Hi,
I'm planning to express a membrane protein in a cell-free system, the Purefrex 2.0 one to be exact. I've tried absolutely everything and failed to do so in E.coli.
They've put in their protocols for membrane proteins to add nanodiscs to the reaction, and in other articles that describe protocols to express membrane proteins in cell-free systems they mention to add liposomes to the reaction.
My question is, if i want the protein to produce antibodies in a rabbit, and don't really care for them being inserted into lipid membranes, can i omit nanodiscs/liposomes from the reaction and get the same or good enough protein yield?
In theory i would think yes but i'm just checking.
Thanks,
Vik
Hi Vik, if you just want to produce antibodies against your protein, you can forgo the whole protein expression altogether and just produce peptides of your protein to make polyclonals.
http://imed.med.ucm.es/Tools/antigenic.pl
http://www.iedb.org/
Steingrimur,
I considered that , but i don't know the prices for peptide synthesis seemed a little high for me.
Firstly, the iedb.org doesn't contain data for phytoplasma
Secondly, let's say i inputed my protein into the antigenic tool , it gave me 6 sequences with a graph, are the higher peaks more antigenic?
And what's the next step i synthesize the most antigenic peptide, even if it is just a 14 AA sequence and inject that? or do i go for 2 or 3 peptides?
Can you recommend a company with low prices and very reliable product for peptide synthesis?
Thank you so much for your help
Also wouldn't i have to have a peptide at least 5-10kDa in size for good antibody production?
The synthesis price for such a big peptide isn't cheap
Ok, you don't need a 5-10K peptide to make polyclonals. From what I have read, you would need 5-10 linear AA to conjugate to something like KL-hemocyanin. People have made antibodies against phosphorylated single amino acids.
These, and other online tools, give you the predictive antigenicity. For choosing a peptide, do a BLAST of the ones that show the highest probability to see if the sequence is unique to your protein and not rabbit proteins.
Unfortunately, I cant help with recommending a company. It has been over 10 years since I had polyclonals made. You would have to shop around.
If the prices are to high for you, you can always express the peptides.
Hi Vicken,
You might get another solution already, but I just would like to share some information.
Technically speaking, you can synthesize membrane protein even in the absence liposomes.
By adding liposomes, you would get higher protein yield (roughly 1.3-fold increase).
Fig2 and S2 of the following paper are good reference.
Comprehensive study of liposome-assisted synthesis of membrane proteins using a reconstituted cell-free translation system
Niwa et al. Sci. Rep. (2015)
https://www.nature.com/articles/srep18025
However, as shown in the above paper, membrane proteins cannot fold properly and make aggregates in the absence of lipid. Although I’m not familiar with antibody production, I don’t think aggregated protein would work for your purpose.
Sawazaki group in Japan published a paper, which you would be interested in.
Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay
Takeda et al. Sci. Rep. (2015)
https://www.nature.com/articles/srep11333
FYI
Hideaki
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