Hello, I am trying to express a fusion protein using a pRSET A vector in C41(DE3) E. coli strains. I have tried many ways and I am trying to see if my sequence is correct. My question is if it is necessary to add methionine (ATG) after the BamH I cloning site in orther to make the expression work or it is enough with the ATG codon included in the vector between RBS and the histidine tag.
Thank you!
The ATG upstream of HIS tag should be enough for optimal expression of your gene. Adding an extra ATG at the start of your gene may not be of any help when you are interested in expressing your gene as a fusion with another peptide upstream of your gene of interest. In my opinion, additional ATG may increase the possibility of your gene being encoded as a mixture of HIS-tagged and un-tagged protein.
As Shyambabu Chaurasiya mentionned, the vector was designed to express your gene of interest with a His-tag. If you add another Start codon after the tag, you may loose a lot of protein when purifying it because some will be tag-less and won't bind to your column, resulting in low yield.
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