We can, though the technology is already in place to just give a batch of e. Coli a chain of DNA to code and let it do the work for us.

You can actually order protein sequences from a company and get it shipped to you in a few days, iirc.

Yes, but it gets tricky to scale up purely chemical processes for protein synthesis (making small quantities is easy, making larger quantities gets expensive and complicated) -- and the problem gets worse, the larger the protein gets.

As already mentioned -- protein expression in bacteria is usually more efficient.

Yes, it is however incredibly cumbersome and difficult to get to any size and the folding is sometimes aided by specific cell organels. So if you have a protein you want, just make it in e.coli. As for the DNA part, we can design DNA de novo aswell. It is also cumbersome and slow, but you can print a bit of DNA. So if you want to make an engineered enzyme just do that, it is for now much easier.

Folding of proteins and post transcription modifications are significant enough and different that for instance insulin can only be made in eukaryotes if you want it to really function well in human systems.

Definitely not a few days. Maybe a few weeks. Shortest turn around I'm aware of for just DNA is 2-4 weeks for whole genes, and for proteins are going to be maybe a week on top of that at a bare minimum.

Source: I work in this area :)

That’s called solid phase peptide synthesis. You start with a single amino acid linked to a solid resin through a cleavable linker. The peptide is built up with amino acids that are selectively protected on the amine (and usually any reactive side chain functional groups) and have a free carboxylic acid. The acid is ‘activated’ into a more reactive form and added to the resin where the growing chain has a free amine to react with it. Then the reactants are washed off and the amine protecting group is chemically removed to expose a new amine and the cycle repeats. When the full peptide has been made there is a global deprotection process that also cleaves the peptide from the resin. It usually needs HPLC purification to separate it from peptides that may have missed a coupling or two.

The major power of this method is that you can readily introduce all sorts of unnatural amino acids or even entirely different kinds of chemical functionalities (e.g. esters instead of amides). While there are ways to do that biochemically, there’s much more flexibility with solid phase synthesis.

As others have noted, there are some limitations to the process. The most important is scale - because it’s being built on a resin you’re limited by the number of sites where a peptide can start from, so you’re generally going to get milligrams to tens of milligrams out. Second, there can be complications depending on how favorable it is for the growing peptide to fold in on itself, which can happen even using strong solvents like DMF. That can keep the end of the peptide from efficiently reacting, so you’ll end up with errors.

With all of that said, it’s relatively trivial to make 20-mers with automated synthesis. Once you get out to the 60-mer range it becomes challenging but not impossible. Much longer than that and you’re probably better off with biochemical synthesis, either in cells or a cell-free extract.

You got the right info below, but I want to comment to elaborate a hair.

As others have noted, you can do this with cumbersome chemistry, but we tend to much more easily use DNA to make protein, just like cells do. Why is that so much easier? Because we're leveraging the molecular machines that already exist in cells for this exact purpose--and they are GREAT at what they do!

It's not a metaphor to call enzymes in the cells machines--they really are, just made differently than machines humans make. So now that we know how to leverage the machines inside cells (or even take them out of cells) as little protein factories, is so much easier than trying to do the chemical steps individually--that's exactly what the existing machines, honed by a billion years of evolution, are built to do. And they are amazing at it.

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yaa ya im aware of that i was just wondering why chemical synthesis is almost never done .

thanks

In short, yes, but not all proteins are equal here. Longer ones are harder to make.

There are techniques for immobilized substrate synthesis. Basically you glue you starting amino acids to a surface, and you wash your next amino acid reaction blend over to attach the first one. Rinse and repeat for your growing peptide.

This is great for smaller peptides, but massive biopeptides become another beast. G-mod yeast is another great tool as its a cheap way to make abunch of a target protein or enzyme. This is awesome if the enzyme you want already exists in nature.

But fully synthetic peptides? Especially enzymes? Very very very tricky. Some of the most powerful computational chemistry goes toward predicting how proteins will fold, and folding into the right shape is crucial. Fold into the wrong shape and you get Mad Cow Disease, for example, or most often just a dud.

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thanks man thats real helpful

Proteins are floppy for lack of a better word. They're long and stringy, and can have multiple results. Think of it like trying to react spaghetti, and each way you bend the spaghetti gives you a different result.

When I was in college in the early 90's I had professors spending lots of supercomputer time just trying to predict protein folding.

IDT's gBlocks have a 5-8 day turnaround for tubes of DNA up to 3000bp, if you're ever in a crunch.

Chemical synthesis takes care of primary structure, but the relatively extreme chemical environment needed typically results in secondary and tertiary structures that do not happen when the macromolecule is synthesized in biological conditions. This is generally due to denaturing (loss of structure). DNA is fairly forgiving when it comes to structure, as nearly all of the information it encodes is in its sequence.

