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Purifying hydrogen bonded peripheral protein


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#1 AJ Burdette

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Posted 07 May 2009 - 12:22 PM

Hello All,

I'm having some trouble purifying a particular protein. I've fused MBP to my AAA+ protein for overexpression and purification in E. coli. Surprisingly, I found that it associates with E. coli membranes and the N-terminal portion of my protein is responsible for this anchoring as other constructs of it fused to MBP remain in the cytosol.
From further experiments, I've found that it can only be removed by high ph and triton x-114, which relocates it to the acqueous phase suggesting a h-bond association.

Here is the problem. High Ph and detergent (above the cmc) kills the atpase activity of the protein. Does anyone have any suggestions to break these bonds without hurting the protein? I'm thinking of possibly sonicating the membranes in triton x-100 at 0.01% to try to solubilize all the membranes without getting above the cmc of the detergent and compromising the proteins activity. Any suggestions are greatly appreciated.

AJ Burdette

#2 LifeTein Peptide

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Posted 12 May 2009 - 05:54 PM

I guess it will be too much work for you to mutate the N terminus and start it over. If you change the hydrophobic aa of your N terminus to hydrophilic, it will not be the anchor.

It is hard to release the protein without using the detergent to destroy the membrane. Have you tried other mild detergent such as 0.5% NP40 or others.


James
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#3 AJ Burdette

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Posted 20 May 2009 - 03:53 PM

I guess it will be too much work for you to mutate the N terminus and start it over. If you change the hydrophobic aa of your N terminus to hydrophilic, it will not be the anchor.

It is hard to release the protein without using the detergent to destroy the membrane. Have you tried other mild detergent such as 0.5% NP40 or others.


James
Understanding Life One Protein At a Time...
www.lifetein.com
A peptide synthesis company.


our data shows that besides detergent, only high ph can release the protein from the membranes so I'm not sure if the interaction is hydrophobic. Unfortunately there is another segment of the protein in the middle of the AAA domain that is also anchoring it to the membranes, so I'm afraid any alteration of the amino acids might compromise the overall protein structure.

The mildest detergent I've tried is triton x-100 at 0.1% which kills the atpase activity and does not really solubilize any of the membranes either at that concentration.

#4 phage434

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Posted 20 May 2009 - 07:01 PM

Have you checked to see if the N terminus has an export/lipoprotein tag which would insert it into the membrane? The tag would have a basic N terminus, a hydrophobic region, and a conserved cysteine about 20 AA into the coding region. Processing would cleave before the cysteine, and then attach lipids to the -SH and -NH2 of the (now N terminal) cysteine.




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