i´m triying to design a expression cassette for a monoclonal antibody, that needs to be produce in a tomato plant.
i have a previous aminoacid sequence of the gene i need, from a previous work that is express in another plant, the sequences start like this: QVQLVQSGAEVKKPG...
as you can see there is no start codon, my expression cassette would be in a plasmid PBI121, flanked by BAMHI-KOZAK- GENEofinterest- SEKDEL-Histag- STOPcodon -SacI.
some researchers had told that i won´t need the start codon, but i still have doubts. Hope someone could help
How do you infer from amino acid sequence, whether there is START codon or not? It would be better to show your DNA sequence.
The Kozak sequence should contain START codon, just beware, that there is also additional G, so make sure that it is in frame with your GOI.
A conventional Kozak does have the ATG in it (but as noted above it ends in ATG G). Double check your reading frame to make sure it is ok.
Out of curiosity, are His tags common in tomato plants? I used to work on Arabidopsis and that species has enough endogenous proteins have His rich regions that you got a ton of background reactivity.
you have probably the sequence of the mature antibody=protein: as it is glycosylated and secreted there is a signal peptide (with a methionine) that is removed during secretion then the mature protein starts with a Q. To express the antibody you will probably have to add the signal peptide. By the way a monoclonal antibody is made of two different chains so you need two genes... or is it a lama nanobody?
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