4 ms·
Can anyone shed light on how complicated, slow, and costly it is to synthesis these proteins in vitro?
by ackbar03 5y ago
Can anyone shed light on how complicated, slow, and costly it is to synthesis these proteins in vitro?
- flobosg 5y agoThis article might help: https://cen.acs.org/articles/89/i22/Making-Peptides-Large-Scale.html https://cen.acs.org/articles/89/i22/Making-Peptides-Large-Sc...
- dnautics 5y agoBy hand a skilled practitioner of t-boc chemistry can couple one amino acid per hour, though t-boc (boc) is disfavored since workup involves using 100% HF. Fmoc is the more popular technique and it's more like two hours. Robots can do this too, there's no real time benefit (but a robot can do more than 10 peptide couplings a day and doesn't need a lunch break -- though I guess neither does a grad student). Chief problems include: you need super concentrated protected peptides, which are highly insoluble in just about any solvent, so your system needs to have tons of filters, plus stuff gunks up your apparatus over time anyways. Then, fmoc craps out at about 20 amino acids if you're lucky, due to exponential decay of yields (99%^20, not to mention later yields can be lower due to peptide chains curling on themselves), boc can go longer. Not to mention separating what you want away from side products gets difficult. Finally the wastefulness: boc chemistry you're throwing away atoms almost 1:1 with what you're putting in to the peptide. Fmoc is typically 3:1. The solvents you use are these nasty greasy fishy smelling solvents (dmf, nmp). And the activating agents are really expensive compounds that you will slowly become allergic to if you keep working with peptides. I personally have, though unpublished work, the first total (artificial) synthesis of ubiquitin, that I did as a grad student in my first year. Although it's 76 amino acids we synthesized it in two parts and stitched them together using a technique called "native chemical ligation", though we did something special called "folding-assisted native chemical ligation".
- robbiep 5y agoI would have imagined labs these days just synthesise the mRNA and then let a ribosome do it’s work?
- dnautics 5y agoYou can't always do that. Suppose you want a peptide with an unnatural amino acid... Or a natural amino acid with a specific post translational modification...
- XorNot 5y agoUnnatural amino acids can be done though - there's been a bunch of work on getting/finding ribosomes which will polymerize all sorts of whacky stuff.
- dnautics 5y agoI'm aware, I used to work next to Floyd Romesberg. Yields are low, suppose you want to put TWO strange things in. Suppose you want to build a depsipeptide (or a dipeptide analog with C=C linkage). Suppose you need a 12-mer (ribosomes are very bad at short peptides).
- cing 5y agoNot saying it's easy but ribosomal synthesis of consecutive non-canonical amino acids has been achieved by some groups (https://www.cell.com/cell-chemical-biology/fulltext/S2451-9456(16)30435-4 https://www.cell.com/cell-chemical-biology/fulltext/S2451-94..., https://pubs.acs.org/doi/10.1021/jacs.8b07247 https://pubs.acs.org/doi/10.1021/jacs.8b07247), and many of these peptides are extremely short. Figure 5 of this manuscript describes successful applications of this for hit identification often in the context of massive peptide library screens, https://pubs.acs.org/doi/10.1021/acs.accounts.1c00391 https://pubs.acs.org/doi/10.1021/acs.accounts.1c00391
- dnautics 5y agoit's not really a generalizable solution, though, because suppose you need an unnatural AA that needs a specialized ribosomal cavity and that cavity hasn't been developed yet. And you need the material by a year's time. You are not going to evolve a ribosome to make your peptide. And, there are post-translational modifications that are too big to fit into ANY cavity, though in theory you could use tricksy chemistry to figure out a downstream processing step, that chemistry may not exist yet, or it may require a pure enzyme that is a pain in the ass to obtain, etc. etc. Finally, charging the correct tRNA is not necessarily easy or efficient. Unlike well-designed programming architectures, biology is usually not "trivially composable". Oddly, SPPS is. Yes, there are corner cases, but those are more the exception than the rule.
- techbio 5y agoI had to Google ubiquitin to verify that this wasn't a dryly elaborate reddit-surprise thread. It is indeed a well-named compound, ubiquitous among eukaryotes, and even if "boc" and "fmoc" are basically just fizz and buzz to me, it sure sounds like you know what you're doing.
- cing 5y agoThere is innovation in this space. From green chemistry initiatives to replace hazardous solvents by CROs/industry invested in large-scale production of peptides, https://www.bachem.com/news/bachem-novo-nordisk-redesign-spps-for-green-chemistry/ https://www.bachem.com/news/bachem-novo-nordisk-redesign-spp..., to routine solid-phase synthesis of peptides greater than 100 amino acids in hours (https://www.science.org/doi/10.1126/science.abb2491 https://www.science.org/doi/10.1126/science.abb2491, being commercialized here https://www.amidetech.com/ https://www.amidetech.com/)
- dnautics 5y agoLol at the first one, replacing nmp with DMSO/EtOAc while an improvement in toxicity, it's still expensive, toxic, and hard to remove/recycle.
- deleted 5y ago[deleted]
- cing 5y agoOur startup routinely orders the synthesis of hundreds of peptides for technology validation and drug discovery research (not suitable for human consumption). Costs for a small quantity through a contract research organizion can be $200-$2000 USD per peptide depending on desired purity, length, and chemical complexity. For some applications peptide arrays are suitable, and can drive the costs down to $10 USD per peptide or lower. In both cases, the turnaround time is 4-6 weeks, even though a peptide chemist could do the job in about half the time for a rush order.