expression construct need the addition of STOP Codon? - (Jan/16/2007 )
I want to use pUAST expression vector to express a fusion protein. Is it necessary to add the stop codon at the end of the fusion construct? In fact, I did't add the stop codon, as I heard there is 3 stop codons with 3 frames at the end of the multiple cloning site. Is that right? Will some extra unrelated AA affect the expression or folding of the protein needed to be expressed? Just a little uncertain. Thanks!
I think you should add a stop codon after your protein if you want to get an authentic protein without an addition of amino acid. And if you express your protein into prokaryote host strain, you can use stop codon UAAU.
"I heard there is 3 stop codons with 3 frames at the end of the multiple cloning site. Is that right?"
Yes, expression systems often have some stop codons after MCS and inframe with fusion partner before MCS.
check if there are stop codons and where (map of MCS). some restriction enzymes encode stop codons (ex XbaI) so if your insert is followed by a stop codon, no need to insert one. if that cant be matched you better design one as extra aas are not needed anyway.