Frameshift in pET vector? - (Nov/09/2007 )
Hello all,
I tried to express a eukaryotic protein in E.coli but couldn`t get it to work. I re-analysed the sequencing data of the plasmid construct. The insert should have been ligated into the NdeI and HindIII restriction sites of pET22b+. What I found was that during ligation into the NdeI site several additional bases have been inserted between the ribosomal binding site and the Nde site containing the start codon. Do you think that this might be a reason for the expression failure?
chalet2
it's possible for a frameshift.
Thanks for your answer. Is there generally a certain number of bases between the RBS and the start codon in prokaryotes? Does the length of this spacer region influence the expression?
chalet2
In general, the SD (RBS) sequence, concensus AGGAGG is located about 6 bp before the start codon (usually ATG).
hi
can u tell me how are u checking expression?
can u tell me how are u checking expression?
By SDS-PAGE and Western Blot, but I do not see an overexpression. Solution will be to subclone the genes again.
Do you know what the extra sequence is? BLAST the sequence to find out or align it to your vector and insert. It's always good to know what the extra sequence is because you may have made a simple mistake in making the construct and knowing what the sequence is will help you avoid doing it again. Maybe you didn't make a mistake but something else fishy happened, either way it is good to know exactly what happened.
Yes, I know the sequence of the extra bases added. I got the construct from someone and am not sure how these bases could have been inserted there.