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How to purify unstable protein?


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10 replies to this topic

#1 alam-tus

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Posted 30 December 2011 - 02:17 AM

I am facing problem with purification of a mutant protein by E. coli expression system. Expression of that soluble protein is good and like as wild type. But after purification of that protein by Ni-NTA column, when I tried by gelfiltration it was denatured. How can I solved this problem? I have purified wild type with same procedure without any problem. Please suggest me. Thanks in advance for your suggestions.

#2 mdfenko

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Posted 30 December 2011 - 09:50 PM

denatured in what way? aggregated? reduced size?

if aggregated, is it in multiples of the formula weight? if so, does your buffer contain reducing agent?

if reduced in size, do you add protease inhibitors to your buffer? is the native protein post-translationally modified?
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#3 alam-tus

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Posted 31 December 2011 - 03:03 AM

Dear mdfenko,
Thank you very much for your kind reply. Yes, problem with size reduction. My buffer contains EDTA, DTT, glycerol, Magnesium Sulphate. The native protein is not post-translationally modified. So, what should I do to recover this problem?

Thank you again and happy new year.

Alam

#4 mdfenko

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Posted 01 January 2012 - 03:36 PM

reduced size may be due to protease activity. you should add protease inhibitors to your purification buffers.

are you sure that the mutant protein is not smaller than the wild type? or that it doesn't fold differently?
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#5 alam-tus

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Posted 01 January 2012 - 06:35 PM

Dear mdfenko,
Thank you again for your kind suggestions. Due to misunderstanding, I could not explain correctly in my previous reply. Actually I have deleted 6 amino acids in two different positions (3+3) of wild type and produced the mutant. And I have got the above result. So, please suggest me, how to recover this problem.

#6 swanny

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Posted 02 January 2012 - 02:15 PM

Hi,
Do you have any gels or chromatogram traces?
What changes did you expect from the deletion? Is it in a loop / hinge etc? Does teh deletion introduce a protease site?
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#7 alam-tus

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Posted 03 January 2012 - 08:04 PM

Yes, I have gels and chromatograms of Superose 6 Gelfiltration chromatography. In gelfiltration chromatography, I found the elution of the protein earlier than the expected position (Like as wild type) and when I ran this protein in SDS-PAGE, I found multiple bands. Although when I ran the Ni-NTA purified protein on SDS-PAGE I found the same as gelfiltration. The deletion is on loop part of the protein. The MW of the deletion mutant is almost similar to wild type, so I expected single band on the same position of the mutant as wild type. I don't know either the deletion introduce protease site or not. Please suggest me, how can I recover the problem.

#8 mdfenko

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Posted 04 January 2012 - 07:34 AM

are you saying the mutant eluted at the same volume as the wild type despite the removal of 6 amino acids?

did you take into account the contribution of the his tag to the molecular weight (do both forms of the protein have a his tag?)?

how do the multiple bands (in sds-page) relate to the protein (ie multiples of size or small differences, higher or lower or both)?

is the protein large or small (does the deletion cause a significant percentage difference in size)?

are you sure that the mutant colony is pure for the 6aa deletion?
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#9 alam-tus

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Posted 04 January 2012 - 07:51 PM

Dear mdfenko,
Thank you for your kind response. Followings are the answer of your questions-

1. No, in gelfiltration, most of the proteins eluted as denatured proteins earlier than the expected position and very small amount (2-3%, also in small volume tha the wild type) of proteins eluted in the expected position. I concentrated this small fractions and ran on SDS-PAGE and found multiple very fade bands where some were higher and some were lower MW including expected MW bands.

2. I removed his-tag from mutant and wild type by TEV-protease.

3. As I explained in the answer of question 1- I concentrated this small fractions and ran on SDS-PAGE and found multiple very fade bands where some were higher and some were lower MW including expected MW bands.

4. The mutant is little bit smaller than the wild type as 6aa of two different positions were deleted.

5. Yes, I have confirmed the deletion of 6aa by sequencing of the mutant colony.

I am waiting for your suggestions. Thanks again.

2.

#10 swanny

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Posted 04 January 2012 - 09:24 PM

You say that the mutant expresses like the wt, and it purifies on the NTA as wt does, right? Can you scan and attach a gel image from the NTA column with both wt and mutant?
Could your NTA column be contaminated? How do prepare the NTA eluate for the GF column?
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#11 mdfenko

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Posted 05 January 2012 - 12:30 PM

the mutant eluting earlier than the wild type means that what you are eluting is greater in molecular weight. since you see multiple bands in sds-page, it is most likely an aggregate.

have you eluted the mutant and wt from nta without removing the his tag? if not then try it and see if you get more reasonable results.

if that gives you the expected result then you may want to see what effect protease treatment has on the proteins in solution.
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