October 19, 2011 at 6:03 pm #15557stalinsmaParticipant
Wondering if anyone here can shed some light on this doozey –
I have a target gene sequence I want to amplify using PCR (LIC) but I’ve been told when designing my primer that I should remove the signal peptide (cleavage site at the 26th amino acid) which corresponds to removing the first 78 or so nucleotides in my sequence.
Why is this necessary? Is it just a matter of not wasting resources on replicating a sequence we don’t need in vitro when all we want is the target gene/protein?
BTW I’m using my designed primer in association with a pET vector. I know my primer must be preceded by a particular string in order to fit the vector so this primer (~25 bp) will be extremely specific. SO i don’t understand how we can have the (idealised) forward primer form:
<overhang code to fit pET vector> <ATG><remove the 78 nucleotides of the signal peptide><the rest of my primer>
Presuming the signal peptide code is removed because we just don’t want it for whatever reason (histag removal??) then I still don’t understand how we can remove a large chunk of amino acids from just after the start codon of my particular gene of interest and then just expect the primer to bond to the gene of interest.
October 19, 2011 at 6:54 pm #107032JackBeanParticipant
The target peptide is removed becuase it could cause some problems in your host. Something like that probably usually sticks out of the protein structure and is cleaved after transport, but in the bacteria there is no transport system, so it can mess up. Or if you used some other system with transport, it can be mislocated.
Of course, the primer won’t bind on the START codon. Imagine it in two parts
I unaligning part (the overhang + ATG and maybe even some more nucleotides)
II aligning part (the part which aligns behind the signal peptide) – this part must be long enough to provide strong binding
October 19, 2011 at 8:17 pm #107033stalinsmaParticipant
Yep I think I know what you mean. It would appear from the NCBI database that the signal peptide was included as part of the particular protein I searched for, although I don’t know why that is (accession number EFF03255). A SignalP analysis showed where the cleavage site was on the submitted amino acid sequence and where the mature protein began. From then on it became clear what was happening here.
Any idea why signal peptides are included in protein searches or was this a once off?
October 20, 2011 at 6:28 am #107046JackBeanParticipant
because they are part of the protein
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