I'm trying to express the protein in E. coli, but not sure which host to choose. BL21(DE3) doesn't express it very efficiently. Expression conditions are 37C/0.3mM IPTG/4h.
Is there a reason you're expressing it in bacteria? It's so easy now to express in mammalian cells... What are you planning to do with the TNF?
Good luck
G
Check with DH5-Alpha E.coli cells, it will be suitable for the expression of TNF-alpha. good luck.
There are modified BL21 (DE3) strains that carry plasmids expressing t RNA for codons rarely expressed in E.coli and therby help overcome the problems due to presence of rare codons in the target gene.There are also expression plasmids which use strong promoters like T7 RNA polymerase to increase the recombinant protein expression.
@Grant: I'm going to use it as target protein. I'm also aware that it can be expressed in mammalian cells, but that part is being handled by someone else. We want to see if there's difference in the response to the same protein with two different origins.
you could try tagging for solubility . else try T7 shuffle cells (variant of BL 21)
@Varun: How can DH5-alpha express a gene in a pET/T7 based expression system?
@Anirudh: Thanks for the info, will check it out.
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