In order to access the function of modular domains of a multidomain protein I decided to amplify and clone separately the gene fragment that codes for each domain of this protein in pET28a vector, to posterior overexpression and purification of each protein domain. I divided the gene in 5 different fragments, all were digested with NdeI/BamHI (the restriction sites were added to the primers sequence), and cloned in pET28a digested with the same enzymes.
After cloning confirmation, the plasmids were transformed into BL21. Three of the proteins expressed normally, but two of them didn’t. Coincidence or not, these two genes were amplified with the same forward primer. I sequenced the inserts, and it looks fine, no frame problems or random mutagenesis. I don’t believe in a problem with codon usage, because the full-length protein expresses well in the same system. Someone have any suggestion about what is happening and what I could do to solve my expression problem?
Thanks in advance,