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Low pET21 expression

Low pET21 expression - Protein Forum

Low pET21 expression - Protein Forum


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Old 05-25-2011, 07:42 PM
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Default 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.
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Old 08-20-2011, 05:49 AM
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Default Re: Low pET21 expression

Protein expression in bacteria is very much depending on whether the desired protein favors the expression host. You can try Terrific broth (TB) -
Recipe for 1L
12 g Bacto tryptone
24 g Bacto yeast extract
4 mL Glycerol
100 mL 0.17M KH2PO4 and 0.72M K2HPO4, sterile, to be prepared separately from the tryptone, yeast extract, and glycerol solution.

TB is a rich media that improves the growth of bacteria and will produce higher amount of expressed proteins.
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