PhD-HappyToHelp
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Some time ago I posted about clinical versus lab grade chemicals
<O
</OSince then, I've done a lot more Follistatin research and wanted to summarize one particular aspect of it for everyone. Specifically, what are the manufacturing differences possible with Follistatin and why should you care?
<O</OThis is going to be complicated – no way around that. But, I will summarize into “Rules” as I go.
First, understand that I am talking about what is normally called Follistatin 344 (FS344). FS344 is a particular isoform (version) that is different from FS288. Specifically, FS344 does not bind activin well (which you do not want), but still binds myostatin (which you do want). I would not suggest anyone take FS288, because although it may have the same myostatin inhibition, it could have side effects not present with FS344.
Second, FS344 turns into FS315. The FS344 protein undergoes post-translational modification that removes a 29 amino-acid signal sequence from the amino terminal end (the “front”), turning a 344 amino acid protein into a 315 amino acid protein.
<O</ORefs:
<O</OFST (follistatin)<O</O
Molecular heterogeneity of follistatin, an activ... [J Biol Chem. 1993] - PubMed - NCBI<O</O
INHIBITION OF MYOSTATIN WITH EMPHASIS ON FOLLISTATIN AS A THERAPY FOR MUSCLE DISEASE
Note that the signal peptide is cleaved off in the golgi, which is inside the cell that produced the FS in the first place. The point of that is that FS344 is actually, with “normal” FS (that is, FS produced by your body), never found in the blood stream. This is because the signal peptide was cleaved off before secretion, and so FS315 is what ends up in the blood.
I previously mentioned that the FS344->FS315 change occurs by chopping a piece off the amino-terminal (also called N-terminal) end. This is important because the N-terminal end is where myostatin binds. (See The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding : Abstract : The EMBO Journal
<O</OConversely, the other end of the molecule, the carboxy-terminal (also called C-terminal) end, is what, in some forms, binds activin and related cell-surface molecules.
<O</OWhat does that tell us? Making a very broad, and perhaps inaccurate, generalization, if you screw up the N-terminal end of Follistatin, you may disrupt its myostatin binding (bad). If you screw up the C-terminal end of Follistatin, you may disrupt its activin binding (not bad – in fact, we don’t care about that at all, and it’s what we want with the FS344/315 isoform anyway.
<O</O
So, FS344 and FS315 are related as FS344 generates FS315 (IN CELLS – more on that later). FS288 is not. FS288 has a precursor protein called FS317, instead of FS344. Don’t think that because FS315 and FS317 sound similar that they are. You do not want FS317 or FS288. You want FS344/FS315. And, IMHO, preferably FS315 (but good luck finding that for a reasonable price – it seems like most companies are selling FS344).<O</O
<O</O
Rule 1: FS344/315. Not FS317/288.
<O</OHowever, I have one outstanding question here: Since FS344 is turned into FS315 *inside* cells, and that isn’t going to happen with injections, why is everyone making and selling FS344 instead of FS315? Has it been proven to work the same? Because when VIRAL studies use FS344, the virus makes the FS344 inside cells, and the cell then turns it into FS315 upon secretion into the blood stream. FS344 in the blood stream never happens in nature, so no one that I have seen has tested FS344 binding to myostatin (even when they use FS344 DNA, the DNA is resulting in FS315 in the bloodstream).
<O</OI would very much like to resolve this question. If anyone has a reference to point me to, that would be great.<O</O
Next, consider that FS344 can be made and purified in more than one way. One distinction, which I am going to gloss over, is whether it is made in bacterial or mammalian cells (e.g., E. coli versus Chinese Hamster Ovary cells). I am glossing over it because, although they do result in a different product (due to lack of glycosylation in bacteria), the literature seems to indicate that glycosylation may not be important (see ScienceDirect - Biochemical and Biophysical Research Communications : Site-specific mutagenesis of human follistatin).
<O</OWhat is important about how it is made though is that fact that E. coli contains endotoxin, and so proteins from E. coli must be purified carefully to eliminate the possibility of anaphylactic shock (see my post on the topic).
<O</O
Rule 2: Product must be tested for endotoxin. This is the absolute bare minimum QA test.
<O</O
Also, consider that endotoxin aside, different manufacturing processes can have different purities, and actually produce somewhat different proteins. For example, FS315 could conceivably be produced from FS344 DNA if you were collecting SECRETED protein from a mammalian cell line. And, although I don’t think it is important, different cell lines could glycosylate or not glycosylate. Also, some companies purify with a HIS tag (also called poly-Histidine). This means they stuck something on to the end of the protein to make purification easier. They HIS tag may or may not be removed afterwards. If they are trying to be cheap, it won’t be.
