I work on the expression vector, PET23a, in E-coli strain BL21 and Rozeta. my insert is 621 bp consisted from 8 epitopes that each epitope repeated twice with AAY linker. but this peptide didn't express in all my trails. I confused on this problem. what is your recommendation?
Hi Heidarali,
You should check whether the protein expressed in inclusion body.
If it is the problem of expression, you need to check the open reading frame of your gene in the pET23a plasmid.
If it is the problem of solubility, you can try other bacterial-expressing plasmid with soluble tag such as pET32 (trx tag), pET42 (GST tag)...
If it is the problem of yield, you can optimize the induction condition or optimize the gene sequence perfer for expressing in bacteria
But I think the 621 kb insert is a huge gene fragment for plasmid construction and bacterial expression.
Many things can happen.
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