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A single point mutation enhanced protein expression but attenuated its function

single point mutation protein expression protein function

Best Answer rkay447, 28 February 2019 - 02:29 PM

A potential dominant negative.  The increase in protein expression is either due to a loss of degradation or gain in transcription and/or translation.  I'd first do experiments with either MG132 to shut down proteolysis versus cyclohexamide to stop protein production.  You'll be able to determine the reason for the increased expression.  

 

A negative feedback loop isn't unusual.  When the function goes down, the cell increases expression to compensate.  But the lack of activity could be due to many, many different reasons.   

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#1 Thomson

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Posted 12 February 2019 - 02:37 AM

Hi, 

 

Recently while working on a alanine mutagenesis scanning, I noticed a single amino acid change (Ala to Phe) of one of the residue in a particular extracellular loop of a multispan transmembrane protein caused multiple folds of increased in total protein expression as observed on WB, and 2-3 folds of increased surface expression as detected in flow cyto (compared to wild type). However, functional analysis showed it attenuates the functionality up to 20 folds as compared to wild type protein. Typically, increased protein (for wild type) expression should increase the function of the protein, we found that increased expression of this mutant follows the same trend (but even 10 times more plasmid used for transfection, its function only increases slightly, still about 10 folds lower activity than wild type). Meaning, the lack of activity is not because of the expression since the mutant is more expressed, it's just that more expresion are probably required for the mutant to have comparable activity to the wild type.

 

I am scratching my head hard lately. What do you think could be the possible reason?

Any opinions are welcome.

 

Thanks.



#2 mdfenko

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Posted 12 February 2019 - 12:08 PM

change in conformation of the protein can affect activity


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#3 Thomson

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Posted 13 February 2019 - 06:00 PM

Thanks, I have considered that too.

So I guess the conformation change of the protein making it lack activity and requires more expression to rescue its functionality. 

But could it be a negative feedback mechanism? I wonder why the expression increased so much for the mutant, yet the activity of the protein is still substandard. 



#4 mdfenko

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Posted 15 February 2019 - 03:38 PM

it could be in greater abundance due to lower susceptibility to proteolysis


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#5 rkay447

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Posted 28 February 2019 - 02:29 PM   Best Answer

A potential dominant negative.  The increase in protein expression is either due to a loss of degradation or gain in transcription and/or translation.  I'd first do experiments with either MG132 to shut down proteolysis versus cyclohexamide to stop protein production.  You'll be able to determine the reason for the increased expression.  

 

A negative feedback loop isn't unusual.  When the function goes down, the cell increases expression to compensate.  But the lack of activity could be due to many, many different reasons.   


Edited by rkay447, 28 February 2019 - 02:30 PM.






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