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protein fractionation

protein fractionation bacteria as spec upec

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4 replies to this topic

#1 salmonela

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Posted 09 May 2013 - 03:31 AM

Hi all

I work on an uropathogenic strain of E.coli. When I artificially express from a plasmid one of the transcriptional regulator, it results in secretion of a protein factor in the media, which has interesting biological roles. My aim is to purify this unknown factor present in the growth media and to identify it by Mass Spec.
But the problem is I am not familiar with the fractionation methods. Looking through the literature, I could see that many different protocols are being employed out of which I don’t know which the best is. I like your suggestion with regard to a good standard protocol for identifying an unknown protein in the growth media.
Again to put it differently, I have protein in the supernatant of the growth media. I just need to identify it.
Thanks for all those who read this and Thank you very much for someone who comments.
Moti

#2 mdfenko

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Posted 09 May 2013 - 04:40 AM

it depends on whether you have a tagged protein, size of the protein and any other proteins in the growth medium, etc.

if it is tagged then you can purify with a matrix that reversibly binds the tag.

if not, and the protein is significantly different in size than other proteins in the solution, then you can use gel filtration.

if you can't get good separation with gel filtration then you can try ion exchange or hydrophobic interaction or a combination of methods.

you can download a handbook on purifying recombinant proteins (as well as others) from this ge healthcare webpage.

Edited by mdfenko, 09 May 2013 - 04:41 AM.

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#3 salmonela

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Posted 09 May 2013 - 05:12 AM

Thanks for the reply.

The protein is not tagged, actually I don't know much about the secreted protein (what I know is that when I doing fractionation based on size - by centricon the proteins remain in the fraction above 100kda).

#4 mdfenko

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Posted 09 May 2013 - 05:35 AM

then you can try gel filtration as a first step.

or ion exchange.

check out the handbook(s).
talent does what it can
genius does what it must
i do what i get paid to do

#5 labtastic

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Posted 14 May 2013 - 08:20 AM

I would recommend ion exchange over gel filtration as a first step because then you don't have to concentrate your media (saving yourself a step). Just run all those liters of media over the ion exchange column, wash thoroughly and elute with a salt gradient. Run 5ug of protein from each fraction on a gel to track where your protein came off the column.

Alternatively, if you want to to do gel-filtration first you could ammonium sulfate precipitate your protein out of the media first. Resuspend the pellet in a minimal amount of low salt buffer and run on the gel filtration (no dialysis needed). The only caveat is the hope that your protein precipitates with ammonium sulfate. Most do in my experience, but not all.

Although now that I think about it, you don't need to purify the protein to identify what it is. Run the media on an SDS-PAGE gel (you may need to concentrate the protein using acetone or TCA precipitation), identify your protein band and do an in-gel trypsin digest. Send it to mass spec for sequencing. Since the genome of e.coli is well known, you should be able to identify the protein and the gene from which it came. Clone the gene into an expression vector and see if you can't over express it in quantities needed for future studies.

Edited by labtastic, 14 May 2013 - 08:21 AM.






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