bashert Posted January 28, 2014 Share Posted January 28, 2014 hi everyone... I'm hoping to get a few ideas about what could be going on with this expression system. I'm currently expressing an enzyme in W3110 cells. We discovered a silent mutation that increases production of the enzyme of interest but it also seems to increase the background expression as well. Any thoughts on if that could be possible? Or is it likely to be an artifact of the experiment? thanks~~~! Link to comment Share on other sites More sharing options...
chadn737 Posted January 28, 2014 Share Posted January 28, 2014 How are you measuring expression, by protein or mRNA? If protein, it could have something to do with the tRNA pool or translational efficiency. There are examples of silent mutations that improve translation. If it is by transcription, the situation seems a lot murkier. Link to comment Share on other sites More sharing options...
bashert Posted January 28, 2014 Author Share Posted January 28, 2014 I've measured expression by protein. I'm currently trying to find examples of silent mutations in one protein that improve global protein expression. I think that is basically what I'm seeing. Link to comment Share on other sites More sharing options...
chadn737 Posted January 28, 2014 Share Posted January 28, 2014 It might be interesting to measure expression at the mRNA level as well. If you see no difference between your mutant and wt by qPCR, then it would seem that the effect is at the translational level. A silent mutation shouldn't inhibit protein stability. Another possibility is that the mutation blocks the effect of small RNAs and so increases/stabilizes the level of mRNA. Link to comment Share on other sites More sharing options...
CharonY Posted January 28, 2014 Share Posted January 28, 2014 hi everyone... I'm hoping to get a few ideas about what could be going on with this expression system. I'm currently expressing an enzyme in W3110 cells. We discovered a silent mutation that increases production of the enzyme of interest but it also seems to increase the background expression as well. Any thoughts on if that could be possible? Or is it likely to be an artifact of the experiment? thanks~~~! Background expression? You mean that your general protein amount is higher prior normalization? If so an artifact is the most likely culprit. Silent mutations can have regulatory effects, depending on locus, but I would first make sure that the values are properly normalized. Link to comment Share on other sites More sharing options...
bashert Posted January 28, 2014 Author Share Posted January 28, 2014 hi CharonY...yeah, by background I mean the other normally expressed proteins produced. I normalized by cell paste concentration prior to lysing the cells. Link to comment Share on other sites More sharing options...
chadn737 Posted January 28, 2014 Share Posted January 28, 2014 hi CharonY...yeah, by background I mean the other normally expressed proteins produced. I normalized by cell paste concentration prior to lysing the cells. So other proteins are being expressed at higher levels as well? Link to comment Share on other sites More sharing options...
CharonY Posted January 28, 2014 Share Posted January 28, 2014 hi CharonY...yeah, by background I mean the other normally expressed proteins produced. I normalized by cell paste concentration prior to lysing the cells. So you do not do an internal calibration. Chances are then that if other proteins are higher you have simply started with a higher total amount. I.e. there is some error in protein measurement. What you can do is assess the variance in your measurements taking all potential issues into account (e.g. due to calibration issues, instrument stability, reagent use, timing etc.) and see whether that covers the observed differences. All in all it really points to technical issues rather than biological effect. Link to comment Share on other sites More sharing options...
bashert Posted January 28, 2014 Author Share Posted January 28, 2014 hi! Yeah, Chad, some of the other proteins are expressed at higher amounts. Thanks Charon, I'm redoing to see if I can repeat and try to be exact with my normalizations. The more I think about it, the more it seems likely. thanks all!! Link to comment Share on other sites More sharing options...
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