Proteins, on the other hand, use their sequence to create structures to do functions. These structures are relatively delicate and after denaturing are unlikely to return to their original, functional shape. You've experienced this with cooked eggs, where the high heat causes the proteins in the egg to denature and tangle in a way analgous to synthetic condition. "Uncooking" your product as part of its processing is an extremely difficult step that I've never had success with, and generally scales poorly.

There's other factors, like the maturity of the processes (DNA > Protein), the ability to incorporate post-translational modification (DNA > Protein), and the ability to rapidly scale up protein production cheaply via fermentation (DNA > Protein). Once you factor in other concerns, like hazardous materials in synthesis, where you do one hazardous synthesis with DNA before using relatively safe fermentation feedstocks compared to a hazardous synthesis for every milligram of protein you need, it's difficult to justify not just transforming organisms or using a cell-free extract to produce proteins.

That being said, as protein synthesis matures, it may have significant advantages on turnaround time, throughout, and the ability to incorporate non-cannonical amino acids. Currently it can take years to develop strains that can incorporate a single non-cannonical amino acid, and this tech could radically increase the possible sequence space for proteins. It's just not there yet, and it'll likely be a while before we get there. We still have issues with DNA synthesis, and that's a technology that's far ahead of protein synthesis.

To add to this. Many proteins require post-translational modifications that bacteria cannot perform. In those cases yeast cells are often used to produce the protein.

We can make peptides but not proteins.

The key difference is length. People typically call oligomers of ~50 amino acids peptides. Typical proteins are 100 amino acids or longer.

As others here point out, you can do solid phase synthesis for peptides. However, the limited coupling efficiency (the success rate of adding an amino acid to an existing peptide) and the tendency of growing amino acid chains to adopt conformations that interfere with coupling make it impractical to synthesize proteins.

For example, let's assume that we have a typical coupling efficiency of 98% for adding a single amino acid. For a 50 AA peptide, the success rate would be 0.98^50 = 36%. For a 100 AA protein, this rate drops to 13%, and 200 AA would imply only 1.7% yield.

The above estimate is likely far too optimistic as coupling efficiencies likely decrease with length.

When biological enzymes make protein, they use a molecular machine with error correction to ensure very high coupling efficiencies.

Even if we could make 100 AA length proteins synthetically, another problem is getting the protein to fold correctly. Proteins have their functions because they adopt a specific 3D shape. This 3D shape results from proteins folding in a specific way as they are being synthesized in the biological environment. When we make peptides by chemical synthesis, they are in a non-biological solvent. If you drop a long chain of amino acids made this way into the biological context, they could well fold into a tangled mess that will never function correctly.

RNA is the same way. Biotech industry typically synthesize short RNA drugs (less than 100 nucleotides) with all sorts of chemical modifications using synthetic chemistry), but for long mRNAs such as those in the Covid vaccines, they use enzymes to do in vitro transcription.

Would it be possible/feasible to do a middle ground, where you take ribosomes outside of a cell, a printed mRNA (AFAIK it's relatively trivial to print DNA and RNA on demand) and those chemically different amino acids on custom tRNA, and have the ribosome assemble the protein?

Or insect, or mammalian, depending on the application.

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To obtain functional proteins it often requires post-translational modifications. Most commonly this is phosphorylation but you also have various structural modifications, glycosylation (adding sugars), adding lipids and more.

Then downstream, depending on its properties it may be difficult to separate it from contaminants from the upstream processes mentioned above. Attachment to some resin based on charge or a binding site can be used in a chromatography setup. Solubility or inertness in some chemical can help. Finally regular filtration or some sort of hollow fibre tff setup.

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Trying to avoid burdening my last comment, there’s also a technique called native chemical ligation that can allow you to make full length proteins semi-synthetically: part of the protein is made in cells, part of it is made synthetically, then the two are stitched together. This relies on peptides called inteins that naturally cleave themselves from pro-peptides. It relies on a cysteine to act as a nucleophile and can splice two sections into a new polypeptide. So under the right conditions if you have one peptide with an intein and another with a terminal cysteine you can get them to link up. There are still some real limitations - it’s easiest when the synthetic part goes at the end so you don’t have to sandwich it with two different reactions. You also need to have a cysteine in the vicinity or be able to make a mutant that tolerates the substitution. And, as others have noted, you still have to be able to get it to fold, which is often not trivial.

