protein expression in BL21 strain

dieter.gerards at uni-koeln.de dieter.gerards at uni-koeln.de
Sat Dec 5 08:28:54 EST 1998


> I cloned 2 fragments (hydrophobic C-terminus of proteins) into the
>vector pTFT74, which is under control of the T7 promoter. The
constructs
>are expressed in BL21(DE3), which is a particular strain of E.coli
used
>for protein production in inclusion bodies. I also transformed
XL-I-Blue
>cells to sequence my constructs.
>When I isolate the DNA from XL-I-Blue, everything looks ok (bands at
>right height on DNA gel). Sequencing is ok (right frame). I
transformed
>then BL21(DE3) cells with this same DNA (same clone), and isolated
the
>DNA to check if everything is still ok.... and problems appeared: the
>constructs seem to be degraded (bands 30% shorter on agarose gel). Of
>course, my protein is not expressed (checked on SDS-PAGE) !!.
>
>Very frustrating ! I repeated the whole many times, and the same
happens
>each time. Can anybody help me ??
>Did anybody already work with BL21(DE3) cells ??
>Any suggestion welcome
>thanks

hi
i have just been expressing two proteins in BL21(DE3)  (two different vektors) 
and the cells didnt grow at all in LB-Medium. May be they are toxic.
i also transformed the vektors in DH5alpha to check the sequences.
as mentioned before, if LB is used, there is a leaky expression
resulting from ß-galaktosides in yeast extrakt.
so i use NZCYM  (Maniatis, no yeast extract) for expression and the
cells grow fine and express my proteins after induktion with IPTG.

hope it helps
regards
dieter.gerards.uni-koeln.de
Dieter Gerards



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