low pET21 expression
am trying to express an 18kDa protein which I had synthesized and codon optimized for ecoli. It has been successfully inserted into pET21a(+) (confirmed via sequencing and restriction digest) but growth in BL21(DE3) cells has been less than optimal. I am following the protocol we have established in our group for this same protein from different species (mouse, bovine, trout...while this new one is zebrafish): 4 hours growth in LB media with AMP at 37 degrees (OD at least 0.6), induction with 1mM IPTG, grow for another 4 hours at 37 degrees. The SDS PAGE gel shows my protein is there, just not over-expressing (I am going to need a lot - and even after purification it is still just a small band). I have been playing with IPTG concentration (0.5-2.0 mM does not seem to change), induction time (4-6 hours after induction does not change anything), and temperature (O/N induction at 24degrees didn't work).
Any suggestions would be great.
|expression , low , pet21|
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