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Mutation in one chain of homodimer

mutation cloning

Best Answer phage434, 08 April 2015 - 06:38 AM

If you do this, you likely will have to denature and renature the proteins after mixing. Otherwise, dimers will already be formed and stable in each of the homodimer populations. This is probably easy to do with buffer conditions, heat, and dialysis, but getting the conditions correct may be challenging. In any case, you will end up with at best 50% of your heterodimer product, and it will be extremely challenging to purify it. So, you'll need to think about working with the mixture in follow on experiments.

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#1 madmaddy77

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Posted 07 April 2015 - 10:46 PM

I am working on a protein which is a homodimer. I have to study the functional abrogation if only one chain of this homodimer is mutated.

How to specifically mutate only one chain of homodimer? 



#2 bob1

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Posted 08 April 2015 - 12:39 AM

I don't know for sure, but I suspect that you would be best off making the mutant and normal proteins in separate expression systems, purifying and then mixing together.

 

You might be able to do it by expressing both parts off two transfected  vectors (or the same vector with an IRES or similar), and then seeing if you can see a reduction in the protein function which could be attributable to a proportion of the final proteins being defective.



#3 phage434

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Posted 08 April 2015 - 06:38 AM   Best Answer

If you do this, you likely will have to denature and renature the proteins after mixing. Otherwise, dimers will already be formed and stable in each of the homodimer populations. This is probably easy to do with buffer conditions, heat, and dialysis, but getting the conditions correct may be challenging. In any case, you will end up with at best 50% of your heterodimer product, and it will be extremely challenging to purify it. So, you'll need to think about working with the mixture in follow on experiments.



#4 madmaddy77

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Posted 10 April 2015 - 04:08 AM

Thanks a ton bob1 and phage434. I will follow same protocol purifying both separately and denaturing and renaturing the protein mixture. Lets hope this one works. I only doubt homodimers and heterodimers concentration and their separation would be the challenging part. That should be critically optimized.



#5 labtastic

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Posted 29 May 2015 - 08:55 AM

This probelm recently came up in lab meeting.

 

The consensus was to co-express both the native sequence and the mutated sequence on a single dual expression vector (e.g. pETduet) with different affinity tags.

 

For example, put a strep tag on the native sequence and his-tag on the mutated sequence. Co-express on the same plasmid. First purify with the his-tag...this will enrich heterodimer and mutant homodimer. Then purify that mixture using the strep tag. What elutes off that column will be pure heterodimer.

 

His and strep tags are short enough you can encode them into primers if need be, so you don't need specialized expression vectors to do this.

 

I think this is better (and less work) than unfoldling/refolding. 


Edited by labtastic, 29 May 2015 - 10:59 AM.






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