Expression vector/host for plant genomic sequence - (Oct/11/2001 )
I need to express a plant protein to be used for antibody production, but have only a genomic sequence not the cDNA. Is there any point in using E.coli (prokaryote host expression system) or should I just use a eukaryote expression system (yeast). I am worried about protein processing.Any suggestions on expression vectors, as well?
I think you shoud use yeast. We do not know the exact process it go through. so, Better use yeast. As for vector, our lab used use pbluescipt .
If you use yeast, you can use you whole genomic sequence. I am not too familiar with vectors to use in yeast, but I think you would have to use a Yeast artificial chromosome to clone you gene into so there is an origin of replication that the yeast cells can recognize. There may be other vectors to use though. An alternative is to use a prokaryote system with a vector ( I think Clontech is a good place to go with to find a good prokaryote expression vector) In this case, you would have to get rid of the introns of course. You could isolate RNA and make cDNA from the plant tissue you are studying and design primer from the 5' and 3' ends of the genomic clone since you know the sequence. Then you could PCR the coding sequenc only from the cDNA you have made. Be sure that you make the primers from the 5' and 3' UTR so you have the whole coding sequence. You should check what the vector you are going to use provides though. (ie. if your sequence needs to start right at the start codon, or if you need to provide your own polyA addition site, or if the vector has it) good luck!
Karen here - I have done some checking around and have been informed that it is unlikely that a plant protein would be processed correctly in either yeast or insect (the apparent better choice to yeast). Any comments?And that therefore the best choice is to make a cDNA.