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purifying proteins with repeats

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#1 Kaire76



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Posted 12 July 2013 - 08:05 PM

Hi all,

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,


#2 labtastic



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Posted 18 July 2013 - 06:07 AM

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.

#3 Adrian K

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Posted 29 July 2013 - 01:21 PM

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.



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#4 labtastic



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Posted 30 August 2013 - 04:37 AM

^ 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?

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