I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli
However I don't know I should only inserting the Coding sequence ,or I can just insert whole cDNA into the plasmid and let the bacteria express.I am curious whether the 5' and 3' UTR will express or not,and if they express ,will they change my interested protein structure? Thank you so much
It very much depends upon the upstream sequence. You need to have a suitable ribosome binding site properly positioned before the start codon to get good expression. So if your sequence does not have one, then it is likely you will not get good expression.
It usually would be best to PCR amplify your open reading frame and clone directly into an E. coli expression plasmid if you want to get good expression. And you can design your primers
to have the correctly positioned restriction sites for easy cloning.
Just amplifying CDS region(From ATG to TAA/TAG) and Kozak squence before start codon(ATG) is enough for overexpression. 3'UTR and 5'UTR would not alter protein structure.
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