Making synthetic human insulin
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- Опубліковано 22 бер 2022
- In this video we explore the traditional way that synthetic, recombinant human insulin is produced, by synthesising the A and B chains separately in separate strains of E. coli, and then combined in vitro to make insulin. We look at how ampicillin and tetracycline resistance genes, and blue-white screening with X-gal are used to identify recombinant colonies, and the role of β-galactosidase in promoting transcription and as a reporter gene. We also look at the reason for creating fusion proteins, as a precursor to the final product, and other questions that are frequently asked about the process..
Images of proteins are generated from www.rcsb.org/3d-view/1MHI and www.rcsb.org/3d-view/ngl/6tbj. All other images © Andrew Douch. biologypodcast@gmail.com
This is brilliant! In my genetics class we were unfortunately unable to delve this deep, you have scratched my itch to know more!
Wonderful video. Thanks for uploading this lecture it was tremendously helpful.
thanks! I'm glad it helped you
you are an articular genius. Thank you so much for creating this content!
You're welcome :-D
this literally was the best way to explain this concept I was so confused for my genetics exam today and now I feel like I fully understand how it works and will be prepared to answer any question on my test like wow thank you and I love your attitude you kept my attention the whole time, kudos to you I really appreciate that you took the time to organize this concept so well. 5 stars. you clearly know what youre talking about so I hope youre a teacher or a prof!
Thanks so much for letting me know that it helped you to understand. I'm chuffed. :)
Brilliant! Thank u so much
You're very welcome!
Hello, great explanation, I have one question - if we decide to synthesize complex peptide (around 130aa length with couple disulfide bridges between subunits) and this is non-natural peptide so we cant extract dna sequence from any natural cells - what is the method to pass the information about disulfide bridges into plasmid? Plasmid seems to contain „flattened” sequence of connected nuclear bases so how rybosome which „reads” plasmid knows where it must attach one aminoacid to another through a disulfide bond? Is it obligatory to biosynthesize every subunit separately and next attach them like u say here? Or there is a method to make whole target peptide „at once” ?
Thank you so much for uploading this! I love your videos so much, you are a such a great teacher
Thanks, it's great😊👍
Hi Andrew! Wonderful Video. I'm doing a presentation about this manufacturing method in a class. Which products on the market today are made using this method?
Humulin (the original) is one - there may be others. But there are several approaches that can be used. All the techniques are based on similar concepts, but perhaps use different plasmids with different antibiotic resistance genes. And of course, now, we know the DNA sequence of the the insulin genes, so cDNA can be used, whereas Genentech created a synthetic insulin gene because at the time nobody knew what the sequences were, or where their loci were.
Hello Andrew, this was an extremely helpful video :). Just an additional question, after the A and B chains are combined to form functioned insulin, how is it purified and if so, are there multiple ways in which it can be purified?
Hi Anastasia. Glad you found it helpful. I'm not really sure to be honest, but purification happens for each chain separately before they are combined, and I understand that once they are mixed together, the two (already purified) chains automatically combine to make a functional Insulin - I don't think that any further purification is needed - but I could be wrong about that.
@@andrewdouch Hi Andrew, Thank you so much for your reply back! After some investigation, the A and B chains can separately be purified through "affinity chromatography, " and then combined to form functional insulin :)
A wonderful video and an interesting and simple explanation... Thank you very much🌸🌸. My graduation project is on improving insulin production with biotechnology. Can you advise me on some references related to this topic or the source that I relied on in this video?
Hi - glad you found it helpful. The main references I used (combined with what I already knew) were these: www.pnas.org/doi/pdf/10.1073/pnas.76.1.106 , dnalc.cshl.edu/view/15928-How-insulin-is-made-using-bacteria.html , www.sciencedirect.com/science/article/pii/S0021925817304052?ref=pdf_download&fr=RR-2&rr=8947131e2d225a98, journals.plos.org/plosone/article?id=10.1371/journal.pone.0206169 . The first one is the most important, I think but the others supplied little bits of information that filled in gaps. All the best for your project.
Thank you, Mr Douch! Just one question, is the B chain the Beta-galac chain or is the beta galac apart of the A chain?
I'm not Douchy but hopefully I can answer your question in the mean time - there are two insulin sub units (A and B) so the B chain is likely referring to the insulin B sub unit gene. The beta galactosidase is attached to the start of both the insulin A and B genes (since this process is duplicated for two different subunits). So "yes" the B-gal is a part of the A chain, but only so the cell doesn't break down the protein until it's ready for purification.
Hopefully this helped - good luck with your exam tomorrow!!
The insulin protein is made of two polypeptide chains (called A and B). Each of them is cloned separately, and produced as part of a fusion protein with β-galactosidase protein. So don't get confused between the B chain and the β-gal protein. Two different things.
@@angusdillon8755 Thank you so much! thankfully I didn't need to know it in the exam!
hi, I just have a question about the transcription of the B-gal/insulin gene, from the plasmid. if there is no stop codon on the B-gal gene, does that mean the stop codon is on the insulin gene? because if there wasn’t, wouldn’t transcription just continue forever? thanks
Spot on! there is still a stop codon at the end of the Insulin gene.
no one explained this far. thank you! my question is ...why did we not put beta galac-Gene in the first place? ..............
Good question. I guess you have to do it in one order or another. If they'd put β-gal in first, we could have asked 'why not put in insulin in the first place?'.
hi douchey I had two questions about insulin:
would the universality of the genetic code also play a role in the making a synthetic insulin subunit gene? - in that the same sequence of nucleobases code for the same amino acid in most organisms.
2nd question
When insulin binds to an insulin receptor which structure is the most involved in allowing it to do so?
Could you make insulin in an apocalyptic zombie world? Walking dead the show has a character that has diabetes and apparently someone knew how to make it…….i call BS 😂 but curious still
If you had access to a well-stocked genetics lab, perhaps. The protocol
is no secret, so it's not that far-fetched.
I am working on how to infect diabetes patients with viruses containing their insulin genes am I weird for this?
Hi, I noticed that if you use cDNA from insulin mrna for your recombinant dna insert, wouldn't you have problems with the orientation of the dna insert when you try to insert the gene in your plasmid? does it even matter, how do yo deal with this problem? to my understanding you would yield approximately half correct dna insert orientation(dna insert alligned properly with operator and promoter) and half of the dna insert will be oriented backwards, thus will not be transcribe properly, is this right?