I am using a pET14b vector (with ampicillin resistance) to express and purify several recombinant proteins but I always have problems on expression of recombinant protein production larger than 50kDa.
I got several success on 20-25kDa size recombinants in LB media with a pET14b vector.
Does anyone have experience on recombinant proteins larger than 50kDa?
Several research papers used Kanamycin resistant vectors and does it help for protein expression in E. coli larger than 50kDa recombinants??
Our lab has been using baculovirus to express proteins over 100 kDa (LEKTI and LBP-32). You can buy baculovirus system from Life tech. It is easy and fast. Good luck!
Hi Huihun,
Did you check that the codon usage of the genes you are trying to clone is compatible with good expression in E. coli ? Rare codons may be the cause of your problem. If so, you could try to introduce aplasmid overexpressing rare codons, like pRARE or pCodonPlus. Producing proteins up to and above 100 kDa in E. coli has been achieved many times, although proteolysis is often a problem. As to kanamycin, its main advantage is that it is not destroyed by resistant cells , unlike ampicillin and other beta lactams, which tend to disappear especially in high density cultures, leading to a loss of selection pressure to maintain the plasmid and invasion of the culture with segregants. But it has little to do with expressing large proteins to my knowledge.
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