Will CMV promoter help nanobit paris with very low activity

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  • Last Post 02 August 2017
MARKBOND posted this 28 July 2017

Just tried all combinations of my known interactors using the HSV-TK promoter vectors but only got at best 2 fold activity above the halo-tag control pair (should be at lest 10 fold). The PKA positive control pair worked fine with 50-60 fold activity above background. Do you think I should subclone my genes to CMV vectors to boost expression? I may have high endogenous levels of the interactors and wonder if these may be competing for binding with the Nanobit tagged versions. High expression may help here. Has anyone else had better luck using CMV rather than TK? I am working in Hela and have about 60% transfection efficiency.

My proteins are also normally nuclear. Not sure if this is a problem?

 

Thanks

 

Mark

alandrem posted this 02 August 2017

Hello @Markbond,

Thanks for your question and sorry for delay in getting back to you.

In general, known interaction pairs are 10-1,000 fold higher than the LgBiT fusion co-expressed with HaloTag-SmBiT. This is only a guideline, however, and we’ve seen cases where the signal is actually dimmer. How much brighter are the different orientations vs. a mock-transfected control? If you’re not well above this control, you could consider a switch to CMV-based constructs. For the PKA pair, is the LgBiT-PRKAR2A:SmBiT-PRKACA pair 50-60 fold brighter than the LgBiT-PRKAR2A:HaloTag-SmBiT control?

For a switch to CMV-based constructs, I’d recommend our new BiBiT-ready vectors, which we have for both MCS and Flexi formats. Once you identify an optimal pair, you can use HygR and BlastR resistance genes on separate plasmids to make a stable cell line. Alternatively, you can digest both plasmids and ligate fragments from each to make a single construct where both fusions are expressed via a bi-directional CMV promoter. This is the “BiBiT” vector, which has the BlastR resistance gene, and this is our preferred approach for stable expression. The BiBiT-ready vectors are currently available via our Custom Assay Services department (Promega CAS). Because they use the CMV promoter, it will be important to dilute the plasmid DNA when using transient expression as described in Section 5.B of TM461. Without dilution, you’ll very likely see low S:B for treatments that are known/expected to modulate the interaction.  If the BiBiT system isn't of interest you could also move your constructs into any CMV vector that you currently have.

I hope this helps but please let us know if you have additional questions.

 

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