)) when expressing the construct in bacterial cells. I've tested a battery of cell lines including bl21, blr (recA-, de) and stbl2 (RecA-, de). I have not tried Rosetta cells yet.
My hunch is that the extra bands are consequence of looping out of repeats.
My basic protocol is as follows;
1. Inoculate small LB culture with single colony and let it go overnight at 30c (temp they recommend for stbl2, i have tried 37c as well)
2. Next morning, inoculate larger LB culture and grow at 30c
3. When reaches OD 600=0.6 induce with 200 uM IPTG
4. Grow cells overnight at 18c
5. Perform standard His chromatography.
Any suggestions would be welcome,
What do you mean by, "My hunch is that the extra bands are consequence of looping out of repeats"?
My first question is- are you boiling your samples prior to SDS-PAGE? If not, then the ladder of protein bands may be the result of only partially denatured protein. I have had situations where I get several bands when not boiling the sample, but when boiled they all turn into a single fat band of the proper size. Also, do you have reducing agent in your SDS samples? The multiple bands could be from intra-molecular di-sulfides that cause the protein to run faster.
The other option is that these are proteolytically cleaved products. If you aren't already, consider adding a protease inhibitor cocktail during cell lysis.
Also, is your his-tag N- or C-terminal? If it's N-terminal, the multiple bands could be the result of translational truncation errors. Using a C-terminal his-tag will ensure you only purify full length protein.
You could also try running your nickle purified protein on a size exclusion column to see if you observe the same polydisperse pattern as you see on your gel. This will tell you whether you really have cleaved/truncated/proteolyzed protein, and if so, you could just purify out the proper peak with your full length protein.
Not sure if you are doing this:
For the inducing steps, I always pellet down the cells, replace the buffer with new buffer (which is already mixed with IPTG) and start induction, instead of just adding the IPTG itself.
^ why do you do that exactly? Seems like a tremendous amount of work, with risk of losing cells and contamination, especially if you're growing multiple liters of culture for expression. The only reason I can think of is if you're trying to label the protein with isotopes or seleno-methionine, and you want to grow in rich media but then induce in minimal media with the label. Is that what you're doing for expression?