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Why is solid phase peptide synthesis limited to 70 amino acids?
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+ Biochemistry
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Nabeel Khan
Why is solid phase peptide synthesis limited to 70 amino acids?
The issue is one of yield. Each step has a certain yield percentage of yield, that the synthesizers will maximize as much as possible. However, given that it is fairly impossible to generate 100% yield all the time, the amount of desired protein you get over time will deteriorate exponentially with length.
For example, say that each step gives you 95% yield. If you have a protein that is ten amino acids long, your final step will give you .95^10 yield, which is about 60%.
However, with the same yield per step, but a 70 amino acid chain, the amount of protein you want drops to 2%.
Even if you are at 99% yield per step (a difficult feat), your final yield will be under 50% for a 70 amino acid chain.
The issue is one of yield. Each step has a certain yield percentage of yield, that the synthesizers will maximize as much as possible. However, given that it is fairly impossible to generate 100% yield all the time, the amount of desired protein you get over time will deteriorate exponentially with length.
For example, say that each step gives you 95% yield. If you have a protein that is ten amino acids long, your final step will give you .95^10 yield, which is about 60%.
However, with the same yield per step, but a 70 amino acid chain, the amount of protein you want drops to 2%.
Even if you are at 99% yield per step (a difficult feat), your final yield will be under 50% for a 70 amino acid chain.
No. You wouldnt use this method at all for a bigger protein. Improving the efficiency would be far too expensive relative to its effectiveness. To make a larger protein, youd just make a bacteria do it.More
Very good answer, so to improve this method for create a bigger protein, we will need to reduce the number of reaction (or using other substance to have more percent of efficiency) ?More
Thank you for your easy to understand answer. But can we reuse the 5% unreacted peptide to use in the next reaction and thus we wont lost anything?More
The yield is not just a function of unreacted peptide. The peptide may have undergone a completely different reaction entirely, or it could be lost in transferring to another beaker. In the lab, the situation is ambiguous enough that we cannot be certain. In solid phase synthesis, we try to give a huge excess the amino acid were trying to react relative to the peptide, but that may or may not work as much as we would want, depending.More
Good answer. If anything, this is on the optimistic side, as for each residue added, there are at least two synthetic steps: deprotection of the terminal end (usually removing Fmoc from the N-terminus) and then the coupling to the next residue.More
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Adding to the already good answers:
Each added amino acid in solid phase peptide synthesis actually consists of three steps:
coupling of the new peptide
capping any unreacted peptide (optional)
deprotecting the newly-added amino group.
The first step is clear. Capping is required because not all free amino groups will be coupled to the new peptide and you want to prevent hard to remove side-products in your mixture. Deprotection then liberates the amino group of the new amino acid so it can form another peptide bond.
This now means that when you couple 70 amino acids, you are in fact looking at 140 or 210 reactions. Even if the deprotection step gives you $99.9\:\%$ yield, you still lose substance every coupling step — and this is where the other answers’ calculations step in.
Each added amino acid in solid phase peptide synthesis actually consists of three steps:
coupling of the new peptide
capping any unreacted peptide (optional)
deprotecting the newly-added amino group.
The first step is clear. Capping is required because not all free amino groups will be coupled to the new peptide and you want to prevent hard to remove side-products in your mixture. Deprotection then liberates the amino group of the new amino acid so it can form another peptide bond.
This now means that when you couple 70 amino acids, you are in fact looking at 140 or 210 reactions. Even if the deprotection step gives you $99.9\:\%$ yield, you still lose substance every coupling step — and this is where the other answers’ calculations step in.
Yeahh, i like this answer, i thought about join multiple fragments today, i think that is the best idea to create a protein without using bacteria.More
@DavidWang technically, yes. In practice, it is a lot easier to create larger peptides either biologically (i.e. letting bacteria do the job) or to synthesise multiple fragments of a length maximum 50 and fuse them together later. (convergent approach)More
Does it mean that its possible to create a bigger protein with this method, but we will need more and more material ? Can i say that : if we have enough material, we can create an unlimited protein length that we want?More
The issue is one of yield. Each step has a certain yield percentage of yield, that the synthesizers will maximize as much as possible. However, given that it is fairly impossible to generate 100% yield all the time, the amount of desired protein you get over time will deteriorate exponentially with length.
For example, say that each step gives you 95% yield. If you have a protein that is ten amino acids long, your final step will give you .95^10 yield, which is about 60%.
However, with the same yield per step, but a 70 amino acid chain, the amount of protein you want drops to 2%.
Even if you are at 99% yield per step (a difficult feat), your final yield will be under 50% for a 70 amino acid chain.
The issue is one of yield. Each step has a certain yield percentage of yield, that the synthesizers will maximize as much as possible. However, given that it is fairly impossible to generate 100% yield all the time, the amount of desired protein you get over time will deteriorate exponentially with length.
For example, say that each step gives you 95% yield. If you have a protein that is ten amino acids long, your final step will give you .95^10 yield, which is about 60%.
However, with the same yield per step, but a 70 amino acid chain, the amount of protein you want drops to 2%.
Even if you are at 99% yield per step (a difficult feat), your final yield will be under 50% for a 70 amino acid chain.
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Adding to the already good answers:
Each added amino acid in solid phase peptide synthesis actually consists of three steps:
coupling of the new peptide
capping any unreacted peptide (optional)
deprotecting the newly-added amino group.
The first step is clear. Capping is required because not all free amino groups will be coupled to the new peptide and you want to prevent hard to remove side-products in your mixture. Deprotection then liberates the amino group of the new amino acid so it can form another peptide bond.
This now means that when you couple 70 amino acids, you are in fact looking at 140 or 210 reactions. Even if the deprotection step gives you $99.9\:\%$ yield, you still lose substance every coupling step — and this is where the other answers’ calculations step in.
Adding to the already good answers:
Each added amino acid in solid phase peptide synthesis actually consists of three steps:
coupling of the new peptide
capping any unreacted peptide (optional)
deprotecting the newly-added amino group.
The first step is clear. Capping is required because not all free amino groups will be coupled to the new peptide and you want to prevent hard to remove side-products in your mixture. Deprotection then liberates the amino group of the new amino acid so it can form another peptide bond.
This now means that when you couple 70 amino acids, you are in fact looking at 140 or 210 reactions. Even if the deprotection step gives you $99.9\:\%$ yield, you still lose substance every coupling step — and this is where the other answers’ calculations step in.
More
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