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pelB leader - how to use

periplasm pet

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#1 Sindit

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Posted 05 October 2011 - 09:15 AM

Hello,

I intend to express a sequence in pET 22 with the pelB signal to send the product to periplasm.
So, when designing the primers, I use to put 4 random bases + restriction enzyme site + start of the gene sequence. In this case, I believe I should use NCO I enzyme restriction, but if I design the primer this way I think that it will change the reading frame from the pelB, since NCO ends CCATGG, I will try to explain it better:

CAG,CCG,GCG,ATG,GCC,ATG,GAT
Pro - Ala - Met Ala Met - Asp
the signal peptidase ends on Ala and if I put my gene sequence after the NCO I sequence, it will be read in a different r. frame:

CAG,CCG,GCG,ATG,GCC,ATG,GAT G GCA TGG...
Pro - Ala - Met Ala Met - Asp

so, how should I design primers for this? should I design the gene sequence after the NCO? should I put some random bases to adjust the frame, or the reading frame doesn't affect it at all? I will repeat the link for pET 22 map here
Please, this is very important to me, thank you.

#2 allynspear

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Posted 05 October 2011 - 11:32 AM

pET22 is designed to have things cloned into either the MscI site (Blunt) or the NcoI site. Using the NcoI site, they are assuming you will use the ATG codon in the NcoI recognition site as the initiating ATG in your gene of interest.

So if your gene starts out:
ATGGCATGG

Then your primer should be:
NNNNNNCCATGGCATGG.................

This should keep your gene in frame with the pelB leader, and the signal peptidase will cleave off the pelB leader after the Ala, leaving your authentic amino terminal end (Met-Ala-Trp-.....).

On a side note, I alway use 6 nucleotides upstream of my restriction sites on primers since nearly all enzymes capable of cutting PCR products will be at maximum efficiency with 6 or more.

Best of luck.




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