Protein Expression in E. coli

Michael Benedik bchs1b at Elroy.UH.EDU
Thu May 13 12:40:55 EST 1993


In article <C6z5H6.C8n at news.cis.umn.edu>, paul-b at molbio.cbs.umn.edu (Paul Bucciaglia) writes:
>hello folks,
>I am trying to express a cDNA in E. coli using the FLAG vectors from IBI.  The
>vector consisits of the tac promoter driving the flag peptide (an 8 aa seq)
>fused to the cDNA of interest.  I have been able to detect soluble protein
>on western blots but the band is very faint and it ~1000 fold less abundant
>than an alkaline phophatase positive control.
>
>growth conditions:  inoculate 50 ml flask 1:100, 2xyt media. grow to 0.8
>OD600, reduce temp to 25 and grow 2-3 hrs.  Spin down cells and 
>French press.
>
>I have also tried the betaine-sorbitol media mentioned here but get no
>detectable protein.
>
>My question:  I am using DH5alpha; are there other strains of Ecoli which
>might be better?  other growht conditons I could try to improve yield
>of soluble protein?  any help would be much appreciated!
>
>paul
>
>paul-b at molbio.cbs.umn.edu
>
>paul bucciaglia

1) Do you need to add IPTG inducer and are you doing so?
2) Perhaps your protein is forming insoluble aggregate. You would miss this
by french press if you discard the junk. Taking 0.1ml of cells or so and
just boil in SDS loading buffer and run a gel. See what you get.


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 Michael Benedik				INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik at uhou
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