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Yes absolutely! If you're really interested you can look into a very famous chemistry professor named Samuel Danishefsky - he carried out the total synthesis of a fully glycosylated Erythropoietin protein. This took probably ~200-400 Phd years (20 people ~ 10- 20 years) to fully complete as there are MANY challenges that come along the way.

Fundamentally, its much much easier to do semi-synthesis or coupling of large chunks of proteins that have been synthesized by cells in cell culture. If you're interested in semi-synthesis you can look at famous chemical biologists like Tom Muir.

The VAST majority of times, when researchers/scientists want a protein, they simply ask bateria/yeast to express it by adding in the DNA that encodes for that protein. The cells natural protein producing machinery produces it, the cells are killed, and that protein is extracted and purified. This is FAR cheaper and more reliable than other methods to date. IF you want to make an unnatural protein - you can simply just change the DNA sequence that you're transfecting the bacteria with.

SYNTHETICALLY we can make proteins from total scratch. Its a TON of work, but has been done before (mostly for the purpose of understanding and optimizing really hard chemistry - not for large scale production).

1. Coupling amino acids in LARGE iterations has poor yields. - if you make a protein with 500 amino acids, and want to couple them step by step, that would be (excluding intermediate steps) 1000 reactions. EVEN at a 99.5% efficiency you're going to have huge problems (0.995^500) = an 8.1% yield. What is EVEN worse is the remaining 91.9% of the material is VERY similar to your product and you will have issues separating it (unless you tag it and use special chromatography).
2. Chemical reactions become HARDER (substantially) as molecules get larger. Fundamentally to reaction kinetics is the "number of productive collisions". If two objects (molecules) collide and they're small a huge fraction of their surface (say 10%) is reactive. If these molecules are much larger the portion of their surface that is reactive decreases with the surface area of the molecule making is much lower (say 0.1%). This slow down in rate makes some reactions on a large scale simply too slow to function.
3. Mammalian Proteins are Glycosylated - this process is inherently heterogeneous. Most of proteins that are produced in humans and other mammals are covered with glycans (sugars) of varying length (1-20 monomer sugars). This post-transnational labeling of proteins is not consisten (same animal, same protein, may exist with a distribution of types/locations of sugars). Sugar chemistry is FAR more difficult that protein synthesis and this proposes a MASSIVE challenge in ensuring you get the "correct/natural" protein that is in your body. The exact source of the protein (yeast, human tissue, bacteria) will produce the "same protein (amino acid sequence)", but variants of their attached glycans.

NOTE* Anything with 50 amino acids or less (very small proteins or peptides) CAN be produced on scale chemically quite easily with a process known as Solid-Phase Peptide synthesis. For large scale, industries actually do liquid phase peptide synthesis as its higher yielding (but very labor/time intensive for a single person).

That's called in vitro translation, and it is an established research method. It's more commonly done with some sort of cell lysate than just ribosomes and charged tRNAs etc. that are directly involved in the reaction.

> > > > > Folding of proteins and post transcription modifications are significant enough and different that for instance insulin can only be made in eukaryotes if you want it to really function well in human systems.

Well, there are ways around it. Some companies produce insulin in bacteria, while others produce it in yeast. It's a tradeoff since yeast is better at making it correctly in the first place, but is otherwise a less efficient and more complicated expression system than bacteria.

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> > > Mammalian Proteins are Glycosylated - this process is inherently heterogeneous. Most of proteins that are produced in humans and other mammals are covered with glycans (sugars) of varying length (1-20 monomer sugars).

Extracellular ones yes, but it's less common for cytoplasmic proteins.

On the other hand, you have a crapton of other modifications that are common on intracellular proteins. Phosphorylation, methylation, acetylation, acylations in general, SUMOylation, ubiquitination, neddylation, succinylation and so on and on.

> Attachment to some resin based on charge or a binding site can be used in a chromatography setup. Solubility or inertness in some chemical can help. Finally regular filtration or some sort of hollow fibre tff setup.

Yep. A very typical workflow would be preliminary semi-purification over an affinity column followed by isolation of your protein of interest over a size exclusion column. Just one, but very common, method.

Where I work we manufacture polypeptides through chemical synthesis, peptides are short amino acid chains. The longest polypeptide we produce is 60 amino acids long which I believe can be considered a protein. Its by no means a large volume production, we manufacture around 500 kg of various polypeptides per year.

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OH BOY. This is actually what I did my PhD and Post doc doing!

We can! In fact we do it a lot.

As many others have pointed out, this is done through Solid Phase Peptide Synthesis (SPPS). Here we take a solid resin that looks just like plasticy sand, and amino acid (or two) at a time, we attach them using essentially fancy activated ester chemistry. We can do this pretty well up to ~80ish amino acids, with some new high flow reactors doing 120-150 amino acid proteins (<1% yield).