<O</OAnd finally, there is simply the issue of general impurities, whatever they may be.
<O</O
Note that I have spoken to some manufacturers who refuse to reveal their manufacturing process, claiming that it is a trade secret. Bullshit. This is basic biotech stuff. You can go out on the web and find a protocol for producing Follistatin easily. It’s the experience to actually do it in the lab, and the equipment and facilities it requires that are the barrier to competition. Anyone who refuses to tell you the cell line they use, what version of Follistatin it is, what QA tests were done, or what the final purity is, are not to be trusted. You wouldn’t even have to ask a “real” company for most of this – it would be on their web site, at least in outline form.
<O</O
They tell you their Follistatin consists of amino acids 30 – 344 (the 29 amino acid signal sequence is, appropriately, gone – and since there are no extra Histidines on the end, you know that they didn’t leave a HIS tag on there), that they used CHO cells to make the Follistatin, how they measured its purity and activity, and that endotoxin has been tested and found to be below 1.0 EU per ug of protein. THAT is what you want on a web page. Not just “1mg Follistatin X dollars,” which should leave you with a ton of nagging questions.
<O</O
Rule 3: You have a right to know how the product was made, including the basic process, and any Quality Assurance tests performed.
<O</O
Note that Rule 2 is really a subset of Rule 3. But, Rule 2 is “absolute bare minimum.” Shoot for good answers to Rule 3. There can still be contaminants that will hurt you, even without endotoxin.
<O</O
So, I know that was a lot to digest. But, it boils down to 3 simple rules:
<O</O
Rule 1: FS344/315. Not FS317/288.
Rule 2: Product must be tested for endotoxin. This is the absolute bare minimum QA test.
Rule 3: You have a right to know how the product was made, including the basic process, and any Quality Assurance tests performed.
<O</O
Rule 3 is tough because it may be hard for you to interpret without a lot of knowledge about biotech. But, hopefully this information will help, as will the example at R&D Systems. If their stuff wasn’t so darn expensive, that is exactly what you would want.
Good luck, and be safe!
<O
</OSince then, I've done a lot more Follistatin research and wanted to summarize one particular aspect of it for everyone. Specifically, what are the manufacturing differences possible with Follistatin and why should you care?<O
</OThis is going to be complicated – no way around that. But, I will summarize into “Rules” as I go.First, understand that I am talking about what is normally called Follistatin 344 (FS344). FS344 is a particular isoform (version) that is different from FS288. Specifically, FS344 does not bind activin well (which you do not want), but still binds myostatin (which you do want). I would not suggest anyone take FS288, because although it may have the same myostatin inhibition, it could have side effects not present with FS344.
Second, FS344 turns into FS315. The FS344 protein undergoes post-translational modification that removes a 29 amino-acid signal sequence from the amino terminal end (the “front”), turning a 344 amino acid protein into a 315 amino acid protein.
<O
</ORefs:<O
</OFST (follistatin)<O</OMolecular heterogeneity of follistatin, an activ... [J Biol Chem. 1993] - PubMed - NCBI<O
</OINHIBITION OF MYOSTATIN WITH EMPHASIS ON FOLLISTATIN AS A THERAPY FOR MUSCLE DISEASE
Note that the signal peptide is cleaved off in the golgi, which is inside the cell that produced the FS in the first place. The point of that is that FS344 is actually, with “normal” FS (that is, FS produced by your body), never found in the blood stream. This is because the signal peptide was cleaved off before secretion, and so FS315 is what ends up in the blood.
I previously mentioned that the FS344->FS315 change occurs by chopping a piece off the amino-terminal (also called N-terminal) end. This is important because the N-terminal end is where myostatin binds. (See The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding : Abstract : The EMBO Journal
<O
</OConversely, the other end of the molecule, the carboxy-terminal (also called C-terminal) end, is what, in some forms, binds activin and related cell-surface molecules.<O
</OWhat does that tell us? Making a very broad, and perhaps inaccurate, generalization, if you screw up the N-terminal end of Follistatin, you may disrupt its myostatin binding (bad). If you screw up the C-terminal end of Follistatin, you may disrupt its activin binding (not bad – in fact, we don’t care about that at all, and it’s what we want with the FS344/315 isoform anyway.<O
</OSo, FS344 and FS315 are related as FS344 generates FS315 (IN CELLS – more on that later). FS288 is not. FS288 has a precursor protein called FS317, instead of FS344. Don’t think that because FS315 and FS317 sound similar that they are. You do not want FS317 or FS288. You want FS344/FS315. And, IMHO, preferably FS315 (but good luck finding that for a reasonable price – it seems like most companies are selling FS344).<O
</O<O
</ORule 1: FS344/315. Not FS317/288.
