rare codon usage in e coli expression very urgent!!!!!!& - (Jun/08/2012 )
hi, i have a big trouble in expressing my plant beta glucosidase. first i tried in yeast but failed. then i try in e coli and it failed too. i learned that from RaCC site, my protein includes ;
Double rare codons at positions:
4 5 66 76 248 (agg)|(aga)|(cga)1.2 307 409 cta2.2 ata3.2 ccc4.2
Triple rare codons at positions:
5 (agg)|(aga)|(cga)1.3 cta2.3 ata3.3 ccc4.3
Could anyone answer me please to solve my problem. Does this means e coli is not a proper organism for my protein to express ? could i have a chance to express my protein?
rare codon dosent mean you can not express your gene .
rare codon means your favorate expression system has a tendency to use one of specific amino acid codon more than others : that can leads for more productivity or even activity of protein .
dont worry about codon usage .
if its possible say more information about your expression vector . i think you should use a vector that consist of HIS taq for detect your protein by western blotting to detect small amount of protein.
unfortunately you didint mention about what kind of technique used for your research .
hi, thank you for your reply. i use fermentas alicator cloning kit plate 51 vector. it is new. before, i tried to clone to pet21a but failed. it has a his tag at n term,nus. now i try rosetta gami2 de3 plys s for expression but i am not certain about the host can express the protein because my vector is amp resistant and it says this strains may express the protein. not should:(
can you get your hands on some Stratagene Codon Plus(DE3) RIPL cells? You have a lot of rare codons, you might want to consider/research other expression system like pichia, S9 insect cell or some plant cell culture expression system. The amount you need for experiments will also determine what system will work for you.
thanks for your advices. i tried pichia before but faied. then i thought that i could not manage expression but now, i am pretty sure that stg is wrong with my gene:( i think plant cell culture is the best way:(( but have no time to try