Basically the issue is the most simple reaction for adding a new amino acid is generally 3 chemical steps. 1) Deprotection of the resin; 2) Activating the incoming amino acid; 3) Coupling the amino acid. Then you repeat it over and over. If your efficiency of any of these steps is under 99.5%, you will have basically zero yield at longer peptides. To combat this, we've generated some amazing chemicals to make the active esters, and have begun using microwave SPPS to make the coupling happen very very fast and efficiently.

Now on to your real question. To make full length proteins, what we do is we do what's called Native Chemical Ligation. This is where we add on specialized termini to our peptides so that once we have a ~40-50 amino acid peptide, we can mix them together and boom, we have a longer peptide. Repeat this a handful of times and you can make longer proteins.

This has been done to make a lot of histone proteins, including histone H1, which is 212 amino acids that included a number of post-translational modifications. I think this had an 8% overall yield? I forget

Now that we know what we're doing, it still takes ~1-2 years for a post-doc to work out all of the kinks for a longer protein before they can make enough to do research with it. For larger amounts of material it is probably not worth it.

> We can make peptides but not proteins.

This is usually true in most cases, but it can be done. I study a relatively small enzyme (~15 kDa) in my research, and there is a paper describing a method to synthetically make the entire enzyme. They even tested the activity of their synthetic enzyme and found it was basically identical to biochemically produced enzyme.

That said, you won’t find me using solid phase peptide synthesis to make this enzyme - E. coli are far better at it than I am!

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That's what... 130-140 AA? Where they able to crystallize the proteins? I'd like to see exactly how close the structures are... with a protein that small, I would assume that there aren't many concerns with folding. But I haven't worked in biochem for over a decade, so I'm constantly surprised.

Yes, it's possible, up to a certain length. But when you don't have enzymes doing the work for you, it's hard to get the exact sequence the way you want it after a certain length due to errors. Enzymes that do this type of work are highly conserved and highly specialized for their jobs. See this for chemical synthesis: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6270108/

Also worth noting is that protein structure is just as important as, if not more important than, protein sequence. Protein structure and folding is what conveys enzymatic activity and function. The tougher challenge in modern chemistry has been getting proteins to fold the way you want it to.

or you could make several pieces on solid phase then combine them together. there are many caveats but this can work

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Yes, so basically you have 2 options to do this. The first is purely chemical using eg temperature and pressure. The problem is that when doing this method you get a lot of throughput but little control on direction the amino acid is added basicly you add v and v together and sometimes you get vv and the other x% of time you get v^. The problem here is that both react differently. Especially as proteins compose as little as 10 amino acids to thousands it can add up.

The second method is to use enzymes (other proteins) to create new proteins. This is actually what most living organisms including bacteria and humans use to make new proteins. It has the benefit of reducing the needed temperature and pressure while also often ligating amino acids in thesame direction. To make this easier we mostly let other bacteria such as E. Coli make those proteins by cloning the gene (DNA) for that protein into the bacteria and letting it produce it in larger quantities. This proces is often referred to as fermentation. The other, more expensive process is biokatalysis where instead of using an organism you use seperate enzymes in sequential order to synthesise your protein. This is usable for smaller/less complex proteins.

Yes, it's called native chemical ligation

See also: Cell-Free Protein Synthesis (wiki) and the PURE system (NEB)

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Ngl, it's amazing to me that pretty much everyone is saying solid-phase peptide synthesis (SPS) when the ring-opening polymerization of N-carboxyanhydrides (NCAs) is a thing and, imho, far superior to the cumbersome, albeit Nobel Prize winning SPS method (shout-out Bruce Merrifield, absolute unit, made the first synthetic insulin using SPS). But NCA polymerization allows you to get polypeptides of several hundred-to-several thousand amino acids long so... In short, yes we can. And it's going to get even better in the near future.

But that actually was not a good answer in the end. We'll never be able to achieve functional proteins through synthetic methods. Insulin sold today is made via pig liver cell lines and it's because nature does it best. Another commenter touched on in vivo post-modification (glycosylation, acetylation, etc.) and that blue-print is basically mother nature's best kept secret and it likely will always remain so.

Excellent thread though, thanks for a thought provoking question.

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That is the essence of protection/deprotection chemistry. The peptide is generally attached to the resin using a functional group is is susceptible to the same cleavage conditions as the side chain protecting groups (HF for Boc peptides, TFA for Fmoc peptides) so that global deprotection and resin cleavage happen simultaneously.

You normally add a tag such as ctag, histag, monoFc or biotin to assist with purification.

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