<O
</OHowever, I have one outstanding question here: Since FS344 is turned into FS315 *inside* cells, and that isn’t going to happen with injections, why is everyone making and selling FS344 instead of FS315? Has it been proven to work the same? Because when VIRAL studies use FS344, the virus makes the FS344 inside cells, and the cell then turns it into FS315 upon secretion into the blood stream. FS344 in the blood stream never happens in nature, so no one that I have seen has tested FS344 binding to myostatin (even when they use FS344 DNA, the DNA is resulting in FS315 in the bloodstream).<O
</OI would very much like to resolve this question. If anyone has a reference to point me to, that would be great.<O</ONext, consider that FS344 can be made and purified in more than one way. One distinction, which I am going to gloss over, is whether it is made in bacterial or mammalian cells (e.g., E. coli versus Chinese Hamster Ovary cells). I am glossing over it because, although they do result in a different product (due to lack of glycosylation in bacteria), the literature seems to indicate that glycosylation may not be important (see ScienceDirect - Biochemical and Biophysical Research Communications : Site-specific mutagenesis of human follistatin).
<O
</OWhat is important about how it is made though is that fact that E. coli contains endotoxin, and so proteins from E. coli must be purified carefully to eliminate the possibility of anaphylactic shock (see my post on the topic).<O
</ORule 2: Product must be tested for endotoxin. This is the absolute bare minimum QA test.
<O
</OAlso, consider that endotoxin aside, different manufacturing processes can have different purities, and actually produce somewhat different proteins. For example, FS315 could conceivably be produced from FS344 DNA if you were collecting SECRETED protein from a mammalian cell line. And, although I don’t think it is important, different cell lines could glycosylate or not glycosylate. Also, some companies purify with a HIS tag (also called poly-Histidine). This means they stuck something on to the end of the protein to make purification easier. They HIS tag may or may not be removed afterwards. If they are trying to be cheap, it won’t be.
<O
</OAnd finally, there is simply the issue of general impurities, whatever they may be.<O
</ONote that I have spoken to some manufacturers who refuse to reveal their manufacturing process, claiming that it is a trade secret. Bullshit. This is basic biotech stuff. You can go out on the web and find a protocol for producing Follistatin easily. It’s the experience to actually do it in the lab, and the equipment and facilities it requires that are the barrier to competition. Anyone who refuses to tell you the cell line they use, what version of Follistatin it is, what QA tests were done, or what the final purity is, are not to be trusted. You wouldn’t even have to ask a “real” company for most of this – it would be on their web site, at least in outline form.
<O
</OThey tell you their Follistatin consists of amino acids 30 – 344 (the 29 amino acid signal sequence is, appropriately, gone – and since there are no extra Histidines on the end, you know that they didn’t leave a HIS tag on there), that they used CHO cells to make the Follistatin, how they measured its purity and activity, and that endotoxin has been tested and found to be below 1.0 EU per ug of protein. THAT is what you want on a web page. Not just “1mg Follistatin X dollars,” which should leave you with a ton of nagging questions.
<O
</ORule 3: You have a right to know how the product was made, including the basic process, and any Quality Assurance tests performed.
<O
</ONote that Rule 2 is really a subset of Rule 3. But, Rule 2 is “absolute bare minimum.” Shoot for good answers to Rule 3. There can still be contaminants that will hurt you, even without endotoxin.
<O
</OSo, I know that was a lot to digest. But, it boils down to 3 simple rules:
<O
</ORule 1: FS344/315. Not FS317/288.
Rule 2: Product must be tested for endotoxin. This is the absolute bare minimum QA test.
Rule 3: You have a right to know how the product was made, including the basic process, and any Quality Assurance tests performed.
<O
</ORule 3 is tough because it may be hard for you to interpret without a lot of knowledge about biotech. But, hopefully this information will help, as will the example at R&D Systems. If their stuff wasn’t so darn expensive, that is exactly what you would want.
Good luck, and be safe!
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