I use a pET vector to express a protein of two amino-acid tandem repeats. I found the protein was truncated half way where there is no stop code. I tried all temperatures, hours and IPTG concentrations for induction and also denatured/native purifications but all comes to the same results. I am just wondering if somebody could help me solve the issue. Thanks!
Do you switch your codons around, and did you make sure you used the codons normally used by E coli?
Repetitive DNA sequences are intrinsically unstable (http://www.pnas.org/content/98/15/8319.full.pdf). If you have money I would recommend to switch for synthetic optimized gene. During optimization codons will be shuffled around, reducing/eliminating repetitive sequences that cause ribosome slippage (for example 5’-AAAAAA-3’ is classical ribosome slippage sequence in E.coli). As well two commercial E.coli strains: BLR and HMS174 have recA mutation, which is claimed to stabilize target plasmids containing repetitive sequences.
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