Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
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I thinking of doing my PhD in manipulating plants to produce antibodies for humans. I’m kind of stuck on the part of working out the gene that makes the antibody?...I know they are companies online that can produce a specific antibody from injecting a mammal with an antigen. I think I can get 20uL for a cost. I assume I derive the DNA sequence from this, thus allowing me to create the gene.....right?..through an automated technique. I guess my question is...what technique I need to determine the DNA of the antibody?.....how much antibody do I need to use with this technique?...any other advice you could give me would greatly appreciated. Also, understand I haven’t started the PhD...I am just preparing the research proposal.
Thanks in advance
A place you are likely to run into problems is in getting the pattern of glycosylation right. B cells & plasma cells attach sugars to antibodies. The pattern of glycosylation might be critical for interaction with other systems in the animal, such as T cells or complement proteins. Can you get the plant to attach sugars in similar places?
as far as I know, glycosylation signals are conserved among all eukaryotes. For example, I just expressed chicken avidin into yeast and it gets glycosylated just fine
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter
There are differences in glycosylation even among eukaryotes, and even among quite closely related species. Yeasts often over-glycosylate their products, although this is not to say they couldn't get it right in many cases. Even such commonly used species as S. cerevisiae and P. pastoris can have differences in glycosylation if you express the same protein in them.
Unfortunately I don't remember what was the case with plants and glycosylation, but like Jonmoulton said, antibodies have very specific places for sugar residues that you need to get right (in some proteins sugars don't play a crucial role, but here you might want them in their right places). Then again, if I recall correctly, they make (humanised) recombinant proteins for therapeutical uses even in E. coli (a species that has no clue about glycosylation whatsoever...)
Another problem you may encounter with plants is protein extraction: it is much more laborous to isolate your specific proteins from plant mass than it is from neatly secreted products that hybridoma cells and bacteria make.
I guess you would have to determine the amino acid sequence of the antibody, translate that into genetic code and synthesise the gene sequence de novo, then clone your synthetic gene into an expression vector and transform it into your plants (several ways to do this).
The bad news - a lot of this is quite hard. Peptide sequencing in particular requires specialist equipment and knowledge that may not be available in most labs. Even then, I get the impression that it can be a long and difficult process.
The good news - you can pay people to do the hardest parts for you.
http://www.proteomefactory.com/proteomi ... index.html
Gene synthesis (a few places do this, here's an example):
http://www.epochbiolabs.com/genesynthes ... e=products
I don't know how much these services would cost, you'd have to ring them up and get quotes. Could be pricey though.
Have you thought about applying for a PhD with a project already set up? I honestly wouldn't envy you in setting out on this project unless you could outsource a lot of it or work with a lab who are very experienced in these techniques. Otherwise it sounds like it would be a pretty extreme learning curve and 3 years could be optimistic. I might be wrong though as I haven't personally attempted any of these procedures.
Also bear in mind that there are a number of patents covering antibody production in plants, it could possibly be worth having at least a rough idea of this from the start if you are looking at an endpoint of commercial production.
8 posts • Page 1 of 1
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