Protein Expression in E. coli

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Thu May 13 17:19:00 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

Hi Paul!
    I have found that DH5alpha is not as good as JM109 for
producing foreign proteins or fusion proteins.  We did some 
comparisons producing pGEX fusions and JM109
was the best.We never got much out of DH5 alpha.
I do agree with Michael Benedik though, you should look at the insoluable
stuff on a gel!  Also try JM109.
______________________________________
R.Daniel Gietz Ph.D. (Dan)
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)788-6458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
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