[DIYbio] RE: On protein expression in eukaryotes

I meant 20,000km not 20km, pardon the typos, my thumbs are dumb today.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

Hello everyone!

Hope all is well in your parts of the world. I had a question that one of the many bright minds on the list may have an answer to. I've been thinking about what's important, rather more important, in the successful expression of eukaryotic proteins. In what order would you list the following (most important 10 to least important 1) when it comes to optimal protein expression:

Promoter strength
Transcription factors
Codon optimization
mRNA stability (UTRs, polyA, etc)
Kozak sequence
Terminator efficiency

The for this question came to me in the shower this morning and I've been stumped since. If you print a ton of functional mRNA via a strong constitutive promoter, but its not the most stable thus degrades quickly, would it compare in terms of total protein content to a weaker promoter with a nicely stabilized (native-like) mRNA?

I see super viral promoters as a quick and dirty way to increase protein content. In plants for example the cauliflower mosaic virus promoter region (35s) is the most widely used (especially since final Monsanto patent expired last year or so), and is the cornerstone for basic transient and stable expression vectors. That's all fine and dandy but no viral promoter-driven protein can match rubisco in terms of total protein by mass. Since its the most important protein in plants, its produced ad nauseum and maintained at a very high concentration. Attempts to replicate that kind of protein concentration have failed and IIRC 10% is the current ceiling in transient plant based protein expression. Simply overdriving the transcription is not enough, but what % of all the factors contributing to protein production does strong transcription rate fall under. On the other hand, would a ton of stable mRNA floating around be detrimental to the system? Its hogging ribosomes, so to speak, and the whole cytosol gets flooded with non stop ticker tape parade of recipes for making this one protein. Could that actually limit the production of other proteins by occupying more ribosomes than the rest, statistically speaking? It seems like we tend to underestimate just how large cytosolic space is in comparison to a strand of rna, but also how crowded it is when you take into account the cytoskeleton and its multi-lane super highway of kinase bound proteins. Not sure how to see it. Kinda like the whole argument about spaceship battle scenes in star wars, especially those involving bobbing and weaving around asteroids, where in reality the average distance between any two bodies in our solar system alone is about 15-20km. Anything closer would begin to interact via gravity and accrete. Digression...

Is there some synergistic property that allows for speed greater than the individual effects combined? Assume we are talking about a simplified or minimal promoter just for arguments sake. No fancy 1000 base upstream enhancer sequence that magically folds onto RNAPII and does amazing things. :P

I'm on a hard promoter design kick but want to toy with UTRs and kozaks to see what changes what. Its a long term project with tons of work but I think its worth it, if anything for personal discovery. Ill share my results if I get anything interesting but until then, could someone spare their two cents on the matter? Thanks!



Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D