You probably can't compare something like RuBisCo to other proteins. What I think you are talking about is how much of a protein can be in a cell. This is a different question then, how much protein can we express.
This is governed:
#1 by promoter & RNAP binding site.
#2 by The protein
Sure, things such as transcription factors and such play a role but let's assume everything like that is the same between RuBisCo and our protein of interest.
Why, how is there so much RuBisCo?
Proteins need to be able to fold properly, not have many off pathway intermediates, not aggregate when folded and not have a penchant to being marked for degradation. They need to not have many nonspecific interactions and play a role in the positive fitness of the organism. I think once promoters and RNAP binding sites are the same this is what it boils down to: How stable is the protein in the cell and what does it interact with? This will keep them around. In protein accumulation staying power is the most important.
People have generated > 10% cell mass of a protein in bacteria and >10% in plants (http://www.plantcell.org/content/25/7/2429.full and http://www.plantphysiol.org/content/148/3/1212.abstract?ijkey=be8dbdf615bc1e3a8de904ba45a8319cb0810bd1&keytype2=tf_ipsecsha) similar to what RuBisCo is, maybe?(it is hard to find definitive numbers but people who say 50% are crazy because that would mean there is more RuBisCo than lignin? but who knows) So it seems to have been done.
In my knowledgeable but sometimes fault opinion it comes down to two things:
How fast can you make the protein?
But More specifically how long can you keep the protein around?
You want protein to accumulate and not be degraded.
Though some people rave about codon optimization I have never seen much of an effect in increasing the quantity of protein overexpression significantly (mostly in bacteria though) I think it's applicability is in unique circumstances.
mRNA stability and Terminator efficiency again probably have their place but I don't think these would be major factors in the majority of cases. If you are talking about squeezing every last ounce of protein out then yes take everything possible into account but the majority of the time I think you can focus on just promoter.RNAP and protein stability.
Josiah Zayner, Ph.D.
NASA Ames Research Center
http://DoItOurselfScience.blogspot.com
On Tuesday, March 11, 2014 6:45:44 PM UTC-7, Sebastian wrote:
-- This is governed:
#1 by promoter & RNAP binding site.
#2 by The protein
Sure, things such as transcription factors and such play a role but let's assume everything like that is the same between RuBisCo and our protein of interest.
Why, how is there so much RuBisCo?
Proteins need to be able to fold properly, not have many off pathway intermediates, not aggregate when folded and not have a penchant to being marked for degradation. They need to not have many nonspecific interactions and play a role in the positive fitness of the organism. I think once promoters and RNAP binding sites are the same this is what it boils down to: How stable is the protein in the cell and what does it interact with? This will keep them around. In protein accumulation staying power is the most important.
People have generated > 10% cell mass of a protein in bacteria and >10% in plants (http://www.plantcell.org/content/25/7/2429.full and http://www.plantphysiol.org/content/148/3/1212.abstract?ijkey=be8dbdf615bc1e3a8de904ba45a8319cb0810bd1&keytype2=tf_ipsecsha) similar to what RuBisCo is, maybe?(it is hard to find definitive numbers but people who say 50% are crazy because that would mean there is more RuBisCo than lignin? but who knows) So it seems to have been done.
In my knowledgeable but sometimes fault opinion it comes down to two things:
How fast can you make the protein?
But More specifically how long can you keep the protein around?
You want protein to accumulate and not be degraded.
Though some people rave about codon optimization I have never seen much of an effect in increasing the quantity of protein overexpression significantly (mostly in bacteria though) I think it's applicability is in unique circumstances.
mRNA stability and Terminator efficiency again probably have their place but I don't think these would be major factors in the majority of cases. If you are talking about squeezing every last ounce of protein out then yes take everything possible into account but the majority of the time I think you can focus on just promoter.RNAP and protein stability.
Josiah Zayner, Ph.D.
NASA Ames Research Center
http://DoItOurselfScience.blogspot.com
On Tuesday, March 11, 2014 6:45:44 PM UTC-7, Sebastian wrote:
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
From: Sebastian Cocioba
Sent: 3/11/2014 9:38 PM
To: diybio
Subject: On protein expression in eukaryotes
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
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diybio@googlegroups.com. To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To unsubscribe from this group and stop receiving emails from it, send an email to diybio+unsubscribe@googlegroups.com.
To post to this group, send email to diybio@googlegroups.com.
Visit this group at http://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/405d4f79-fc37-47c7-9ff2-d53d40a9632f%40googlegroups.com.
For more options, visit https://groups.google.com/d/optout.
0 comments:
Post a Comment