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Recombinant protein aggregation after purification


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#1 Lauren T

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Posted 05 August 2010 - 09:02 PM

Hi all,

I am having some big troubles with recombinant protein aggregation after purifying the protein. So let me start from the beginning...

1) Recombinant protein with MBP tag made in E.Coli
2) Protein isolated (normal methods, expression, cell lysis, centrifugation etc)
3) Protein ran though a maltose column with SDS rinse to strip purified protein from column, dialysed with PBS buffer.
4) SDS-PAGE gel ran expected MW of protein ~67kDa (including MBP tag)
5) Observed several larger proteins (140kda - dimer of original protein, and even maybe a trimer in the HMW range).
6) I need to isolate the single 67kDa protein, tried putting through 100kDa spin column, but the majority of the 67kDa protein did not pass through, which means that the protein is aggregating in solution and not passing through the filter.

So... I really need to disrupt the aggregation and then run it straight through the 100kDa spin column. How can I disrupt the aggregates? I am thinking a quick burst of sonication (like 3 x 20 second pulses), or perhaps some kind of detergent like either 0.1% SDS or 0.01% triton X?

Advice please!!!

Lauren

#2 HomeBrew

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Posted 06 August 2010 - 05:26 AM

Do you know they're aggregates (by western blot, for example) or could the larger bands be host proteins that bind maltose?

#3 mdfenko

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Posted 06 August 2010 - 08:02 AM

if you confirm aggregation then you can try using dtt or 2-mercaptoethanol along with sds to break them up (like with the sample buffer for sds-page).
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